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1.Soluble Expression of HCV Core Protein by Auto-induction and the Identification of Its Biological Function2.Analysis of HBV Infectionwith Different Genotypes by High Throughput Sequencing

Author: GongXuYang
Tutor: HuangAiLong
School: Chongqing Medical University
Course: Internal Medicine
Keywords: Hepatitis C virus Core protein Auto-induction Soluble expression Hepatitis B Virus genotype ThroughputSequencing
CLC: R512.63
Type: Master's thesis
Year: 2013
Downloads: 20
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Abstract


Soluble Expression of HCV Core Protein by Auto-inductionand the Identification of Its Biological FunctionObjective: A recombinant prokaryotic expression plasmid containingthe full-length gene of Hepatitis C virus (HCV) Core protein (protein C)was to be constructed, following by transformation into expressing E.coli.To obtain the recombinant HCV protein C and detect its biological function,the expression in vitro by Auto-induction to a high concentration wascarried on.Methods:H/FL plasmid as template, a PCR was performed to obtainthe full-length HCV protein C DNA (573bp), followed by a doubledigestion to be cloned into pET28a prokaryotic expression vector.Recombinant prokaryotic expression vector containing HCV protein C gene was transformated into E. coli BL21(DE3) pLysS, which expressedrecombinant C protein of high concentration by Auto-induction.Recombinant protein C was purified by Ni-NTA affinity chromatographycolumn,following by HCV NS3protein binding assay to detect thebiological activity of recombinant protein C.Result: A large number of recombinant protein C was present in thesonicated supernatant of bacteria. The antigenic of protein C was detectedby Western blot. The recombinant protein C can not be purified by Ni-NTAaffinity chromatography,but co-purified with HCV NS3protein.Conclusion: The soluble recombinant protein C was successfullyexpressed,and the protein interacts with the HCV NS3protein wasconfirmed, which proved the biological activity of recombinant protein C.Our study lay a foundation for the further study of HCV core protein’sbiological functions,crystal structure and antigen detection in clinic.Analysis of HBV Infectionwith Different Genotypes byHigh ThroughputObjective: Previous studies have shown that HBV genotypes cancoexist in HBV infection. Genotypes’ research has become a new hot spot.But the study for that is not a lot. In order to investigate the B and Cgenotype infection in patients with HBV, high-throughput sequencing with large number of sample was used.Methods: Extracted serum DNA of532cases of patients with chronicHBV infection.436with treatment,60infants and18pairs of mother andchild serum samples.PCR amplification followed by Solexa sequencing.Anewgenotyping method was used.Sanger method andcloningsequencingrepeat the results. The statistical analysis of the relationship between mixedinfection and different clinical data was performed.Result:532specimens are basically of two mixed genotypes. Ofwhich96cases of samples by direct sequencing using the Sanger method toverify the results of the two methods.The Solexa sequencing samples in40cases, the use of cloningsequencing further validate the results of the twomethods.

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CLC: > Medicine, health > Internal Medicine > Infectious disease > Viral infections > Viral Hepatitis > Hepatitis C (non- A non-B )
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