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Table Of Contents

1.1 Genomic organisation of HIV-1
1.2 The HIV-1 lifecycle
1.2.1 Binding and entry
1.2.2 Reverse transcription
1.2.3 Nuclear transport and integration
1.2.4 Expression of viral proteins
1.2.5 Virion assembly, budding and release
1.3 The preintegration complex (PIC)
1.3.1 The composition of PIC
1.3.2 Nuclear transport of PIC
1.4 Structure and function of reverse transcriptase
1.5 Structure and function of integrase
1.6 The RT – IN interaction, and its study
1.6.1 Coimmunoprecipitation
1.6.2 The yeast two-hybrid system
1.6.3 Physical chemical methods
1.6.4 Enzyme Linked Immunosorbent Assays (ELISA)
1.7 Aims of study
2.3 Antibodies
2.4 Bacterial strains
2.6 Solutions and buffers
2.6.1 Produced solutions and buffers
2.6.2 Purchased solutions and buffers
2.7 Molecular weight standards
2.8 Chemicals
2.10 Electrical equipment
2.11 Miscellaneous
2.12 Bacteriological methods
2.12.1 Preparation of electro-competent E.coli cells
2.12.2 Transformation of cells
2.12.3 Isolation of plasmids from bacteria
2.13 PCR amplification of the RT and IN coding regions
2.13.1 The PCR reaction
2.13.2 Purification of the PCR products
2.14 Construction of in vitro transcription and translation vectors encoding IN
2.14.1 Digestion with restriction enzymes
2.14.2 Electrophoresis in low meltingpoint (LMP) agarose
2.14.3 Ligation in LMP agarose
2.14.4 Sequencing of constructs
2.15 In vitro transcription and translation
2.15.1 Production of reverse transcriptase and integrase in vitro
2.16 Protein methods
2.16.1 SDS-PAGE
2.16.2 Western blotting
2.16.3 Detection of proteins on Western blots
2.17 The ELISA procedure
3 Results
3.1 Production of in vitro translation vectors
3.1.1 Amplification of coding regions by PCR
3.1.2 Cloning of RT and IN into the vector pCITE-5b(+)
3.1.3 Determination of the concentrations of pCITE-RT and pCITE-IN
3.1.4 Sequence analysis of recombinant constructs
3.2 Description of the anticipated proteins
3.3 In vitro transcription and translation
3.3.1 Production of protein batches
3.3.2 Determination of protein concentrations
3.4 Results with ELISA
4.2 In vitro transcription and translation
4.2.1 Labelling of proteins
4.2.2 Production of proteins
4.3.1 Direct ELISA
4.3.2 Other ELISA formats
4.4 RT-IN interactions
4.6 Future strategies
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Published by tweety_sweety192001

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Published by: tweety_sweety192001 on Sep 06, 2010
Copyright:Attribution Non-commercial


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