JC病毒小包膜蛋白VP2抗原及鼠多克隆抗体的制备

目的 构建JC病毒小包膜蛋白VP2的原核表达载体,表达纯化VP2蛋白,并制备其抗体.方法 用PCR方法从患者脑脊液中扩增JC病毒小包膜蛋白VP2基因,测序正确后克隆入pET-32a(+)原核表达载体,转化BL21大肠埃希菌,用异丙基-β-D硫代半乳糖苷(IPTG)诱导表达该蛋白并纯化.纯化的VP2蛋白免疫BALB/c小鼠,制备鼠多克隆抗体.结果 将pET-32a(+)-VP2重组表达载体分别转化BL21大肠埃希菌,经IPTG诱导后,表达出相对分子质量为58.5×10~3左右的重组融合VP2蛋白.聚丙烯酰胺凝胶电泳分析显示,IPTG诱导6～10 h重组蛋白表达水平较高.Western印迹证实其具有良好的抗原性,且免疫BALB/c小鼠后成功制备了鼠多克隆抗体.结论 成功构建原核表达载体pET-32a(+)-VP2并获得纯化VP2融合蛋白,并制备JC病毒小包膜蛋白VP2抗体,为进一步对VP2蛋白的功能及JC病毒流行病学调查等研究奠定了坚实的基础.

Expression and purification of JC virus VP2 fusion protein and preparation of its polyclonal antibody

Objective To obtain the antigen and antibody of JC virus(JCV)VP2.Methods The JCV VP2 gene were amplified from a cerebrospinal fluid sample by polymerase chain reaction (PCR)and confirmed by sequencing.Then,the gene was cloned into plasmid pET32a(+)to construct recombinant prokaryotic expression vector pET-32a(+)-VP2.The recombinant plasmid was transformed into the competent E.coli BL21.Induced with isopropyl-β-D-1-1 thiogalactopyranoside (IPTG),E.coli BL21 were subsequently crushed by ultrasound.The gene expression in the supernatant was analyzed by Western blot.Thereafter,the expressed protein was purified by isoeleetric point method.The polyclonal antibody against JCV VP2 protein was obtained from the BALB/c mouse immunized with the purified protein.Results The VP2 fusion protein was expressed in the E.coli BL21.The recombinant fusion protein was expressed by IPTG induetion with relative molecular mass of 58.5×10~3.Sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDSPAGE)analysis showed that the expression level was highter after 6-10 h of IPTG induction.The recombinant protein had good antigenicity which was confirmed by BALB/c mice immunized with the protein.Conclusions The successful expression and purification of VP2 fusion protein and the antibody will be valuable for the study on the biological function of VP2 and JCV epidemiologieal investigation.