| Summary: | 碩士 === 國立臺灣大學 === 微生物學研究所 === 89 === Hepatitis C virus (HCV) is an enveloped virus. The viral genome is a single-stranded, 9.6 kb long RNA molecule of positive polarity. The plus-stranded RNA genome encodes a polyprotein of about 3010-3033 amino acids. Generation of mature proteins from the polyprotein precursor is mediated by cellular and viral proteases.
Amino acid sequence analysis revealed that HCV NS5B protein contains the conserved -GDD- motif and is the viral RNA-dependent RNA polymerase. In our previous studies, two independent subdomains from amino acid residues 83 to 194 and from 196 to 298 were demonstrated to be important for the NS5B protein to bind to the viral RNA. In addition, the conserved motifs of RNA-dependent RNA polymerase for putative RNA-binding (220-DxxxxD-225) and template/primer position (282-S/TGxxxTxxxNS/T-292) are present in the NS5B(196-298). In this study, additional NS5B mutant plasmids were constructed and transformed into E. coli DE3(BL21). NS5B mutant proteins were partially purified following polyacrylamide gel electrophoresis and electro-elution. Their binding activities to the HCV RNA were investigated. The result demonstrated that NS5B(207-298) mutant protein retained the ability to interact with the viral RNA.
On the other hand, plasmid pcDNA3-NS5B(1-570)-Cr1 encoding a fusion protein containing NS5B from amino acid residues 1 to 570 linked to the constant region of the immunoglobulin heavy chain (Cr1) was injected into mice skeletal muscle to induce immune response. The mice antisera were collected over a period of 12 weeks. However, the specificity of the mice antisera to the NS5B need to be further evaluated. In addition, procedures used in the DNA immunization may be modified in the future in order to evaluate the potential to develop DNA vaccine for HCV infection.
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