重组丙型肝炎病毒核心蛋白的原核表达、纯化和抗体制备

Prokaryotic expression and purification of hepatitis C virus core protein(HCV-C)and preparation of polyclonal antibodies against HCV-C

本文旨在获得纯化丙型肝炎病毒(HCV)核心蛋白(HCV-C)及抗HCV-C多克隆抗体,为深入研究HCV-C与肝细胞相互作用的分子机制奠定基础。首先以HCV1b亚型HC-J4-91全基因组质粒为模板,聚合酶链反应(PCR)扩增HCV-C基因,构建重组质粒pQE31-HCV-C。融合蛋白经原核表达、纯化后,免疫BALB/c小鼠,制备抗HCV-C多克隆抗体。利用酶联免疫吸附试验(ELISA)检测抗体效价,蛋白免疫印迹(Westernblot)和间接免疫荧光染色鉴定抗体特异性。结果显示,表达HCV-C的原核表达质粒pQE31-HCV-C构建正确,获得相对分子质量约22000的纯化融合蛋白。ELISA检测重组蛋白免疫小鼠的抗血清效价达1:12800。结果显示,自制的抗HCV-C多克隆抗体能特异性识别HCV-C。本研究获得了纯度较好、原核表达的HCV-C,并成功制备了抗HCV-C多克隆抗体,为深入研究HCV-C的致病机制提供了有实用价值的研究工具。

To investigate the interaction between hepatitis C virus core protein（HCV-C）and liver cells,purified HCV-C was obtained and polyclonal antibodies against HCV-C were prepared in the present study.The plasmid containing HC-J4-91whole genome of HCV1bsubtype was used as the template and the HCVC gene was TA-cloned by polymerase chain reaction（PCR）.The prokaryotic expression plasmid containing 6×His and HCV-C gene was constructed and named as pQE31-HCV-C.After expression in Escherichia coli and purification,the recombinant HCV-C was used to immunize BALB/c mice to obtain the antiserum.The titer and specificity of the antibody were determined by enzyme-linked immunosorbent assay（ELISA）,Western blot and indirect immunofluorescence staining.The results demonstrated that the prokaryotic expression plasmid pQE31-HCV-C expressing a 22 000fusion protein was successfully constructed.ELISA showed that antiserum against HCV-C from immunized mice had high antibody level,with titer at 1：12 800.Immunofluorescence staining and Western blot indicated that the antibody had good specificity and could recognize natural and unnatural HCV-C.It is concluded that the purified HCV-C protein and anti-HCV-C polyclonal antibodies obtained in this study might be useful for studying the molecular interaction mechanism between HCV-C and liver cells.