丙型肝炎病毒多表位抗原的融合表达及抗原性分析

Expression of HCV multi-epitope antigen and its immunogenicity assay

目的表达HCV核心蛋白Core、非结构区NS3、NS4和NS5区的部分优势表面抗原的融合基因,为丙型肝炎病毒的检测提供合适抗原。方法通过RT-PCR从HCV全长基因组中分别克隆Core、NS3、NS4和NS5区的部分优势表面抗原的基因片段,运用重叠延伸PCR技术将它们拼接成融合基因,将融合基因再克隆到表达载体pBVIL-2中,转化大肠杆菌JM109,阳性克隆经42℃热诱导融合蛋白的表达,表达产物经SDS-PAGE及Westernblot鉴定。重组蛋白经阳离子交换和分子筛纯化。结果42℃诱导可高表达分子量约为43kD的融合抗原,重组蛋白主要以包涵体形式存在。经过纯化目的蛋白纯度达90%以上的,表达量约为886μg/mL。ELISA结果表明纯化蛋白具有良好的抗原性。结论HCV复合多表位抗原融合蛋白可作为HCV诊断抗原提供了靶抗原。

[ Objective ] To express and observe the immunogenicity of hepatitis C Virus （HCV） multi-epitope antigen, and to explore the possibility of detection HCV antigen by antibody. [ Methods ] DNA fragments of HCV Core fragment, NS3 fragment, NS4 fragment and NS5 fragment have been obtained from HCV genome by RT-PCR, then all the fragments were linked by Overlap Extension PCR （OE-PCR） and formed a chineric gene Core-NS3- NS4-NS 5 （C-N5）. The chimeric gene was then ligated to expression vector pBVIL-1 and the recombinant protein was expressed in Escherichia coli. The recombinant protein was purified by by DEAE column and molecular sieve column. [Results] The recombinant protein was correctly expressed, a band of 43 kD was detected by 15% SDSPAGE and western blot analysis. After purification, its yield amounted to 886ug/mL. ELISA result showed that the chimerical antigen had good antigenicity. [ Conclusion ] The chimerical antigen had good antigenicity, which could play an important role in anti-HCV detection.