SINH TNG HP PROTEIN

TS.BS. Th Thanh Thy

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GENOMICS V PROTEOMICS
GENOME B gen (h gen) GENOMICS Nghin cu v b gen (h gen)
PROTEOME B protein ca t bo hay m PROTEOMICS Nghin cu v b protein ca cc loi

T GENE N PROTEIN CHC NNG

B PROTEIN NGI
Rt phc tp c tnh >100.000 phn t protein Do v tr splicing khc nhau Do bin i sau dch m

02/2008:48891 cu trc trong PDB

Central Dogma of Biology

Transcripti on DNA RNAtranslation Protein

LUN THUYT TRUNG TM

DNA
Replication

RNA viruses
Reverse Transcription (Retrovirus)

Transcription

Chuyn m (sao chp)

ARNm

RNA
(David Baltimore v Howard Temin)

Gii m (phin m)

Protein

Francis Crick

Prions: scrapie v mad cow Stanley Pruisner:1997 Nobel Prize

TO HNH PROTEIN CHC NNG

Protein s gp v xon ( helices, sheets) v bin i (phosphoryl ha, acetyl ha, glycosyl ha ).

TO HNH PROTEIN CHC NNG

Protein c khuynh hng gp v xon thnh cu hnh c nng lng t do thp nht. Protein bt u gp v xon t khi ang c tng hp. Protein a cc phn k nc vo trong to dng helix. Hu ht protein u c dng cu trc bc 2: helice v sheet. 7

TO HNH PROTEIN CHC NNG

S PHN CA PROTEIN SAU GII M 50%

- AA t bin
- Nhit ,
nng cht tan, pH

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TO HNH PROTEIN CHC NNG

Chaperonin

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THOI HA PROTEIN

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PROTEIN MISFOLDING DISEASES

Amyotrophic lateral sclerosis

SOD1 (Superoxide dismutase 1) gn Cu v Zn, phn hy cc gc superoxide t do. t bin (>100) gy bnh Amyotrophic lateral sclerosis gia nh (ALS, bnh Lou Gehrig): X ct bn teo c nh vt l ni ting th gii Stephen Hawking: bc x ca l en

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PROTEIN MISFOLDING DISEASES Alzheimers Disease: A protein

Tch t cc protein lin quan mt thit vi nhau c gi l Abeta peptit, to thnh nhng mng Amyloid bt thng v cc nhm m x si cun ri nm trong hoc bao xung quanh TB thn kinh, lm cht t bo no v hu qu l mt kh nng ti to cc t bo thn kinh.

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PROTEIN MISFOLDING DISEASES Alzheimers Disease: A protein

Mng beta-amyloid tch t xung quanh v bn ngoi cc t bo thn kinh. Amyloid: 1850s do nhum bt mu vi iodine. Thnh phn chnh l protein.

An actual AD plaque

cun ri x si thn kinh pha bn trong t bo thn kinh

An actual AD tangle
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PROTEIN MISFOLDING DISEASES

Alzheimers Disease: A protein

Mng amyloid

Amyloid precursor protein (APP) l tin cht ca cc mng bm dng bt

1. APP xuyn mng TB thn kinh.

1.

2. Enzyme ct APP thnh cc on, c on gi l Amyloid.

3. Cc on Amyloid c khuynh hng to thnh cm gi l mng
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2.

3.

PROTEIN MISFOLDING DISEASES Alzheimers Disease: A protein

Tau protein:
Gi h thng vi ng bn vng.

Trong AD, Tau protein b thay i, tch t li vi nhau to thnh cc cun ri x si thn kinh
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PROTEIN MISFOLDING DISEASES

Mad Cow Disease - Prion Protein

Stanley Ben Prusiner, 1997
Transmissable spongiform encephalopathies (TSEs), gm bnh b in (BSE) v bnh Creutzfeld Jakob (CJD) ngi. Bnh xy ra khi protein tn PRION xon sai lch (binh thng l thnh phn mng TB TK) Khi xon sai, gi l infectious prion, gy hiu ng domino ti cc prion bnh thng khc, truyn qua ng n ung.

PrPc (Prion Protein cellular)

PrPsc (Prion Protein scrapie)

B mt no ca Bn CJD

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PROTEIN MISFOLDING DISEASES Mad Cow Disease - Prion Protein

PrPsc

PrPc (Prion Protein cellular)

PrPsc (Prion Protein scrapie)

Hiu ng domino ca cc prion (proteinaceous infectious particle)

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BNH L LIN QUAN N PROTEIN TO HNH SAI

Bnh l
Alzheimer

Protein
Amyloid-

Parkinson
Diabetes type 2 Amyotrophic lateral sclerosis Haemodialysis-related amyloidosis Cystic fibrosis Sickle cell anemia Hungtington Creutzfeldt-Jakob Amyloidosis

-Synuclein
Amylin Superoxide dismutase 1 2-microglobulin Cystic fibrosis transmembrane regulator Hemoglobin Huntingtin Prion protein 10 protein khc
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BIN I TO PROTEIN CHC NNG

Sau khi tng hp hoc ng thi trong khi gii m
Ct b mt hay nhiu on peptid

INSULIN
Tng hp di dng PREPROINSULIN - Loi b signal sequence (PRE) - Loi b on peptid (PRO) - Cu disulphide ni 2 on peptide
H2NH2NH2NH2N-

disulfide bonds

ZYMOGEN

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BIN I TO PROTEIN CHC NNG

Bin i ha tr: thng xy ra sau khi gii m
Collagen: Proline Hydroxyproline (cu trc bn) (hydroxylation) Cn Vitamin C O Thiu Vitamin C khng Hydroxyproline C R Scurvy
R N HN OH H OH

O C OH

Prothrombin:
O Glutamate gamma- Carboxy Glutamate O H2N CH2 CH2 CH2 CH C O O C OC OO CH C OH H2N CH C OH

(carboxylation) Cn Vitamin K Thiu vitamin K RL ng mu

O-

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BIN I TO PROTEIN CHC NNG

modification Phosphorylation Glycosylation Methylation Hydroxylation Sulfation Prenylation Palmitoylation Myristoylation Acetylation Sulfation Amidation Ubiquitylation Truncation target site Ser, Thr, Tyr Asn, Ser, Thr Arg, Lys, His, Glu Pro, Lys, Asn, Asp Tyr Cys Cys N-terminus Lys, N-terminus Tyr C-terminus Lys various cellular process signalling, activation protein activity prot. repair, chemotaxis collagen structure protein-protein interan signalling membrane association membrane association gene expression protein-protein interan bioactive peptides degradation activation 24

PCR REALTIME PCR

NGUYN TC V NG DNG

PCR
POLYMERASE CHAIN REACTION

Kary Mullis m t 1983 t Nobel Prize ha hc 1993 Polymerase Chain Reaction (PCR) l phn ng khuch i mt on ADN bng enzym polymerase, thc hin trong ng nghim, c nhy rt cao.

PCR
POLYMERASE CHAIN REACTION

PCR POLYMERASE CHAIN REACTION

Denaturation
Bin tnh 92-96oC

100 90 Temperature ( C) 80 70 60 50 40 30 20

Extension
Ko di 72oC

Lp li 30 ln (chu k)

Annealing
Bt cp 45-55oC

10
1 2 3 4 5 Time (minutes) 6

PCR NGUYN TC PHN NG

MT CHU K NHIT
Giai on bin tnh (92-96oC)

35-40 chu k nhit

Giai on bt cp (45-55oC)

Giai on ko di (72oC)

S copies = 2n x y n: S cycle y: s copies ban u

THNH PHN TRONG PCR

3 5

DNA ch
3

primers

Nucleotide t do

Taq DNA polymerase

2+ Mg2+ Mg 2+ 2+ Mg Mg Mg2+ 2+ Mg

Dd m cha magnesium

NGUYN LIU CHO PHN NG PCR

1. DNA ch (on DNA quan tm) T mu, cht tit (m, dch mng phi, dch khp, tc, pht nim mc m, pht c t cung) on DNA 1kb n 10kb (thng <1 kb) 2. Mi (primers) on n DNA thng c 18-22 nucleotide, b xung vi on DNA ch. Mi xui, mi ngc (F, R). 3. DNA polymerase: enzym chu nhit, xc tc tng hp si DNA mi. 4. dNTP (deoxy ribonucleotid): dATP, dGTP, dCTP, dTTP 5. Ion Mg++: cofactor ca enzyme 6. Dung dch m: Tris pH 8.3-8.8

THC HIN PHN NG PCR

(I) My lun nhit (THERMOCYCLER) Thi lin tc vo bung nhng lung kh nng sinh ra t mt n pht nhit, hay lung kh lnh sinh Ra t mt dn lnh ca my lm lnh (LightCycler Roche).
(II) Da theo hiu ng Peltier ngc. Hai thanh kim loi ni vi hai cc ca ngun in v c iu chnh t ng chiu dng in i qua c th thay i: bn ny nng, bn kia lnh. Khi thay i chiu dng in th nhit ca hai thanh cng thay i ngc li: bn ny lnh, bn kia nng

PHT HIN SN PHM PCR

in di trn gel agarose
(submarine agarose gel electrophoresis)

phn loi DNA da vo kch thc 1. Trong in trng, DNA tch in m s di chuyn v cc dng, on DNA c kch thc ngn di chuyn nhanh hn so vi on c kch thc ln. 2. Trong khi in di, ethidium bromide s an xen vo cu trc si i DNA v lm si i DNA pht sng khi quan st di n UV

Chun b gel agarose

PHT HIN SN PHM PCR

Chun b mu in di :
ng DNA cn in di (cha 1 hoc nhiu on DNA c kch thc khc nhau) ng cha thc o (ladder, marker) ng cha dd m c mu (loading buffer) . Cht pht quang (EtBr, SYBR G,)
Loading buffer Theo di s di chuyn ca cc on DNA Glycerol/buffer gip ko cc on DNA lng xung y ging.

PHT HIN SN PHM PCR

PHT HIN SN PHM PCR

Chp hnh gel (n UV)

CC YU T NH HNG PCR 1. 2. 3. 4. 5. Nhit bin tnh v thi gian Nhit bt cp v thit k on mi di ca on mi Nhit giai on ko di v thi gian S chu k thc hin Lu khi thc hin PCR 1. Chng m (Negative control) Khng cho DNA (thay bng nc ct) Mc ch: Phn bit c ngoi nhim hay khng 2. Chng dng (Positive control) C mt lng DNA bit trong phn ng Mc ch: m bo phn ng tt

PCR V GII HN CA PCR

1. Pht hin im cui (End-Point Detection) 2. c hiu km 3. nhy thp 4. phn gii km 4. Ch phn bit da trn kch thc sn phm PCR 5. Phi x l sau PCR, d ngoi nhim 6. Chy gel nh Ethidium bromide (cht gy ung th)

REALTIME PCR
Real-time PCR l phn ng PCR m qu trnh nhn bn DNA c theo di trc tip trn my lun nhit theo tng chu k nhit. Kim sot tn hiu hunh quang pht ra trong mi chu k phn ng (t l vi lng sn phm PCR to thnh trong tng chu k nhit - Real time). Pht hin v nh lng nng sn phm sau mi chu k nhit da trn cng pht hunh quang.

10 102 103 104 105 106

Cycle 11
Cycle 13 Cycle 15 Cycle 19 Cycle 23
C.41 C.39
C.36 C.34

Mu cha bit [C]

Standard curve

Cycle 26
Cycle 29 Cycle 31 Cycle 33 Cycle 34 Cycle 36

Knh lc kch thch

C.33
C.31 C.29

Cycle 39
1

10

102

103

104

105

Cycle 41

THI IM PHT HIN TN HIU

- Threshold: im bt u quan st c tn hiu hunh quang pht ra, ti thi im ny tn hiu do sn phm PCR > tn hiu nn.

- Ct (Threshold cycle): l s chu k m ti tn hiu hunh quang ca sn phm khuch i PCR c pht hin.
- Realtime PCR nh lng s copies trong giai on tng ly tha.

TN HIU HUNH QUANG TRONG REAL TIME PCR

1. Cht nhum khi chn vo si i DNA s pht hunh quang: Ethidium bromide SYBR green 1 2. Probe c hiu c gn cht pht hunh quang Molecular Beacon Taqman probe...

Primers Taq polymerase dNTP PCR buffer MgCl2 Cht pht hunh quang

Target DNA (Template)

Real-time PCR MIX

Real-time PCR mix

NG DNG CA PCR-REALTIME PCR

NG DNG TRONG Y HC
1. Pht hin v nghin cu cc tc nhn gy bnh 2. Chn on v nghin cu cc bnh l di truyn 3. Chn on v nghin cu mt s bnh ung th 4. Php y, quan h huyt thng

NG DNG TRONG DC HC
1. Sn xut thuc
2. Sn xut vaccin

PCR pht hin tc nhn VSV gy bnh

Bnh phm Chun b mu Ly trch acid nucleic acid

PCR
Pht hin sn phm PCR

PCR pht hin tc nhn VSV gy bnh

- Tc nhn khng th nui cy thng qui - Tc nhn virus ( virus gy vim gan B, C, SXH, H5N1, H1N1) - Tc nhn vi khun (Chlamydia, Legionella,) - Tc nhn VK nui cy thng qui cho kt qu chm (M. tuberculosis) - Tc nhn VK nui cy tht bi v c mt rt t trong bnh phm, hay b iu tr khng sinh trc ( lao nui cy tht bi, vim mng no m ct u, lu ct u)

NG DNG TRONG DC HC

Yu t di truyn ngoi chromosomal

Sn xut Insulin

One cell with the recombinant plasmid

A fermentor used to grow recombinant bacteria.

NG DNG PCR TRONG PHP Y, QUAN H HUYT THNG

Blood Semen

Tissue

Teeth

Urine Hair Saliva

Bone

Short Tandem Repeats (STR): Cc trnh t lp li ngn t 2-5 bp, dng phn bit DNA profile ca tng ngi . S a hnh trong STR th hin s copy ca cc on lp li khc nhau trong qun th.

Ex: locus TH01 c 6 trnh t lp li TCAT. CCC TCAT TCAT TCAT TCAT TCAT TCAT AAA - C hn 20 alleles cha TH01 c bit. - Mi ngi c hng 1 allele t cha hay m

AATG

7 repeats

8 repeats

Realtime PCR l cng c pht hin tc nhn gy bnh tt nht trong mu bnh phm
n gin, nhanh chng, d phn tch kt qu. Trnh c ngoi nhim v khng cn phn tch sau PCR. C th nh lng trong phm vi ln (log 7 n log 8) nhy cao hn so vi PCR. c hiu cao hn so vi PCR
Thch hp cho lm sng Khng ngoi nhim nhy tt

c hiu tt

Cng c tt nht

Realtime PCR l cng c nh lng tc nhn gy bnh tt nht trong mu bnh phm

Xc nh s lng virus (HIV, HBV, HCV...) Xc nh s lng khi u ca cc tc nhn gy bnh khn cp trong mu bnh (St xut huyt, H5N1, H1N1..)

Theo di iu tr

Tin on mc nghim trng v tin on kt qu