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UNIVERSITI TEKNOLOGI MALAYSIA
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ALI ADEL DAWOOD 1st NOVEMBER 1966 CLONING OF INFLUENZA B NS1 GENE IN Escherichia THE EFFECT OF ETHNO/LINGO DIVERSITY ON coli
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DR. CHAN GIEK FAR
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3rd DECEMBER 2010
3rd DECEMBER 2010
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: ..................................................................... : Dr. CHAN GIEK FAR : 3rd DECEMBER 2010
CLONING OF INFLUENZA B NS1 GENE IN Escherichia coli
ALI ADEL DAWOOD
A dissertation submitted in partial fulfillment of the requirements for the award of the degree of Master of Science (Biotechnology)
FACULTY OF BIOSCIENCES AND BIOENGINEERING UNIVERSITI TEKNOLOGI MALAYSIA
... The thesis has not been accepted for any degree and is not concurrently submitted in candidature of any other degree.... : Ali Adel Dawood : 3rd DECEMBER 2010
Signature Name Date
Hereby....... I declare that this thesis entitled “Cloning of influenza B NS1 gene in Escherichia coli” is the result of my own research except as cited in the references.......
Thanks for the dim of light when all I see were darkness… Thanks for giving me the best things in my life …and finally Thanks for your sacrifices to make me a better person each day….iii
To my beloved parents& my wife.
persistency and his blessings throughout my completing this project.
I am deeply indebted and grateful to my family especially my mother. guidance. support and praying for my success in every time. advices and care helped me in all the time of research and writing of this thesis.
Sincere appreciation and thanks are also extended to all staff of Mosul College of Medicine especially members of the Department of Anatomy for their encouragement. advices even in some words.
Special thanks to all the staffs of the Faculty of Biosciences and Bioengineering and members of the labs for not only helping me with my study but also making my stay in the lab a very pleasurable and memorable one.iv
In the Name of Allah.
. the Most Benevolent. father and my wife for their love.
Last but not least. Chan Giek Far. No word can express my appreciation for their love. support. guidance. for encouragement. I would like to thank to people and everyone who has helped me direct or indirectly towards completing this research project. I would like to express my utmost gratitude and special appreciation to my supervisor Dr. their patience and kindness in helping and guiding me in every part of this project. Most Merciful
Firstly. I thank Allah for giving me the patience.
respectively using restriction digestion enzymes (SacI. NS1B was extracted and attempted to be cloned into prokaryotic expression vectors pET-32b. recombinant DNA was attempted to be transformed into Escherichia coli strains BL21(DE3) and DH5α.
The non-structural NS1 protein of influenza B virus is a multifunctional virulence protein which is involved in the transport of viral RNA. NS1 protein is utilized as a target for diagnostic of influenza viruses in infected animals. NS1B antagonizes and inhibits the α/β interferon system which is induced as host antiviral response. pET-32a. Inside the host cell. it prevents the activation of double-stranded-RNA activated protein kinase (PKR) by binding to dsRNA and inhibits the maturation of GAS8 (gene of tumor suppressor). PstI and HindIII). pQE-81L and pQE-80L. pUC57 carrying NS1 synthetic gene of influenza B was attempted to be transformed into Escherichia coli strain BL21(DE3). Then.
PET-32a. pUC57 membawa gen NS1 sintetik influenza B diklonkan ke dalam Escherichia coli strain BL21(DE3). Selain itu. PstI dan HindIII). Kemudian DNA rekombinan ditransformasikan ke dalam strain Escherichia coli BL21 (DE3) dan DH5α. NS1B menghalang sistem interferon α/β yang menyebabkan tindakbalas antivirus oleh perumah. protein NS1 mencegah pengaktifan doublestranded RNA-aktif protein kinase (PKR) dengan cara mengikat dsRNA dan menghalang pematangan GAS8 (gen tumor supresor). Di dalam sel perumah. pQE-81L dan pQE-80L masing-
masing dengan menggunakan enzim pembatas (SacI. NS1 protein digunakan sebagai target untuk diagnostik terhadap virus influenza pada haiwan yang dijangkiti.vi
Protein non-struktur NS1 virus influenza B adalah virulensi pelbagai fungsi dan terlibat dalam pengangkutan RNA virus.
. NS1B diekstraksi dan diklon ke vektor ekspresi prokariotik pET-32b.
4 4 4
Overview of influenza
2.2 Influenza virus 1.3 Problem statement of the study 1.vii
TABLE OF CONTENTS
DECLARATION DEDICATION ACKNOWLEDGEMENTS ABSTRACT ABSTRAK TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF SYMBOLS/ABBREVIATIONS
ii iii iv v vi vii xii xiv xvi xix 1 1 1 2 2 3 3
LIST OF APPENDICES 1
INTRODUCTION 1.1 Influenza 1.5 Scope of the study
1.1 Influenza infection
.1.6 Significant of the study
LITERATURE REVIEW 2.4 Objective of the study 1.
3 Viral genome
Types of influenza virus
Lifecycle of influenza virus
NS1 protein prevents activation of PKR 24
Isolation of influenza virus
Preventative measures for human
7 7 8 8 8 10 11 14
NS1 protein interacts with GAS8 22
2.4.3 Influenza B virus 2.5
Previous studies of cloning and expression of NS1 gene 25
MATERIALS AND METHODS 3.2 2.1 2.viii
2.4.2 2.1 NS1B binds to ISG-15 protein 2.4.2 Influenza virus 2.3
NS1 inhibits nuclear export of mRNA 21
function and influences of NS1 protein 2.2.1
Nonstructural protein NS1 of
Influenza B virus
22.214.171.124 NS1B inhibits and antagoni-sts α/β interferon system 2.2.5
NS1 protein inhibits premRNA 23
126.96.36.199 Nonstructural (NS1) protein 2.4.
12 Transformation of recombinant product 3.14.1
Liquid and solid media preparation
3.3. coli BL21(DE3) competent cells 41 42 43 44 44 44 45 46 46 47
3.3 Methods 3.ix
3.2 3.1 3.2 Preparation of ampicillin
3.11 Ligation NS1B with pET-32b 3.10 Determination of DNA concentration
3.2.1 Luria Bertani agar and
35 35 35 35 37 38 40 40
Chemicals pET vectors pQE vectors NS1B Synthetic Gene
Isolation of pUC57-NS1B plasmid Agarose gel electrophoresis Isolation of pET-32b plasmid Single digestion
3.2 Polymerase chain reaction 3.2.1 Primer design 3.2.15
47 47 48
Double digestion using PstI and HindIII restriction enzymes in one step 49
3.9 DNA Extraction
3.4 3.3 3.14 Amplification of NS1B gene using
Polymerase Chain Reaction (PCR)
Double digestion using restriction
Preparation of competent cells
Transformation pUC57-NS1B into E.2 Materials 3.2 188.8.131.52 Isolation of pQE-81L plasmid 184.108.40.206.
Cloning of pQE-81L-NS1B into E. transformation into E.20
Isolation of pET-32a plasmid Amplification of NS1B gene using Polymerase Chain Reaction (PCR)
Isolation plasmid (pQE-80L) PCR amplification.24 3. coli DH5α 61
4.17 3. coli BL21 (DE3) and and screening of colonies 57 57
3.18.19 3. transformation into DH5α and screening of colonies
RESULTS AND DISCUSSION 4.18. double digestion.1 Chelex 100 extraction method 3.2
Cloning of pET-32b-NS1B into E. coli BL21(DE3) 65
Double digestion using SacI and HindIII restriction enzymes in one step 55
enzymes in two steps
Cloning of pQE-80L-NS1B into E. coli BL21(DE3) 59
Double digestion using SacI and HindIII restriction enzymes in two steps 55
3.20.1 Primer design
3.2 PCR amplification and gel
electrophoresis to detect NS1B
3. coli BL21(DE3) 59
50 51 52 52
Ligation and transformation into DH5α Screening of positive colonies
Transformation of pUC57-NS1B into E.4
Cloning of pET-32a-NS1B into E.
1 Conclusion 5.xi
Confirmation of ligation
CONCLUSION AND FUTURE WORKS 5.2 Future works
71 71 72
REFERENCES Appendices A-B
Double digestion components in PCR tube using PstI(2nd step) 51 53
The pUC57 primers for PCR of NS1B gene from pUC57-NS1B 48 48 49
Single digestion components in PCR tube using HindIII 44
LIST OF TABLES
Double digestion components in PCR tube using HindIII(1st step) 50
PCR reaction components of pUC57-NS1B PCR cycle Double digestion components in PCR tube using PstI and HindIII
Double digestion components in PCR tube using SacI and HindIII 55
Ligation mix components of pET-32b and NS1B 46
The pQE primers for PCR Primers containing SacI and HindIII restriction enzyme sites used to amplify NS1B gene
Double digestion components in PCR tube using HindIII(1st step) 56
Double digestion components in PCR tube using SacI(2nd step) 56
Primers containing SacI and HindIII restriction enzyme sites used to amplify NS1B gene 57
LIST OF FIGURES
2.1 2.2 2.3 2.4 2.5
Life cycle of influenza virus Influenza virus structure Electron microscope image of influenza B Influenza virus particles on cells lining Homology modeling of RNA binding domain of NS1B
10 11 13 14
Flow chart of experimental design Cloning of pET-32b-NS1B into E. coli BL21(DE3)
Cloning of pQE-81L-NS1B into E. coli DH5α Cloning of pET-32a-NS1B into E. coli BL21(DE3)
33 34 36 37 38 39 60
3.5 3.6 3.7 3.8 3.9 4.1 4.2
Cloning of pQE-80L-NS1B into E. coli DH5α pET-32 vectors pQE-80L vector pQE-81L vector pUC57-NS1B gene construct Restriction digestion by HindIII. Transformation of pET-32b-NS1B into competent E. coli BL21(DE3)
Plasmid isolation and digestion of pUC57-
NS1B and pQE-81L 4.4 4.5 PCR amplification of NS1B Transformation of pQE-81L-NS1B into competent DH5α 4.6 4.7 PCR of recombinant pQE-81L-NS1B Miniprep of pUC57-NS1B and PCR amplification of NS1B 4.8 Transformation of pET-32a-NS1B into competent E. coli BL2(DE3) 4.9 4.10 PCR of recombinant pET-32a-NS1B Transformation of pQE-80L-NS1B into competent DH5α 4.11 4.12 PCR of recombinant pQE-80L-NS1B PCR amplification
68 69 70
LIST OF SYMBOLS/ ABBREVIATIONS
AMP ATP bp BSA α/β ˚C cDNA del NS1 dH2O dNTP DNA dsRNA E. coli EDTA ELISA g GAS GST HA His-Tag IFN IPTG IRF
Adenosine monophosphate Adenosine triphosphate Base pairs Bovine Serum Albumin Alfa / Beta interferon Degree Celsius Clone deoxyribonucleic acid Deletion nonstructural 1 Deionized water Deoxynucleoside triphosphate Deoxyribonucleic acid double-stranded RNA Escherichia coli Ethylene diamenetetraacetate Enzyme linked immunosorbent assay Gram Growth arrest specific gene Glutathione S-transferase Hemagglutinine Histidine tagged Interferon Isopropyl β-D-1-thiogalactopyranoside Interferon regulator factor
ISG kb KDa LB LPAI M mM MCS ml mg min. µg µl µm MgCl2 mRNA NA NEP NES ng NLS NMR NS1 OD PACT
Interferon stimulate gene Kilo base Kilo dalton Luria Bertani Low pathogenicity avian influenza Molar Milmolar Multi cloning site Milliliter Milligram Minutes Microgram Microliter Micromter Magnesium chloride Messenger ribonucleic acid Neuraminidase Nuclear export protein Nuclear export sequence Nanogram Nuclear localization sequence Nuclear Magnetic Resonance Nonstructural 1 Optical density Protein activator of the interferon-induced protein kinase
PCR PKR RNA
Polymerase chain reaction Protein kinase Ribonucleic acid
Polyacrilamide Gel Electrophoresis
RNP rpm RT-PCR SDS-PAGE
Ribonucleoprotein Rotation per minute Reverse transcription polymerase chain reaction Sodium Dodecyl Sulphate. SIV ssRNA TAE Tris U6 SnRNA UV V
Seconds Simian immunodeficiency virus Single strand RNA Tris-acetate-EDTA 2-hydroxymethyl-2-methyl-1.3-propanediol U6 small nuclear ribonucleoprotein Ultraviolet Volts
LIST OF APPENDICES
NS1B gene sequence (870 bp) of Influenza B virus (B/Taiwan/45/2007)
NS1B protein sequence (281 amino acids)
2006. 2009).. weakness and general discomfort.1
Influenza is a contagious respiratory viral illness of global importance. Influenza B viruses
. muscle pains. Some influenza viruses can cause more severe diseases than the common cold like pneumonia. They have been classified into three distinct types: A.1
1. but have not been responsible for pandemics. Spickler et al. B and C. The most common symptoms of the flu are chills. sore throat. coughing.. This can also be transmitted by direct contact with infected animals or humans (Metreveli et al. fever. The disease was caused by influenza viruses known as flu. These viruses can cause epidemics in human populations. Influenza B viruses are mainly found in humans. sneezes. severe headache. creating aerosols containing the virus. Influenza viruses spread around the world and can be transmitted through the air by coughs.
Influenza viruses have unique features of reverse sense single strand RNA.
though it occurs more slowly than in influenza A viruses (Metreveli el al.. coli hosts.
. 2006. pQE-81L.3
Problem statements of the study
The main problem of this study is to clone NS1B gene in pET-32b.2
comprised of single group of hemagglutinin and neuraminidase antigens since their first isolation in 1940 (Nerome et al. pET-32a. 1998). and pQE-80L vectors for transformation into Escherichia coli BL21(DE3) and DH5α. Twelve antigenic variants were distinguished by a panel of monoclonal antibodies appeared to circulate in the 1981–1982 epidemic season in Japan.. pET32a. Spickler et al. and pQE-80L vectors. Influenza B viruses undergo antigenic drift. 2009).
1.. The evolutionary lineages of influenza B viruses since 1988 have been represented by two epidemic strains B/Victoria/2/87 and B/Yamagata/16/88 (Nerome et al. Influenza B viruses are categorized into lineages rather than subtypes and are also classified into strains. The recombinant constructs were transformed into E. pQE-81L. 1998)..4
Objective of the study
The objective of this research was to clone of NS1B synthetic gene into pET-32b.
Successful cloning and overexpression of NS1 gene are useful for specific diagnostic and further applications. This reverse genetic system will allow studies to explore the functions of NS1B domains during the replication cycle and to assess their contributions to the pathogenesis and virulence of influenza B virus.
Scope of the study
The scope of this study encompassed the cloning of NS1B gene of influenza B into pET-32b.
1. coli BL21(DE3) and DH5α. pET-32a. which are subsequently transformed into competent E.6
Significant of the study
Recombinant NS1 fusion protein of high purity is more significant for detection of antigenicity. pQE-81L. and pQE-80L vectors.3
Historically. this influenza pandemic killed more people than the World War 1 (Metreveli et al. During the years 1918-1920.1.. and in 1968 (H3N2). Pandemic influenza A viruses emerged three times during the last century: in 1918 (H1N1 subtype).1
Overview of influenza
2. It resulted in the death of an approximately 50-100 million people thus.
2. It was then replaced by another human influenza virus called the Asian flu virus. 2006). influenza virus caused one of the most destructive disease outbreaks in world history and later become known as the Spanish flu pandemic. the diseases occurred in the United States in 1924-1925. This virus continued to circulate in humans until 1957. and then emerged in 1929 in a much milder form.
. It’s often associated with considerable morbidity and mortality. in 1957 (H2N2). Influenza A is responsible for occasional pandemics affecting millions of people worldwide.
. 2009).. Transmission of this virus across the placenta may also be possible (Spickler et al.
. The severity of zoonotic avian influenza varies with the virus. and occasionally to swine and rare cases from person-to-person spread. Infected adults usually begin to shed influenza A viruses the day before the symptoms appear. Low pathogenicity avian influenza (LPAI) viruses generally cause asymptomatic infections and mild respiratory. 2009). Humans can transmit influenza viruses to ferrets.. and infections remain limited to individual animals or small groups (Spickler et al. Fecal shedding of the avian H5N1 virus has been documented in a child with diarrhea. Influenza C viruses infect mammals only and generally without cause disease. Severely immunocompromised individuals may remain infectious for weeks or months. Young children can shed virus up to six days before.. including a localized outbreak among recruits at a military base which have been reported in humans infected with swine influenza viruses. and are infectious for 3-5 days after initial signs. avian influenza viruses do not spread efficiently in mammals. 2006).
Influenza B can also cause similar symptoms as Influenza A.
The human influenza viruses are transmitted from person to person. High pathogenicity avian influenza (HPAI) viruses cause severe disease that can kill up to 90-100% of a poultry flock. 2006).
Currently both H3N2 and H1N1. They are genetically distinct from A and B types (Metreveli et al. No cases of sustained transmission have been reported in humans infected with the avian influenza viruses. there are two forms of disease. and 10 days or more after they become ill. Generally. but generally in a milder form. a late fifties variants that re-appeared in 1977and remained co-circulate in humans (Metreveli et al.
were most recently reported in 1918. 1957 and 1968 (Spickler et al.2
Antiviral drugs are available for influenza treatment in the United States..6
Other avian influenza viruses can also undergo cross-species transmission. resulting from antigenic shifts.
. As of January 2009. if treatment begun within the first 48 hours. Treatment usually results in milder symptoms and recovery one day sooner. Laboratory studies have shown that influenza viruses can also become resistant to zanamivir and oseltamivir. human H9N2 infections have been significantly less severe than those caused by avian HPAI H5N1 viruses. However. Human pandemics.. H9N2 viruses have been infected humans. 2009). during the 2006-2008 flu seasons. Zanamivir and oseltamivir are used effective for both influenza A and influenza B. Drug resistance develops rapidly in viruses exposed to amantadine or rimantadine and may emerge during treatment. Side effects including neuropsychiatric events may appear. Recently. LPAI H9N2 viruses have become endemic in poultry in parts of Asia and the Middle East may be of particular concern. they were found in pigs with respiratory disease and fatal paralysis in China. Epidemics occur every few years. For example. due to small changes in the influenza viruses. this appears to be less common than resistance to adamantanes (Spickler et al.
2.1. In addition. Amantadine and rimantadine (adamantanes) are active against human influenza A viruses. 2009). human influenza viruses circulating in the United States and Canada exhibited high resistance to amantadine and rimantadine.
besides the serological tests which include complement fixation.1.. Antigens can be detected in respiratory secretions by immunofluorescence or enzyme-linked immunosorbent assays (ELISA). and is updated annually (Spickler et al. It contains the viral strains which are most likely to produce epidemics during the following winter. Reverse transcription polymerase chain reaction (RT-PCR) techniques are also available. Both inactivated (injected) and live (intranasal) vaccines may available. hemagglutination inhibition and immunodiffusion.
Isolation of influenza virus
Human influenza A and influenza B infections can be diagnosed by virus isolation. The vaccine is given in the fall before the flu season. RT-PCR can be used for the diagnosis of influenza C and avian influenza viruses (Spickler et al.3
Preventative measures for human influenza viruses
An annual vaccine is available for influenza A and B. 2009). 2009). Commercial rapid diagnostic test kits such as (Directigen® Flu A test) can provide a diagnosis within 30 minutes.
. The viruses can be isolated in cell lines or chicken embryos. and also can be identified by hemagglutination inhibition tests.. A rising titer is necessary to diagnose human influenza when using serological tests. or retrospectively by serological test.1. detection of antigens or nucleic acids.
swine. B and C. These two
. NS1 and NEP are involved in various aspects in the process of taking over the host cell (Metreveli et al. the hemagglutinin (H) and neuraminidase (N) proteins.2
Types of influenza virus
Influenza viruses have been classified into three distinct types A. and release of virus particles. M1 is a matrix protein that covers the inner surface of the viral membrane. The 5th segment encodes a nucleoprotein (NP) that protects the viral RNA. Influenza viruses are found in a number of species including birds. The last segment encodes two proteins: non-structural protein 1 (NS1) and nuclear export protein (NEP). dogs beside humans. The 7th segment encodes the M1 and M2 proteins. The three largest segments (1.2. 2006).8
2. swine. equine and canine influenza viruses. 2 and 3) encode polymerase proteins. Influenza A viruses were classified into subtypes based on two surface antigens. PB2 and PA that are responsible for RNA synthesis.1
The viral genome consists of eight segments of reverse single stranded RNA or called negative polarity. There are 16 hemagglutinin antigens (H1 to H16) and nine neuraminidase antigens (N1 to N9).
2.2. as well as the human influenza A viruses. but also has other functions. A total of 11 known proteins are encoded by the genome. PB1.. Influenza A viruses include the avian. Segment numbers 4 and 6 encode surface proteins which are hemagglutinin (HA) and neuraminidase (NA) that are involved in attachment to the host cell and fusion between the viral envelope and cellular membrane. horses.2
H5N1 H5N2. place of first isolation. Influenza A viruses were also classified into strains. and antigenic subtype. 2009). Human viruses can also replicate in the nasal epithelium of experimentally infected horses. H2N2 viruses circulated in the human population between 1957 and 1968 (Spickler et al. The H1N2 viruses appeared most recently.. but are classified into strains. Experimental infections have been reported in raccoons. strain number. H4N2. Each strain is antigenically stable. 2009).
Human influenza A viruses are mainly found in humans. H9N2. H4N6. 2009). infections with high pathogenicity subtypes containing H7 or H5 can also occur (Spickler et al. H1N1.. However.9
proteins are involved in cell attachment and release virions from cells. H10N4 and H10N7. 2009). These viruses were first seen in human populations in 2001. Strains of influenza viruses are described as their types. year of isolation. H7N3. and they can also infect ferrets and sometimes swine.
. hosts. Limited subtypes are found in each species of mammal. probably as a result of genetic reassortment between the H3N2 and H1N1 viruses. Subtypes that have been found in ratites include H3N2. For example. H5N9.. Isolates from cage birds usually contain H3 or H4. and accumulates few changes over time (Spickler et al.. H1N2 and H3N2 viruses are currently in general circulation in humans.
Limited information is available on the subtypes found in other species of birds.
Influenza C viruses are not classified into subtypes. the prototype strain of the H7N7 subtype of equine influenza virus which first was isolated in Czechoslovakia in 1956 is A/eq/Prague/56 (H7N7) (Spickler et al. H7N1. and are also major targets for the immune response.
Then RNP is transported into the nucleus.10
Life cycle of influenza virus (Metreveli el al. strains vary in their affinities for different sialyloligosaccharides. Progeny virion is formed and then released from the cell by budding (Metreveli el al.3
Lifecycle of influenza virus
Life cycle of influenza virus is started through attachment to sialic acid receptor of the host cell.. The new viral RNA is encapsidated by the nucleoprotein and transported to the cell surface where envelope hemagglutinin and neuraminidase components are incorporated from the cell membrane. 2006)
. Hemagglutinin is a viral glycoprotein binds to the cell receptor sialic acid. New viral proteins are translated from transcribed mRNA and the viral RNA is replicated from the negative stranded templates.2. Viral envelope fuses with the lipid bilayer of the vesicle and releasing the viral ribonucleoprotein (RNP) into the cell cytoplasm. 2006). The virus receptor bound is taken into the cell by endocytosis.. However.
1998.magnet.like variants and B/Yamagata/16/88. 2008).html)
Influenza B viruses comprised of a single group of hemagglutinin and neuraminidase antigens since the first isolation in 1940. Until now.3
Influenza B virus
Influenza B virus is an envelope virus.11
2. it has been shown that numerous influenza A viruses circulate in wild and domestic
. Hai et al. 2007). 2008).like strains (Nerome et al.. B/Victoria/2/87.2
Influenza virus structure
(http://micro..edu/cells/viruses/influenzavirus.. It contains 8 segments of negative sense single-stranded RNA virion (Palese et al.
Figure 2. it is still unknown why influenza B does not have many natural hosts like influenza A and can be antigenily drifted (Hai et al. Influenza B viruses have only one subtype and evolve under three major lineages: B/Lee/40virus like variants. belonging to the family Orthomyxoviridae..fsu. For this difference.
Indeed. The evolutionary rate of influenza B viruses was reported to be lower than of human influenza A viruses. However. 2009). 1998). Thus. seals.12
animals such as wide variety of bird species. respectively.
The first demonstrated of influenza B viruses have evolved in multiple lineages which can cocirculate for considerable periods of time. The continuing analysis of recent epidemic viruses from evolutionary point of view showed that there have been two distinct lineages of influenza B viruses since 1988. twelve antigenic variants distinguished by a panel of monoclonal antibodies appeared to circulate in the 1981–1982 epidemic seasons in Japan. since has reported in early 1970s. Particularly. the segment number 8 encodes nonstructural
. swine. evolution of influenza B virus is definitely characterized by coexistence of several antigenic variants in the same epidemic period of influenza A virus in which a single dominant virus predominates in a single epidemic period (Nerome et al.
Despite the lack of antigenic shift. 1998). It is still uncertain why the former virus is able to survive for a long period of time without antigenic drift in the hemagglutinin glycoprotein (Nerome et al. These differences in epizootic background may lead to evidence that influenza B viruses have no antigenic shift observed (Nerome et al. being represented by the two epidemic strains B/Victoria/2/87 and B/Yamagata/16/88. it is noteworthy that a higher number of monoclonal variants of influenza B viruses have been detected in the same epidemic season than influenza A viruses. On the other hand. whales and mink. a number of antigenic variants of B viruses have been isolated during periods of widespread influenza virus activity since its first isolation in 1940. equine. some cases lead to severe diseases but rarely morbidity (Spickler et al. 1998).. For example. Most virion segments encode structural protein from reverse RNA.
Common cold is caused by influenza B and is milder than type A viruses.... This is in sharp contrast that influenza B viruses have not been isolated from animals other than humans.
proteins typically 890 nucleotides which encode for two different proteins: nonstructural protein NS1 (26 KDa) and nuclear export protein NEP (11 KDa) which was previously known as NS2 (Spickler et al.ac.
Until now. systematic deletion and insertion mutations have occurred in the same nucleotide regions of hemagglutinin molecule of influenza B viruses (Nerome et al. Recently. antigenic analysis along with investigation of genomic structure. Palese et al. 1998). it is still uncertain why former virus is able to survive for a long period time without antigenic drift.3
Electron microscope image of influenza B
Influenza virus particles on cells lining
(http://www. the influenza NS1B protein comprises of 281 amino acids.uk/Education-resources/Teaching-and-education/BigPicture/All.issues/Epidemics/WTD028128.4
Nonstructural (NS1) protein
Influenza NS1A protein consists of 230-237 amino acids. The first 93 amino acids is N-terminus. On the contrary.wellcome.14
Figure 2. which are divided into two major domains: N-terminal RNA-binding domain. which comprises the first 73 amino acids residues forming symmetric homodimer insolution.ac.htm)
2. This domain is responsible for
Other domain is located in C-terminal of protein body. 2008).7 KDa and the three-dimensional structure has not yet been determined. 2002). except that there are large loops between the three α. The RNA binding domain has a molecular weight of approximately 22..
2..helices in each chain (Yuan et al. this may path the way of utilizing NS1 protein as a target for diagnostic of influenza viruses. sequence alignment of the RNA-binding domains of the NS1A and NS1B proteins suggested that the RNA binding domain of the NS1B protein exhibits a dimeric six-helical chain fold similar to that of the NS1A protein.. Hence. 2004). Computer modeling based on the crystal structure of the N-terminal domain of the NS1A protein has been allowed alignment of the N-terminal RNA-binding domains of the two proteins. these vaccinated animals are not producing NS1 protein specific antibodies and this is allows differentiation between vaccinated and natural infected animals (Fang-kun et al ..
The crystal structure of the NS1B protein has not yet been solved.
. Nonetheless. which are thought to possess similar α-helical structures (Donelan et al. This domain is known as an effector domain which interacts with many viral and cellular factors.15
binding with nucleolin.1
Nonstructural protein NS1 of influenza B virus
The RNA-binding domain (N-terminus domain) of the NS1B protein is 93 amino acids in length and thus 20 amino acids larger than that of the NS1A protein. 2008).4. so that.
NS1 protein persists only in the infected animal cells and not in vaccinated animals. It involves in translation and post-transcriptional processing of RNA (Manasatienkij et al.
R50.5: Homology modeling of RNA binding domain of NS1B R53. R53׳and R50 ׳are the RNA binding residues (Yuan et al.. 2002). 2002)
. The large surface loops between the α-helices in the NS1B model contain 21 amino acids residues.
Figure 2.5). The ribbon representations of the predicted structure of NS1B (1-93) were shown in (Fig. Because these loops appear as insertions in the sequence alignment. so that homology modeling approach does not predict a specific structure for these loops. 2..16
The three-dimensional structure of the NS1B (1-93) RNA-binding domain is based on NMR solution structure of the NS1A (1-73) RNA-binding domain and a sequence alignment of NS1A. of the chain 2. Loop 2 (residues 64–71) of polypeptide chain 1 is located near the N-terminus of the same chain. and by symmetry loop 2 of chain 2 is located near the N-terminus of chain 2 (Yuan et al. The loop 1 (residues 24–36) of polypeptide chain 1 is located between the N and C-terminus domain respectively. these amino acids are left unconstrained in the structure calculations.
The nonstructural NS1A protein (26 KDa) of influenza virus has been shown to enter and accumulate in the nucleus of virus-infected cells independently of any other influenza viral protein (Greenspan and Palese. and a nuclear export sequence (NES). It is difficult to describe a function to this sequence with regard to viral replication. the nucleolar function of NS1 is unknown (Hale et al. First. including UL69 of human cytomegalovirus and human immunodeficiency virus that promote nuclear export of the respective viral mRNAs.
The NLSs of the influenza B virus NS1 proteins are located within the RNA-binding domains at the N-terminal 93 amino acids. RNA binding and
.4. Concurrent with NLS2 is a functional nucleolar localization signal (NoLS).. 1988).
NLS mediate the active nuclear import of NS1 via binding to cellular importin-α. NS1A contains two nuclear localization sequences (NLS1 and NLS2). which includes additional basic residues. Despite this.2
Functions and influences of NS1 protein
Previous studies suggested two nonexclusive explanations for the strong general impact of the NS1B gene deletion on viral replication.17
2. Translocation of NS1 into the nucleus is extremely rapid.. A nucleolar localization sequence (NoLS) has been reported for some strains. 2008). the NS1B protein may play an important role in counteracting the activity of antiviral gene products that are expressed in Vero cells in an IFN-independent manner. NLS2 is absent from the NS1 proteins of a large number of virus strains. 2008). NLSs overlapping with an RNA-binding domain have been described for other viral regulatory proteins. monopartite. and is concomitant with NLS2 (Hale et al. NLS1 is highly conserved. A second possible explanation for restriction of the NS1B virus in Vero cells might be a critical contribution of the NS1B protein to the principal viral replication process rather than an effect on the host cell (Dauber et al.. 2003). and involves three residues also involved in binding dsRNA.
2001 and Krug et al.. 2008).
2. However. This site is located in one of the surface loops of this domain. The N-terminal region of the NS1B protein binds not only dsRNA but also a specific cellular protein (Yuan et al. 2008).1
NS1B binds to ISG-15 protein
The RNA-binding domain at the N-terminal residue and the adjacent 94–103 regions are required for the binding of interferon stimulate gene (ISG-15) (Yuan et al. 2002). Unique biological activities of the NS1B protein are indicated by its deficiency to inhibit pre. the NS1B protein is involved in the transport of viral RNA.2. NS1B protein revealed a highly dynamic intracellular localization and accumulates in nuclear speckle which is an important for efficient virus replication.. 2002). At the late stage of infection.. which is required the whole N-terminal domain (Schneider et al.. thereby ensuring that exported viral mRNAs do not return immediately to the nucleus. The NS1B proteins are required for dsRNA binding (Yuan et al.
At the early infection. ISG-15 protein does not have detectable effect on the binding of dsRNA. NS1B protein accumulates in the cytoplasm which depends on the interaction with another transported proteins and RNAs which could be regulated by an exposed export signal (Schneider et al.4.18
nuclear imports are mutually exclusive in the Rev protein. The NLS of the NS1B protein was necessary but not sufficient to mediate speckle association. 2001). which has ability to bind to the antiviral response gene product ISG-15 and to inhibit its conjugation to cellular proteins (Yuan et al.mRNA processing.. it has not yet been determined whether. It is possible that this overlap indicates a similar regulatory mechanism. Although.. 2003).. ISG-15 and dsRNA can bind to the N-terminal 103 amino acid long fragment of the NS1B protein.
. However. 1992). The specific function of this dsRNA binding during virus infection remains elusive. 1998).. or whether it contains part of the binding site for the ISG-15 protein.. 2002). Moreover. NS1 protein was showed bind non-specifically to all ssRNAs and has greater binding activity (Hatada et al. Garcia-Sastre et al. The NS1 proteins are at a distinct disadvantage in competing with these cellular proteins for dsRNA (Yuan et al. which is an important part of the immune responses of vertebrates (Dauber et al.
The major goals is to determine the role of the N-terminal. and hence proper folding of the NS1B protein. the binding dsRNA to the N-terminal domains of the NS1A and NS1B proteins are well established in vitro (Hatada et al. whether it is required because it maintains the dimerization. 2004). 93 amino acid RNA-binding domain in ISG-15 binding. Efficient replication of influenza viruses and most other viruses necessitates are suppressed of antiviral responses mediated by the α/β interferon system.
The NS1B protein is identified as a viral factor that antagonizes interferon induction and boosts viral replication (Dauber et al. which may additionally benefit the activation of such antiviral reactions (Donelan et al...2. 2003).2
NS1B inhibits and antagonists α/β interferon system NS1 protein is involved in virulence and inhibition of the α/β interferon
system during virus infection (Donelan et al. 2006). 1992. On the contrary.. 1994). 2004..
NS1B protein has prevented the conjugation of the ubiquitin-like ISG-15 protein to cellular target proteins.. Dauber and his coworkers found that analysis of these viral mutants demonstrated inhibition of α/β interferon production is largely independent of the dsRNA binding activity of NS1 but critically relies on the presence of the C-
. Lu et al..4.
By using in vitro binding and reporter gene assays. Dauber (2003) has generated a set of isogenic influenza B viruses with mutations that either affected NS1 dsRNA binding or introduced a large C-terminal truncation. 2006). NS1B protein
prevents the nuclear translocation of interferon regulator factor (IRF-3) and especially β interferon induction in virus-infected cells. the expression of the NS1B protein complements the growth of influenza A virus with NS1A deleted (Donelan et al. 2003).
NS1B protein is not related to its ability to antagonize the interferon (IFN) induced host antiviral response. 2006). a recombinant influenza B virus with NS1 deleted induces higher levels of IRF-3 activation.20
terminal part of the protein. Further analyses of the intracellular RNA and protein targets of the NS1 domains appear to be promising strategy not only to learn more about details of the IFN suppressive activities of influenza viruses but also about the factors and mechanisms that drive cellular responses to virus infections in general (Dauber et al. 2004). it was established in previous studies that the larger Cterminal part (amino acid 94 to 281) of NS1B protein contains can suppress activation of the IFN-β promoter by Sendai virus infection (Donelan et al.
. such as VERO cells and 6-day-old eggs (Dauber et al..
The roles of dsRNA binding by NS1B protein are to promote viral replication and to inhibit of antiviral responses. The dsRNA shielding does not appear to be sufficient to prevent IFN induction by influenza B virus (Dauber et al. which presumably involves an RNA-independent mechanism. In addition. 2004)... since NS1/B/Lee virus is significantly impaired in its ability to replicate even in IFN-deficient systems. Deletion of the truncated NS1B gene residues from (1-93) protein showed that RNA-binding activity correlated with β interferon promoter inhibition.. It was shown that.
The observed lack of activity of the N-terminal domain of NS1 towards IFN inhibition in influenza B virus-infected cells underscores the important role of the C-terminal part of the protein in this process..
Specifically. The blockade of PKR activities by both of the NS1 A and B is a common theme in many virus families. so that deletion of this region would result in a virus that is more attenuated than the wild-type virus. Interactions between proteins that regulate nuclear mRNA export and their nuclear targets will be varied and complex (Qian et al.
On the other hand.21
Inhibition of IFN. This study found that the C-terminal domain of the NS1B protein possesses IFN antagonist activity. dsRNA binding was necessary for inhibiting protein kinase (PKR) activation and enable efficient viral replication.2.. 1993).. the role of dsRNA binding by the influenza B virus NS1 protein is to promote viral replication and to inhibit of antiviral responses.
Cytoplasmic as well as nuclear localization of the NSI protein occurred only when the effector domain was present and was inactivated by a point mutation.. 1993).
2. 2006). the small
. A possible explanation from the observation even wild-type NSI protein has some affinity for one or more cytoplasmic targets. 2004). Viruses containing mutations within the NS1B N-terminal domain that adversely affect binding to dsRNA may also prove to be promising live attenuated vaccine candidates (Donelan et al.β synthesis correlates with the inhibition of IRF-3 nuclear translocation.. The RNA-binding domain binds to the poly (A) sequence in mRNAs. highlighting that this enzyme is a key factor of the cellular antiviral response (Dauber et al.4.3
NS1 inhibits nuclear export of mRNA
The NS1 protein also functions inhibition rather than facilitation of nuclear export of mRNA (Qian et al. However. The effector domain and RNA binding domain of NS1 protein represses transcription.
the function of GAS8 has not yet been determined. Eleven GAS genes have been identified from a variety of biological functions.. the effect of the association between GAS8 and NS1 on virus infection is unknown. 1993).3 and was found to be a common deletion present in breast and prostate carcinomas..2.22
amount of the wild-type NS1 protein that is present in the cytoplasm has been shown to be associated with ribosomes and polysomes. Deletion analysis revealed that the N-terminal 260 amino acids of GAS8 were able to interact with NS1.4. GAS8 is located at 16q24. nerve cell growth or differentiation. apoptosis.4
NS1 protein interacts with GAS8
Growth arrest-specific (GAS) genes are expressed preferentially in cultured cells that have entered a quiescent state following serum deprivation or growth. GAS8 protein associates with microtubules in vitro and in vivo (Zhao et al.
Zhao and coworkers (2009) found that neither RNA binding domain nor the effector domain of NS1 protein interacts with GAS8. and negative and positive control of the cell cycle. It has not been established whether this ribosome associated NS1 protein has any function.
2. 2009). The Golgi apparatus localization is dependent on intact microtubules and in cell-cycle regulated. It was shown as a potential tumor suppressor gene. The GAS8 gene consists of 11 exons spanning approximately 25 kb. Although. This study
showed that interactions between proteins that regulate nuclear mRNA export and their nuclear targets will be varied and complex depending on the interaction of transcription factors with their protein targets (Qian et al. This phenomenon was even observed in the same types of cells. Examination of the localization of GAS8 protein in cells revealed that two types of localization existed: Golgi and cytoplasmic. including the control of microfilament organization. tyrosine kinase receptor activity. The mammalian GAS8 gene is a possible tumor
NS1 protein inhibits pre-mRNA
Lu and coworkers (1994) found the NS1 protein inhibits pre-mRNA splicing both in vitro and in vivo by associating with the spliceosomes that are formed from pre-mRNA. This is consistent with the fact that transient expression of NS1 in mammalian cells leads to retention of polyadenylation RNA in the nucleus and inhibition of pre-mRNA splicing (Zhao et al. Genetics of these processes have been best studied in yeast by the isolation and characterization of pre-mRNA processing.. A fraction of NS1 was also detected in association with ribosomes and polysomes in cytoplasmic fractions of infected cells (Lu et al.23
suppressor that was previously identified as one of several genes that are upregulated upon growth arrest. as shown by the relative accumulations of NS1 and NS2 mRNAs (Fortes et al. However. GAS8 was expressed in HeLa. 2009). 1994). CV-1. He found that NS1 protein associates with U6 snRNA (one of the basic protein of spliceosome) in infected cells. inactivation of the NS1 gene led to a substantial increase in the splicing efficiency. A549.2.. and DU145 cells. NS1 inhibited the maturation of GAS8 mRNA. NS1 mRNA is poorly spliced to yield NS2 mRNA. 1994). These studies found that some of the protein factors involved in the splicing reaction might act as RNA helicases and suggesting that dsRNA unwinding processes are necessary for spliceosome assembly and release of the mRNA from the splicing complex.
The nuclear export and splicing of pre-mRNAs are competing processes.
Activation of PKR results in down regulation of cellular translation and is believed to be part of an antiviral defense strategy in mammalian cells. NS1-I interaction should reveal whether one or more of the temperature.
PKR needs to be activated via a conformational change that is brought about by binding to either of its activators. double-stranded RNA (dsRNA) or the PACT protein.
. Hence.sensitive mutations and identified NS1 protein can be correlated with reduced ability to bind NS1-I (Wolf et al. Overexpression of a truncated of NS1-I protein would affect the normal course of infection and detailed mapping of the interacting domains of NS1.6
NS1 protein prevents activation of PKR
NS1 protein prevents the activation of double-stranded-RNA (dsRNA) activated protein kinase (PKR) by binding to dsRNA. They demonstrated that the direct binding of the NS1A protein to the N-terminal region of PKR can serve as such a common mechanism and this binding does not require the RNA binding activity of the NS1A protein (Li et al. despite their different modes of activation. Host proteins interacting with the NS1 protein during infection have been characterized..4.. 1996). 2006). The identification of such proteins is of great interest. It was demonstrated the conservation of the interaction of NS1-I with six different human and avian influenza virus NS1 proteins.2. they may influence the host range and virulence of influenza virus strains. it has identified NS1-I by its ability to bind to NS1. Such a hypothesis is supported by studies of influenza viruses with mutated or heterologous NS1 alleles which showed altered growth characteristics in different cells. Other studies were carried out a series of in vitro experiments to determine whether the NS1 protein could be utilized a common mechanism to inhibit PKR activation by both PACT and dsRNA. suggesting that there is a role for the NS1–NS1-I interaction during the virus life cycle. By using the yeast interaction trap.24
Previous studies of cloning and expression of NS1 gene
NS1 gene from various influenza viruses had been cloned.25
2. ELISA results on control animals’ positive showed 60% of detection level which were similar to the 79% of detection for naive animals.. This gene was successfully expressed in Escherichia coli (Binns et al. ELISA was carried out on sera experimental samples to test for the presence of anti-NS1 antibodies. NS1 protein from equine influenza virus its entirety or in part as a fusion protein with glutathione S-transferase (GST) in a diagnostic tests for detection this type of virus (Binns et al. Antibody of NS1 was detected in serum sample from ponies’ experimental infected cell with influenza virus without the animals being vaccinated with whole inactivated virion. 1996. ELISA diagnostic was usefulness depending upon duration of the antibodies response to NS1 following the initial infection. 1996). NS1 protein appears only in infected cells.
NS1 gene was isolated from equine influenza virus A/equine 2/Suffolk /89(H3N8). Hatada et al. 1983. 1997)... Using NS1 protein as a diagnostic marker for influenza virus infections in the presence of high levels of antibodies of influenza hemagglutinin generated by recent vaccination is an attractive alternative.
Electrophoresis in slab gels (12. Recombinant protein was observed by SDS-PAGE and immunoblot analysis was carried out to detect the antigens. However. Young et al. coli TG1 competent cell. cloned into pGEX-3X vector and transformed in E.5%) in the presence of SDS was used to observed NS1 protein. Detection efficiency of about 70% would be useful for batch testing training yards (Birch-Machin et al. 1996). 1992). While determination of NS1 antigens were appeared to be located in the C-terminal half of the protein... while in vaccinated animals was 26% (Binns et al.
was used to express NS1 protein of influenza A/Chicken/TH/KU14/04 (H5N1). NS1 gene from Influenza A (H9N2) was cloned and highly expressed to about 37. The amount of NS1 protein purified by the
. The antigenicity of the recombinant protein was demonstrated in Western-blotting test by using positive sera from pigs. coli. 2008). the other type of vector with 6xHis-tag. IPTG was used to induce E.4% of the total cellular protein. The transformants were screened by PCR using specific primers. This way was not only laying the foundation for development of ELISA antibody test for differentiation diagnosis between vaccinated and naturally infected pigs. Ligation product was transformed into subcloning efficiency DH5α competent cell by heating shock of the cells at 42°C for 20 seconds. The NS1 gene was inserted into pQE-80L using T4 DNA ligase. The recombinant NS1 proteins were purified by precipitation and affinity chromatography and characterized by SDSPAGE and western blot analysis. Utilizing IPTG inducer besides lactose inducer had no effect on the growth of host cells (Fang-kun et al.2008). but also facilitating the monitoring and eradication of SIV....26
The antigenic determinants of NS1 protein are situated in C-terminal domain.
pQE-80L. Combination antibody response with a cytotoxic T –cell response if elicited would be indicated that NS1 protein could be an important component of future vaccines in equines and other species (Birch-Machin et al. and nearly 95% after purification. coli was plated on LB agar containing 100μg/ml ampicillin. Highly homologous regions are present at the position of the Miami/63 and Suffolk /89 NS1 proteins and clearly indicate that these proteins should be capable of eliciting a similar immune response.
pET-28 vector was employed to express the NS1 gene under the control of the T7 promoter and fused with His·Tag sequences. This procedure focused on the antibody titer in pigs infected with influenza virus without interference by vaccinated antibody titers and this allows the differentiation between the vaccinated and the naturally infected pigs (Fang-kun et al . 1997). The transformed E.
Fusion proteins were expressed in E. coli protein. the fusion protein was reacted with anti-histidine monoclonal antibodies and was indicated specifically with antibodies raised against the NS1 protein (Manasatienkij et al. It was unknown whether the lack of modification in the NS1 protein would enhance its serological activities (Manasatienkij et al. 2004). these cells support del NS1A virus replication. 2007). On the other hand. In order to determine whether expression of the NS1B protein complements the growth of del NS1A virus.. 2008. The wild type NS1 cDNA was subcloned into the pGEX-5X-1 vector between the XhoI and EcoRI restriction sites.. the results indicated that the NS1 protein of influenza B virus can functionally replace the NS1 protein of influenza A virus in this assay (Donelan et al.NS1B encodes a protein consisting of GST fused to the NS1 protein of influenza B/Yamagata/1/73 virus. in some cases. Hence. del NS1A transfected MDCK cells with pCAGGS-NS1B plasmid.
. coli were not post-translationaly modified... Similarly.
The plasmid pGEX. pGEX-NS1 (94-281) was constructed by fusing the GST ORF to a cDNA encoding from 94 to 281 amino acids of the NS1B protein. To confirm the expression. 2008). NS1A was expressed in trans by transfecting MDCK cells with plasmid pCAGGS as a result. 2008).. However. Spencer et al. coli BL21 and purified using glutathione-sepharose beads. Expression of NS1B protein was able to complement the growth of the del NS1A virus to levels slightly lower than those seen when the NS1A protein was expressed in trans. The recombinant proteins produced in E.27
precipitation method was less than that purified under denatured conditions and contained a small amount of E. The pGEX-NS1B (1-93) plasmid was constructed by in frame insertion of the GST ORF in front of the coding region from 1 to 93 amino acids of the NS1B protein. it may be more effective antigens than the recombinant proteins produced in insect cell systems (Manasatienkij et al. NS1B mutant cDNAs were also cloned into pCAGGS vectors for expression in mammalian cells.
It is a highly sensitive way to detect NS1 protein (Ozaki et al. 2001)..
Other rapid diagnostic method for detection of antigens in nasal swabs of test animals and immune-PCR were established.
1 shows the general flow chart of experimental design for each vector that started from plasmid isolation.5). NS1B gene containing SacI and HindIII restriction enzyme sites were PCR amplified from pUC57-NS1B. which was amplified in E. plasmid miniprep was done for pUC57-NS1B and pQE-81L. ligation. Ligation. transformation and screening colonies were done on clones of DH5α. In the second attempt (Figure 3. agarose gel electrophoresis. Then. Double digestion was done using SacI and HindIII enzymes. In the third attempt (Figure 3. Double digestion. Figure 3. plasmid miniprep was done for pUC57-NS1B and pQE-80L. pET32b was amplified in E.29
MATERIALS AND METHODS
3. Plasmid miniprep was done for pUC57-NS1B and pET-32b. transformation and screening of colonies.3). single digestion was done using HindIII enzyme. coli strain BL21(DE3). Ligation.1
pUC57 plasmid carrying NS1B synthetic gene was cloned and transformed into competent E. In the first attempt (Figure 3. transformation and screening colonies were done on clones of BL21(DE3). coli strain BL21(DE3).4). Double digestion was done using SacI and HindIII
. In the fourth attempt (Figure 3.2). restriction digestion. coli strain DH5α. transformation and screening colonies were done on clones of BL21(DE3). was done using PstI and HindIII enzymes. Ligation. plasmid miniprep was done for pUC57-NS1B and pET-32a.
Flow chart of experimental design
Isolation of pUC57-NS1B and expression vectors from E. PstI and HindIII enzymes
DNA agarose gel electrophoresis and extraction
Ligation of NS1B gene with vectors and transformation into E. Ligation. coli strains
Restriction digestion using Hind III Restriction digestion using SacI. transformation and screening colonies were done on clones of DH5α. gel electrophoresis
Figure 3. coli competent strains
Screening of colonies and PCR amplification
Cloning of pET-32b-NS1B into E. coli BL21(DE3)
Screening of colonies
Figure 3. coli BL21(DE3)
NS1B gene HindIII
Single digestion with HindIII
Single digestion with HindIII
pET-32b vector (5899bp)
NS1B gene (870bp)
NS1B HindII I T7 promoter HindII I
pET-32b – NS1B
Transformation into E.
. coli DH5α Screening of colonies Cloning of pQE-81L-NS1B into E. coli DH5α
PCR amplification of NS1B gene using pUCfor and pUCrev primers
NS1B gene PstI
Double digestion with PstI and HindIII
pQE-81L vector (4753bp)
NS1B gene (870bp)
NS1B PstI HindIII
pQE-81L – NS1B
Transformation into E.
PCR amplification of NS1B gene using NS1BforSacI and NS1BrevHindIII primers
NS1B gene SacI HindIII
Double digestion with SacI and HindIII
pET-32a vector (5900bp)
NS1B gene (870bp)
NS1B HindIII SacI T7 promoter
pET-32a – NS1B
Transformation into E.4
Cloning of pET-32a-NS1B into E. coli BL21(DE3)
Screening of colonies
coli DH5α Screening of colonies Figure 3.34
PCR amplification of NS1B gene using NS1BforSacI and NS1BrevHindIII primers
HindIII SacI NS1B gene HindIII
Double digestion with SacI and HindIII
pQE-80L vector (4751bp)
NS1B gene (870bp)
pQE for pQE-80L – NS1B Transformation into E. coli DH5α
.5 Cloning of pQE-80L-NS1B into E.
Ligase enzyme was obtained from Yeastern Biotech. Yeastern Biotech and GeneAll. Merck. The pET and pQE vectors were ordered from Novagen and QIAGEN. pET-32a (5900bp). All restriction enzymes.2. QIAGEN.1
Two strains of Escherchia coli were used in this study . Promega.BL21(DE3) and DH5α. 2006). GenScript.3
pET is a bacterial plasmid designed to enable quick high quantity production of desired protein.2
3. Bhd.2. and pET-32b (5899bp) contain
. DNA polymerase were purchased from Promega. Primers used for PCR amplification were synthesized by 1st Base Laboratories Sdn.
3. Plasmid miniprep and gel extraction kits were purchased from GeneAll and QIAGEN. use of E.
3.2. coli as a host enables large quantity production of recombinant proteins (Leonhartsberger. These strains are commonly used as a host for cloning and protein expression in molecular biology labs because they have the simplest genomic and foreign DNA can be transformed easily.35
Most chemicals were of analytical and molecular biology grade obtained from Sigma-Aldrich. On the other hand.
1993). T7 promoter which is specific to T7 RNA polymerase..1998).lablife.6
pET-32a is designed for cloning and high-level expression of peptide sequences comprise of 109 amino acids Trx• Tag™ thioredoxin protein.36
several elements like: LacI gene. Cloning sites are available for cloning and producing fusion proteins.nm7d74YBP4q6Utqf6tNu_Ho-).org/p?a=vdb_view&id=g2..
Figure 3. The plasmid also contains cleavable His• Tag® and S• Tag™ sequences for detection and purification (LaVallie et al.
. f1 origin of replication. ampicillin resistance gene and colE1 origin of replication (Blaber et al. Lac operator which is poly linker.
The pQE series have been designed to allow tightly regulated 6xHistagged protein expression in any E. coli host strain. strongly suppressing protein expression from the lac promoter before induction with IPTG (QIAGEN.lablife. pQE-80L and pQE-81L vectors have a cis-lacIq gene that overexpresses the lac repressor. The vectors are based on the pQE-30 series and include a lacIq repressor gene. 2001).37
Fragment was cloned into pUC57 into EcoRV site.5
NS1B Synthetic Gene
The synthetic NS1B gene was installed from synthetic oligonucleotides and PCR products by GenScript (USA).
MCS of pUC57
Figure 3. 2009)
pUC57-NS1B gene construct (GenScript.
3.1g was dissolved in 50 ml of sterile deionized water and filtered by using Sartobind nylon filter of size 0. For LB broth or agar containing 100µg/ml ampicillin.3. For LB broth or agar containing 50µg/ml ampicillin. 20 ml of ampicillin from stock solution of 2mg/ml was added to the media. The ampicillin solution was added to the medium after it was cooled to 55°C.1
Liquid and solid media preparation
3. yeast extract (2g) and sodium chloride (4g) in deionized water and it was made up to 400 ml.1
Luria Bertani agar and broth
LB medium was prepared by dissolving tryptone (4g). For LB agar. 10 ml of ampicillin from stock solution of 2mg/ml was added to the media.3.40
Preparation of ampicillin stock solution
Ampicilin powder (Sigma-Aldrich) of 0.2µm.1.1. The contents were mixed and autoclaved at 121 °C for 20 minutes. 2% of agar was added.
3.1M calcium chloride. The cells were resuspended and plated out evenly using a sterile glass spreader at 100µl on LB agar containing 50µg/ml ampicillin. Immediately. E.41
3. coli BL21(DE3) and DH5α hosts were used in transformation.2
Preparation of competent cells
DNA is very hydrophilic molecule that does not normally pass through a bacterial cell membrane. the cells were centrifuged at 4°C for 10 minutes at 3000 rpm. Each colony was streaked on LB agar containing 100µg/ml ampicillin and the plates were incubated overnight at 37°C. Then.1M calcium chloride plus 15% glycerol and aliquoted into 0.
. It is the best to use fresh competent cell for transformation. The plates were incubated overnight at 37°C.4 cells
Transformation pUC57-NS1B into E. Bacteria need to be made "competent" in order to uptake foreign DNA.2ml suspension and stored at -80°C. The cells were spun again at 4°C for 10 minutes at 3000 rpm. The cells were very briefly spun and 700μl of supernatant was removed. Then. Then. After that. coli BL21(DE3) competent
pUC57-NS1B (2µl) was added to competent cells and kept on ice for approximately 20 minutes and this was heat shocked at 42°C for 45seconds. cells were incubated for 20 minutes on ice. Two strains of E. 1ml of LB broth was added and incubated with agitation at 37°C for 1 hour at 200rpm. the cells were resuspended in 1ml of 0.
3. the supernatant was discarded and the pellet was gently resuspended in 10ml of 0. coli was grown overnight in 10ml of LB broth at 37°C with shaking at 200rpm. the tube was placed back on ice for at least 2 minutes. The cells were chilled for 10 minutes on ice. For long term storage.
Additional centrifugation was done at 14000rpm for 1 minute. The product was run on 1% agarose gel to check whether the plasmid DNA is successfully isolated from E. Next. The cells were harvested by centrifuging at 4000rpm for 5 minutes. The column was then centrifuged at 8000rpm for 30 seconds. 250µl of S2 buffer was added. The flowthrough was discarded and the PD column was placed back in the collection tube.5ml microcentrifuge tube. The plasmids were isolated using plasmid miniprep kit from GeneAll. The supernatant was removed and the pellet was resuspended by pipetting thoroughly in 250µl of buffer S1. The flow-through was discarded and the PD column was placed back in the collection tube.42
3. Then.5ml of microcentrifuge tube. Clear solution was transferred to the PD column by pipetting while white pellet formed remained in the microcentrifuge tube. After 2-minutes stand at room temperature. coli BL21(DE3) containing pUC57-NS1B was cultured overnight in LB broth containing 100µg/ml ampicillin at 37°C with shaking at 200rpm. The solution was transformed to a new 1. The solution was gently mix by inverting tube for 10 times and incubated for 2 minutes until the cell suspension became clear. The PD column was spun at 8000rpm for 30 seconds at room temperature. coli before being kept at -20°C. PD column was transferred into a new 1.
DNA was eluted by adding 50µl of elution buffer to the centre of the PD column. 500µl of buffer AW was added to the PD column and spun at 8000rpm for 30 seconds. Then the PD column was washed by adding 700µl of buffer PW.
Isolation of pUC57-NS1B plasmid
E. PD column was placed in a collection tube. the tube was centrifuged at 14000rpm for 1 minute. 350µl of buffer S3 was added to the solution and immediately inverted gently for 10 times before centrifuged for 10 minutes at 14000rpm. The flowthrough was discarded and the PD column was placed back in the collection tube.
After the gel was solidified in 30 minutes.6
Agarose gel electrophoresis
Agarose gel electrophoresis is widely used in molecular biology labs to separate DNA strands by size.
. 5µl of the DNA was mixed with 1µl of 6X loading dye. The agarose gel was poured into the gel casting tray. The final pH was 8. 6µl of 1kb DNA ladder was used as a marker.1ml). After gel running. Tris base (242g). and to estimate the size of the separated strand by comparing with the known fragments (DNA ladder). pH 8).5 and autoclaved at 121°C for 20 minutes. 1X TAE buffer was diluted from 50X TAE buffer by mixing 20ml of 50X TAE buffer with 980ml of distilled water. the comb was removed carefully. The gel was removed from the casting tray and was viewed on a UV transilluminator and documented by using GeneFlash. The gel casting tray was assembled in gel tank and well forming comb was inserted into slots on casting tray.
To prepare sample.43
3. and the solution was made up to 1000ml with deionized water. power supply was turn off. The tray containing agarose gel was placed into electrophoresis chamber and 1X TAE buffer was added to cover the gel. This is achieved by pulling negatively charged DNA molecules through an agarose matrix with an electric field.5g of agarose powder was added to 50ml of 1X TAE buffer and was boiled in microwave for 1 minute and then left to cool down.
50X of TAE buffer was prepared from EDTA (186g. The sample and the marker were loaded separately into wells and the gel was run at 100V for 50 minutes. 0. and glacial acetic acid (57. The concentration of the agarose depends on the size of the DNA fragments to be separated. 5µl of ethidium bromide was added to the agarose gel.
Following electrophoresis. The gel slices were
.1: Single digestion components in PCR tube using HindIII
pUC57-NS1B Acetylated BSA Buffer E HindIII enzyme Total
3.5 for plasmid miniprep was used to isolate pET-32b from E.
3. the NS1B band with the size of 870bp and pET-32b at size nearly 6000bp were cut out with a clean sharp scalpel from the gel.2µl 2µl 1µl 20µl
pET-32b Acetylated BSA Buffer E HindIII enzyme
16.8µl 0. coli BL21(DE3).
Isolation of pET-32b plasmid
The same protocol as described in section 3.2µl 2µl 1µl 20µl
The reactions were incubated at 37°C for 5 hours.8
pUC57-NS1B and pET-32b were digested using HindIII and the following components were mixed in PCR tube:
Table 3.8µl 0. The digestion products were then purified by gel extraction to remove the buffer and restriction enzymes.9
The digestion product needs to be purified before further use.
Then. It was stood at room temperature for 2-5 minutes and then centrifuged at 13000rpm for 1 minute. 1 µl of distilled water was used to initialize the instrument and 1µl of elution buffer was used as a blank. This step was repeated till all the sample was passed through the column. The solution was incubated at 50ºC for 10 minutes and vortexed at each 2 minutes. The column was transferred to a new 1. The flow-through was discarded. The product was kept in 20ºC. Then. the column was placed back. The tubes were weighed before and after putting in the gel.10
Determination of DNA concentration
The measuring of DNA concentration was carried out by using Nanodrop Spectrophotometer.8 and 2.7ml of PE buffer and centrifuged at 13000rpm for 1 minute. An OD260: OD280 ratio of purified plasmid is to be found in the expected range of 1. The sample was applied to the QIAquick column and centrifuged at 13000rpm for 1 minute.
.5ml microcentrifuge tube and 50µl of elution buffer was added to the centre of column. One volume of isopropanol was added to the sample and mixed. 0.
3.5ml of QG buffer was added to column and centrifuged at 13000rpm for 1 minute. Three times volume of QG solution buffer was added to one volume of gel as mentioned in the protocol of QIAGEN gel extraction kit. The flow-through was discarded and the column was placed back.45
separately put inside 2ml microcentrifuge tube. The DNA concentration and purity was measured by using 1 μl of the sample.0. The flowthrough was discarded and centrifuged again to remove all PE buffer. washed by adding 0.
NS1B and digested pET-32b with HindIII were ligated and the following components were mixed in PCR tube:
3. The ligation mix was kept at 4°C for transformation.46
3. DNA ligases catalyze the formation of a phosphodiester bond between adjacent nucleotide with the hydrolysis of ATP to AMP and inorganic phosphate.2: Ligation mix components of pET-32b and NS1B 1st mix pET-32b NS1B 10X ligation buffer 10X ligation buffer YEA T4 DNA ligase dH2O Total A B 1µl 3µl 1µl 1µl 1µl 3µl 10µl 2nd mix 2µl 4µl 1µl 1µl 1µl 1µl 10µl
The reactions were incubated in thermocycler at 22°C for 20 minutes followed by inactivation at 65°C for 10 minutes.11 was transformed into E. It is better to directly transform the ligation mix into competent cells.
Ligation NS1B with pET-32b
T4 DNA Ligase was used in ligating the vector and the insert gene. The ligation mix (10μl) was added to 100µl of competent cells and incubated on ice for 20 minutes.12
Transformation of recombinant product
The constructed recombinant plasmid prepared as described in section 3. coli BL21(DE3) competent cells.
3. Then. Some cells were plated out on LB agar as a control.5 for plasmid miniprep was used to isolate pQE-81L from E.
. 1ml of LB broth was added to the tube.
3. The plates were incubated at 37°C for overnight. IPTG (5µl) and X-gal (10µl) were spread on the plates for 1 hour prior to use.000 rpm for 30 seconds. Cells were collected by centrifugation at 10. tube was incubated at 37°C for 1 hour with shaking at 200rpm. 0.13
Isolation of pQE-81L plasmid
The same protocol as described in section 3.47
The cells were heat shocked at 42°C for 45 seconds.1
Primers were designed based on the upstream and downstream region of NS1B gene of pUC57 vector.14. Tube was immediately placed back on ice for at least 2 minutes. The cells (100µl) were spread out on LB agar containing 50µg/ml ampicillin. coli DH5α.7ml was removed from supernatant and the cells were resuspended.14 (PCR)
Amplification of NS1B gene using Polymerase Chain Reaction
3. The characteristics of the primers are as described in Table 3.
2µl 32.14.3: The pUC57 primers for PCR of NS1B gene from pUC57-NS1B
Primers Sequences (5)׳3-׳
% of GC Content
Melting Temp.NS1B Total
0. a PCR reaction was prepared by pipetting the following reagents into PCR tube:
Table 3.4: PCR reaction components of pUC57-NS1B
Go Taq DNA polymerase 5X Green buffer dNTP MgCl2 Forward primer (pUCfor) Reverse primer (pUCrev) dH2O pUC57.35µl 1µl 50µl
Polymerase Chain Reaction (PCR)
3.25µl 10µl 1µl 5µl 0.(°C)
pUC for 5׳GTAAAACGACGG CCAGTGA-3׳ pUC rev 5׳CAGGAAACAGCT ATGACC-3׳
The above cycle was run for 25 cycles.15 step
Double digestion using PstI and HindIII restriction enzymes in one
NS1B amplified gene and pQE-81L were digested using PstI and HindIII restriction enzymes and the following components were mixed in a PCR tube:
. Then. The process continued with the denaturation.9 prior to digestion with PstI and HindIII restriction enzymes.
3. annealing and extension steps.6. 30 sec. The PCR products were purified by using the protocols as mentioned in section 3. the sample was run on gel electrophoresis as described in section 3. 1 min. Then the cycle was continued by further extension at 72ºC for 7 minutes.5: PCR cycle
Steps Denaturation Annealing Extension
Temperature 94ºC 50ºC 72ºC
Time 30 sec.49
The PCR process was initiated with initial denaturation step at 94ºC for 5 minutes. The reaction conditions for each step in PCR cycle were as follows:
Table 3.8µl 0. Then. the reaction was observed by gel electrophoresis and extracted using the same protocols as
.8µl 0. Then.
3.2µl 2µl 1µl 1µl 20µl
The reactions were incubated at 37ºC for 5 hours.9.2µl 2µl 2µl 20µl
The reaction was incubated at 37ºC for 5 hours. gel electrophoresis was done and extracted using the same protocols mentioned in sections 3. The extracted product was used for ligation reaction.16
Double digestion using restriction enzymes in two steps
The following components were mixed in PCR tube for the first digestion for NS1B amplified gene and pQE-81L: Table 3.7: Double digestion components in PCR tube using HindIII(1st step)
pQE-81L or NS1B gene Acetylated BSA Buffer E HindIII enzyme Total
15.6 and 3.6: Double digestion components in PCR tube using PstI and HindIII
pQE-81L or NS1B gene Acetylated BSA 10X Multicore enzyme buffer HindIII enzyme PstI enzyme Total
.6 and 3. the reaction was observed by gel electrophoresis and extracted using the same protocols as mentioned in sections 3. The following components were mixed in PCR tube:
Table 3.11 and 3. The extracted product was used for the second digestion. Then. coli DH5α
competent cells were done using the same protocol as mentioned in sections 3. The extracted product was used for ligation reaction.12.6 and 3.17
Ligation and transformation into DH5α Ligation and transformation of pQE-81L-NS1B into E.8: Double digestion components in PCR tube using PstI(2nd step)
First digestion product Acetylated BSA Buffer H PstI enzyme Total
15.2µl 2µl 2µl 20µl
The reaction was incubated at 37ºC for 5 hours.9.9.51
mentioned in sections 3.8µl 0.
It is composed of styrene divinylbenzene copolymers with paired iminodiacetate ions. One benefit of using the Chelex extraction is that divalent heavy metals which introduce DNA damage at high temperature can be removed.18. 100µl of chelex solution was added into 1.
3. The samples were kept in 4ºC for further studies.org/pdi/Subject03/pdi_s03_m03_01. The supernatant was used for PCR amplification. Then.
. Each tube was labeled as the number of colonies. without altering the concentration on nonmetal ions. and most suitable for DNA applications. as the Chelex 100 suspension was prepared in TE buffer (http://www. the samples were centrifuged at 12000rpm for 5 minutes. Chelex 100 is very effective in binding metal contaminants with a high selectivity for divalent ions. Colony PCR was used as the screening method.
10% Chelex 100 was used to extract DNA. The plates were incubated at 37°C for overnight.htm). The samples were incubated at 95ºC for 5 minutes with agitation 400rpm.5ml microcentrifuge tube and the number of tubes was prepared according to the numbers of colonies to be screened. The extraction was set up under aqueous alkaline conditions. resuspended and vortexed with the Chelex suspension for 5 to 10 seconds.nfstc.52
Screening of positive colonies
Colonies grown on LB agar containing 30µg/ml ampicillin were isolated and further streaked on LB agar containing 100µg/ml ampicillin.1
Chelex 100 extraction method
Chelex 100 resin is highly purified. A scrap of each colony was removed using toothpick.
Isolation of pET-32a plasmid
The same protocol as described in section 3.5 for plasmid miniprep was used to isolate pET-32a from E. sequencing primers of pQE-81L vector were used the characteristics of the primers used are as in Table 3.3
Table 3.9: The pQE primers for PCR
Primers Sequences (5)׳3-׳
% of GC Content
3.2 and 3.2
PCR amplification and gel electrophoresis to detect NS1B
For screening of colonies.(°C)
pQE for 5׳CGGATAACAATTTC ACACAG-3׳ pQE rev 5׳GTTCTGAGGTCATT ACTGG-3׳
. coli BL21(DE3).0
56. The PCR
amplification and gel electrophoresis were as mentioned in sections 3.
NS1B forSacI 5-׳GAGCTCATGGCGAA CAAT-3׳ NS1B revHindIII 5-׳AAGCTTCTAATTGT CTCC-3׳
Amplification of NS1B gene using Polymerase Chain Reaction
PCR amplification of NS1B gene from pUC57-NS1B was done using the same protocol mentioned in section 3.10: Primers containing SacI and HindIII restriction enzyme sites used to amplify NS1B gene
Primers Sequences (5)׳3-׳
% of GC Content
Melting Temp.20. The characteristics of the primers design are shown in Table 3.9
The forward and reverse primers were designed to contain SacI and HindIII restriction sites for endonucleases digestion.0
2µl 2µl 1µl 1µl 20µl
The reaction was incubated at 37ºC for 5 hours. The extracted products were used for ligation reaction.9.11: Double digestion components in PCR tube using SacI and HindIII
pET-32a or NS1B gene Acetylated BSA 10X Multicore enzyme buffer HindIII enzyme SacI enzyme Total
Double digestion using SacI and HindIII restriction enzymes in two
The following components were mixed in PCR tube for the first digestion for NS1B amplified gene and pET-32a:
.6 and 3.8µl 0.21 step
Double digestion using SacI and HindIII restriction enzymes in one
NS1B amplified gene and pET-32a were digested using SacI and HindIII and the following components were mixed in PCR tube:
3. the reaction was observed by gel electrophoresis and extracted using the same protocols as mentioned in sections 3.
8µl 0. The extracted product was used for the second digestion.2µl 2µl 2µl 20µl
The reaction was incubated at 37ºC for 5 hours.6 and 3.8µl 0.12: Double digestion components in PCR tube using HindIII(1st step)
pET-32a or NS1B gene Acetylated BSA Buffer E HindIII enzyme Total
. The following components were mixed in PCR tube: Table 3. the reaction was observed by gel electrophoresis and extracted using the same protocols as mentioned in sections 3. the reaction was observed by gel electrophoresis and extracted using the same protocols as described in sections 3. Then. Then.2µl 2µl 2µl 20µl
The reaction was incubated at 37ºC for 5 hours. The extracted product was used for ligation reaction.9.13: Double digestion components in PCR tube using SacI(2nd step)
First digestion product Acetylated BSA Buffer J SacI enzyme Total
15.6 and 3.
3. The primers used for PCR were T7 promoter and T7 terminator primer.2
3. coli BL21(DE3) and colony screening were done using the same protocols as mentioned in sections 3.3
TAATACGACTCACA CAAGGG-3׳ T7 terminator 5׳19 52.0
56.18. coli BL21 (DE3) and screening of
Ligation. transformation into E.5 for plasmid miniprep was used to isolate pET-32a from E. transformation into E.(°C)
Ligation.14: Primers containing SacI and HindIII restriction enzyme sites used to amplify NS1B gene
Primers Sequences (5)׳3-׳
% of GC Content
Melting Temp.5 60.24
Isolation plasmid (pQE-80L)
The same protocol as described in section 3.
. The characteristics of the primers used are shown in Table 3. coli BL21(DE3).12 and 3.
double digestion.21. transformation into
DH5α and screening of colonies
The same protocols as described in sections 3. 3.25
PCR amplification.18 were used. 3. 3.
3.22.17 and 3.20.
RESULTS AND DISCUSSION
Transformation of pUC57-NS1B into E. coli BL21(DE3) was successfully done.
Cloning of pET-32b-NS1B into E. pET-32b was also linearized and its presence was verified by the band of 5. coli BL21(DE3) was done using GeneAll miniprep kit and the presence of pUC57-NS1B was verified by agarose gel electrophoresis. coli BL21(DE3)
Transformation of pUC57-NS1B synthetic gene construct into competent E.
The restriction digestion of pUC57-NS1B was successful and NS1B gene (870 bp) was separated and this is shown in Figure 4.1. The colonies were maintained using LB agar containing 100µg/ml ampicillin and incubated overnight at 37°C. Plasmid isolation of pUC57-NS1B from E.
4.1. coli BL21(DE3)
Single restriction digestion was done for pUC57-NS1B and pET-32b by using HindIII enzyme which recognizes 5׳A▼AGCTT 3 ׳sequence.9kb in Figure 4.
Lane 3: pET-32b
Subsequently. Lane 1. After overnight incubation.87kb)
. 2: pUC57 and NS1B gene. coli BL21(DE3).1
Restriction digestion by HindIII.2.9kb)
M: 1 kb Marker.60
8000 6000 5000 3000 2500 2000 1500 1000 750
pET-32b (5. the NS1B gene was cloned into pET-32b vector and transformed into competent E. colonies were observed and this is shown in Figure 4.7kb)
Colonies were streaked on LB agar containing 100 µg/ml ampicillin and spread with IPTG and X-gal. However.61
Cloning of pQE-81L-NS1B into E. All colonies were observed to be blue in color. Double digestion was done for pQE-81L by using PstI and HindIII enzymes.2 BL21(DE3)
Transformation of pET-32b-NS1B into competent E.fermentas. X-gal is used to indicate whether a cell expresses the β-galactosidase enzyme. X-gal dye is cleaved by βgalactosidase yielding galactose and 5-bromo-4-chloro-3-hydroxyindole
(http://www. As for pUC57-NS1B. This may due to recircularization of the vector. the cloning steps were not successful. it was first digested with HindIII enzyme and the result is shown in Figure 4. coli DH5α
We attempted the directional cloning approach of NS1B gene into pQE81L vector.com/en/products/all/reagents/r094-xgal). Hence. The attempts to increase the ratio of gene: vector insert was done.
. in a technique called blue/white screening or X-gal screening. the insertion of NS1B gene into pET-32b vector gave negative result. which is encoded by the lacZ gene.
Lane 1 and 2 shows the successful digestion of pUC57-NS1B with HindIII enzyme and the band of NS1B is of correct size (870bp).87kb)
Figure 4.6 shows the isolation and digestion of the vector and gene.8kb)
NS1B (0. Lane 4: Double digestion of pQE-81L.8kb) but in this way still unknown whether the digestion correct complete or not. Plasmid miniprep for both pUC57NS1B and pQE-81L were verified in lane 3 and 5. so PCR amplification was done to increase the concentration of NS1B by using primers designed for pUC57. Lane 3: Miniprep of pUC57-NS1B. Figure 4. Lane 4 shows the double digestion of pQE-81L by using PstI and HindIII and the band is of correct size (4.3
Plasmid isolation and digestion of pUC57-NS1B and pQE-81L
M: 1kb Marker. PstI enzyme recognizes the 5 ׳CTGCA▼G 3' sequence and HindIII enzyme recognizes 5׳A▼AGCTT 3 ׳sequence.
Because the concentration of NS1B gene extracted was low (4. A band
.7 ng/µl) and this would affect the next step of digestion.62
6000 5000 3000 2500 2000 1500 1000 750
pQE-81L (4. Lane 1 and 2: pUC57-NS1B digested with HindIII. Lane 5: Miniprep of pQE-81L.
of the correct size was shown in lane 1 of Figure 4. indicating successful amplification.
M Lane 2 5 6
Lane 1 7
Figure 4. The transformed colonies were shown in Figure 4.5. double digested with PstI and HindIII and used for cloning into pQE-81L.4
PCR amplification of NS1B
.4. PCR product was extracted.
Transformation of pQE-81L-NS1B into competent DH5α
After transformation, colony PCR was carried out with pQEfor (5׳CGGATAACAATTTCACACAG-3') and pQErev (5'-
GTTCTGAGGTCATTACTGG-3') primers. However, all the colonies screened gave negative result. As shown in Figure 4.6, the size of amplified band is smaller than 300bp, indicating that only the pQE-81L vector was transformed to cells without the cloning of NS1B gene. The right band size to be expected is nearly 1000bp.
M clone 21 22 23 24 25 26 27 28 29 30 31 32
PCR of recombinant pQE-81L-NS1B
Cloning of pET-32a-NS1B into E. coli BL21(DE3)
PCR amplification was done on pUC57-NS1B to amplify NS1B gene with a newly designed primers NS1BforSacI (5-׳GAGCTCATGGCGAACAAT3 )׳and NS1BrevHindIII (5'-AAGCTTCTAATTGTCTCC-3') which introduced SacI and HindIII restriction sites to NS1B for directional cloning of the gene into pET-32a. The amplified NS1B gene is shown in Figure 4.7.
M 3 4
3000 2500 1000 NS1B
Miniprep of pUC57-NS1B and PCR amplification of NS1B
M: 1kb marker, Lane1: Miniprep of pUC57-NS1B, Lane 2: PCR amplification of NS1B gene The HindIII enzyme recognizes 5׳A▼AGCTT 3 ׳sequence while SacI enzyme recognizes 5 ׳GAGCT▼C 3 ׳sequence. The gene was extracted, double digested with SacI and HindIII and used for cloning into pET-32a. The transformed colonies were shown in Figure 4.8. The colonies were screened by PCR. pET-32a vector’s universal primers, namely TAATACGACTCACTATAGGG-3') and T7 T7 promoter (5'terminator (5'-
GCTAGTTATTGCTCAGCGG-3'). All colonies screened gave negative result. As shown in Figure 4.9, the size of amplified band is smaller than 750 bp, indicating that only the pET-32a vector was transformed into the cells without the cloning of NS1B gene. The right band size to be expected is nearly 1500 bp.
PCR of recombinant pET-32a-NS1B
M: 1 kb Marker. coli
M 1 2 3 4 5 6 7 8 9
10 11 12 13 14
Figure 4.8 BL2(DE3)
Transformation of pET-32a-NS1B into competent E. Lane 1-14: Clone from 62-75
After transformation into DH5α.68
4. This result showed that only the pQE80L vector was transformed into cells without the cloning of NS1B. The result is shown in Figure 4.11. colony PCR was done using pQEfor and pQErev primers. All screened colonies yield negative result.
Transformation of pQE-80L-NS1B into competent DH5α
. The right band size to be expected is nearly1000bp. coli DH5α
The amplified NS1B gene containing SacI and HindIII sites was extracted.5
Cloning of pQE-80L-NS1B into E. digested and cloned into pQE-80L vector. The transformed colonies were shown in Figure 4.10.
. coli BL21(DE3) and pQE-80L-NS1B into E. PCR amplification was done on the ligation. The result shown in Figure 4.6
Confirmation of ligation
Ligation and transformation of pET-32a-NS1B into E.11
PCR of recombinant pQE-80L-NS1B
M: 1kb Marker.69
Clone 83 84 85 86 87 88 89 90 91 92
Figure 4.12 indicated that the fragments without insert were amplified. Clone number from 83-92
4. to confirm whether ligation had happened. coli DH5α yield negative result. 1µl of ligation mix for pET-32a-NS1B and pQE-80L-NS1B were amplified.
Lane 1: fragment without gene insert from pET-32-NS1B. Lane 2: fragment without gene insert from pQE-80L-NS1B
M: 1kb Marker.70
83 84 85 91 92
1993).. coli BL21(DE3). pET-32a-NS1B and pQE-80L-NS1B. Vaccinated animals have not produced nonstructural protein (Fang-kun et al.1
Nonstructural protein NS1 is used for identification and differentiation between the vaccinated and naturally infected animals...71
CONCLUSION AND FUTURE WORKS
5. Similar negative results were observed when screening colonies of pQE-81L-NS1B. The function of NS1 protein during viral multiplication remains unclear. 2003). 8002).
pUC57 plasmid carrying NS1B gene was successful transformed and isolated from E. Dauber and coworkers (2003) found that reverse genetic system studies may allow to explore the function of NS1B during the replication cycle and to assess their contributions to the pathogenesis and virulence of influenza B (Dauber et al.
First screening of pET-32b-NS1B colonies using white/blue method gave negative result. Screening of colonies for pQE-81L-NS1B and pQE-80L-NS1B revealed a band under the size
. Designed primers used for PCR of NS1B showed successful amplification. although a possible regulatory role in viral replication has been suggested from studies of temperature sensitive mutant of NS1 (Binns et al.
Screening of colonies for all vectors used showed only plasmids without any insertion of the NS1B gene. pQE-81L using double restriction digestion with (PstI and HindIII).2
A further construction of NS1 gene into pET32a.72
300bp. pET32a using double restriction digestion with (SacI and HindIII).
Cloning NS1B into pET-32b using single restriction digestion with HindIII. which is not as the expected band size of 1500bp. it is suggested that the overexpression of the constructed recombinant into expression system are being studied and work on purification can also be performed using immobilized affinity chromatography resin. pQE-80L using double restriction digestion with (SacI and HindIII) gave unexpected result.
. NS1B protein can be analyzed by Sodium Dodecyl Sulphate –Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis. This result may relate to re-ligation of digested vector for single digestion and uncompleted digestion for vectors of double restriction digestion. which is not as the expected band size of 1000bp. The NS1B gene can also be cloned into other expression vectors according to the open reading frame and use other host. Screening of colonies for pET-32a-NS1B showed a band of the size nearly 750bp.
In the future.
5. pET-32b. pQE-80L and pQE-81L vectors should be performed with best precaution of procedures to avoid any contamination and the use of appropriate restriction enzymes with high efficiency to get recombinant construction.
More studies to determine immunogenicity of influenza NS1B fusion protein by using Western blotting or Enzyme-linked immunosorbent assay (ELISA) methods should be carried out.
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NS1B gene sequence (870 bp) of Influenza B virus (B/Taiwan/45/2007)
aagcttctgcag atg gcg aac aat atg acc aca aca caa att gag gtg ggt ccg gga gca acc aat gcc acc ata aac ttt gaa gca gga att ctg gag tgc tat gaa agg ctt tca tgg caa aga gcc ctt gac tac cct ggt caa gac cgc cta aac aga cta aag aga aaa tta gag tca aga ata aag act cac aac aaa agt gag cct gaa agt aaa agg atg tcc ctt gaa gag aga aaa gca att gga gta aaa atg atg aaa gta ctc cta ttt atg aat ccg tct gct gga att gaa ggg ttt gag cca tac tgt atg aaa agt tcc tca aat agc aac tgt acg aaa tac aat tgg acc gat tac cct tca aca cca ggg agg tac ctt gat gac ata gaa gaa gaa cca gag gat gtt gat ggc cca act gaa ata gta tta agg gac atg aac aac aaa gat gca agg caa aag ata aag gag gaa gta aac act cag aaa gaa ggg aag ttc cgt ttg aca ata aaa agg gat atg cgt aat gta ttg tcc ttg aga gtg ttg gta aac gga aca ttc ctc aaa cac ccc aat gga tac aag tcc tta tca act ctg cat aga ttg aat gca tat gac cag agt gga agg ctt gtt gct aaa ctt gtt gcc act gat gat att aca gtg gag gat gaa gaa gat ggc cat cgg atc ctc aac tca ctc ttc gag cgt ctt aat gaa gga cat tca aag cca att cga gca gct gaa act gcg gtg gga gtc tta tcc caa ttt ggt caa gag cac cga tta tca cca gaa gag gga gac aat tag aagcttctgcag
a c g t
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Adenine Cytosine Guanine Thymine
NS1B protein sequence (281 amino acids)
MANNMTTTQIEVGPGATNATINFEAGILECYERLSWQRALDYPGQDRLN RLKRKLESRIKTHNKSEPESKRMSLEERKAIGVKMMKVLLFMNPSAGIEG FEPYCMKSSSNSNCTKYNWTDYPSTPGRYLDDIEEEPEDVDGPTEIVLRD MNNKDARQKIKEEVNTQKEGKFRLTIKRDMRNVLSLRVLVNGTFLKHPN GYKSLSTLHRLNAYDQSGRLVAKLVATDDITVEDEEDGHRILNSLFERLN EGHSKPIRAAETAVGVLSQFGQEHRLSPEEGDN
A C D E F G H I K L
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Alanine Cysteine Aspartic acid Glutamic acid Phenylalanine Glycine Histidine Isoleucine Lysine Leucine
M N P Q R S T V W Y
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Methionine Asparagine Proline Glutamine Arginine Serine Threonine Valine Tryptophan Tyrosine