Title of Thesis

Cloning And Expression Studies Of Structural Genes Of Pakistani Hepatitis C Virus Genotype 3a

Author(s)

Muhammad Zubair Yousaf

Abstract
Not Available

1

2.1 History of HCV infection
2.2 Classification and prevalence of HCV
2.3 Organization of the genome of hepatitis C virus
2.4 Natural course of HCV infection
2.5 Natural course of chronic HCV infection
2.6 Replication cycle of hepatitis C virus
2.7 Replication rate of hepatitis C virus
2.8 Transmission of Hepatitis C
2.9 Screening and diagnosing HCV infection
2.10 Treatment of Hepatitis C
2.11 Vaccine development for Hepatitis C
2.12 Significance of HCV core gene
2.13 Cloning and expression of HCV core gene
2.14 Significance of HCV E1 gene
2.15 Cloning and expression of HCV E1 gene
2.16 Significance of HCV E2 gene
2.17 Cloning and expression of HCV E2 gene
2.18 HCV infection and Pakistan-Present and Future challenges

3.1 Sample collection
3.2 Genotyping of HCV serum samples
3.3 Primer designing and primer synthesis
3.4 RNA Extraction from serum specimens
3.5 Complimentary DNA (cDNA ) synthesis
3.6 Confirmation of synthesized cDNA
3.7 PCR amplification of structural genes of HCV
3.8 Bacterial strains and plasmids
3.9 Enzymes and reagents
3.10 Agarose gel electrophoresis
3.11 Purification of DNA fragments from agarose gel
3.12 TA cloning of PCR amplified product
3.13 Transformation in E.coli
3.14 Screening of Positive colonies
3.15 Small scale preparation of plasmid DNA
3.16 Restriction digestion of plasmid DNA
3.17 Preparation of sequencing grade plasmid DNA
3.18 Automated DNA sequencing
3.19 Purifying extension products
3.20 Sample electrophoresis
3.21 Homology search
3.22 Construction of pGEX 4t2 Recombinant Expression vector
3.23 Gene expression and purification
3.24 Determination of protein concentration
3.25 Western blot analysis of recombinant proteins
3.26 ELISA

4

4.1a Genotyping of HCV serum specimens
4.1b HCV genotype distribution
4.2 Establishment of assay conditions
4.3 Primer designing and synthesis
4.4 Confirmation of complimentary (cDNA) synthesis
4.5 PCR amplification, characterization and expression of  core gene of HCV
4.6 Construction of recombinant expression vector pGEX4t2C
4.7 Expression of HCV core recombinant protein
4.8 PCR amplification, characterization and expression of E1 gene of HCV
4.9 Construction of recombinant expression vector pGEX4t2E1
4.10 Expression of HCV E1 recombinant protein
4.11 PCR amplification, characterization and expression  of E2 gene of HCV
4.12 Construction of recombinant expression vector pGEX4t2E2
4.13 Expression of HCV E2 recombinant protein
4.14 Optimization and production of recombinant structural  proteins of HCV 3a in E.coli
4.15 Optimization of expression and recombinant protein production
4.16 Purification of recombinant structural proteins of HCV 3a  through affinity chromatography
4.17 Validation of anti-HCV screening assay via known sera
4.18 Validation of anti-HCV screening assay via unknown sera

5

6