柯萨奇病毒B组5型非结构蛋白3C表达纯化及多克隆抗体的制备

目的 表达和纯化柯萨奇病毒(CVB5)非结构蛋白3C,免疫雄性大鼠制备多克隆抗体.方法 通过酶切质粒获取3C基因序列,构建到原核表达系统大肠埃希菌中诱导表达重组的3C蛋白,使用镍柱亲和层析法纯化重组蛋白,十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和考马斯亮蓝染色法(BSA)对重组蛋白的纯度与浓度进行分析,重组的3C蛋白作为抗原,免疫雄性SPF级大鼠获得多抗血清,用ELISA测定抗体效价并Western印迹检测抗体特异性,用该抗体检测病毒在细胞内的复制.结果 构建了重组表达载体命名为3C-pET-28a,并在大肠埃希菌BL21(DE3)中诱导表达重组3C蛋白.ELISA测定制备的多抗血清效价为1:500 000,Western印迹检测在EV71、CVA16中都有交叉反应,并确定病毒感染细胞后24 h开始复制.结论 实验成功地利用原核表达系统表达柯萨奇病毒非结构蛋白3C,为进一步研究柯萨奇病毒的感染机理以及疫苗制备提供了基础.

Expression and Purification of Non-Structural Protein 3C of Coxsackievirus B5 (CVB5) and Preparation of Its Polyclonal Antibodies

Objective To express and purify 3C of coxsackievirus B5 (CVB5) non-structural protein,and to prepare its polyclonal antibodies by immunizing male rats.Methods The 3C gene sequence of CBV5 was amplified by digestion with the restriction enzyme and then inserted into the E.coli prokaryotic expression vector.The recombinant protein 3C was purified by the Nickl column affinity chromatography and evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie Brilliant Blue.Thereafter,male rats were immunized with the purified protein of 3C.ELISA was used to evaluate the potency of rat anti-serum,and Western blotting to measure its specificity.Viral replication was detected in cells using this antibody.Results The prokaryotic expression vector 3C-pET28a was successfully constructed and the recombinant protein was highly expressed in BL21 cells.The titer of anti-serum measured by ELISA was 1:500000.Western blotting showed that there were cross-reactions in EV71 and CVA16 and replication began 24 h after virus infection.Conclusion The non-structural protein 3C was purified and the polyclonal antibody against 3C was prepared in the study,which provides the basis for the further research of coxsackievirus infection mechanism and the preparation of vaccine.