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目的原核表达、纯化草鱼呼肠孤病毒(Grass carp reovirus,GCRV)HZ08株VP4蛋白,并制备其多克隆抗体。方法采用PCR法扩增GCRV HZ08株S6基因部分节段,插入原核表达载体pET-32a(+),构建重组表达质粒pET-32a-S6,转化大肠杆菌BL21(DE3),经IPTG诱导表达。表达的重组蛋白纯化后免疫新西兰白兔,制备多克隆抗体,采用间接ELISA法测定其效价,Western blot鉴定其特异性。结果重组表达质粒经双酶切及测序证实构建正确;表达的目的蛋白相对分子质量约为51 000,表达量占菌体总蛋白的73%,主要以包涵体形式表达,反应原性良好;制备的多克隆抗体效价约为1∶8 000,且具有较高的特异性。结论成功制备了GCRV HZ08株VP4蛋白高效价多克隆抗体,为VP4蛋白功能的深入研究、抗原表位分析、草鱼出血病诊断方法的建立及相关基因工程疫苗的研发奠定了基础。
Objective To express and purify the VP4 protein of HZ08 strain of Grass carp reovirus (GCRV) and prepare its polyclonal antibody. Methods The partial S6 gene of GCRV HZ08 strain was amplified by PCR and inserted into the prokaryotic expression vector pET-32a (+). The recombinant plasmid pET-32a-S6 was constructed and transformed into E. coli BL21 (DE3) for expression under induction of IPTG. The expressed recombinant protein was purified and immunized New Zealand white rabbits to prepare polyclonal antibody. The titer was determined by indirect ELISA and its specificity was identified by Western blot. Results The recombinant plasmid was confirmed by double enzyme digestion and sequencing. The relative molecular mass of the expressed protein was about 51 000, accounting for 73% of the total bacterial protein. The recombinant protein was mainly expressed in inclusion bodies and had good reactivity. Polyclonal antibody titer of about 1: 8000, and has a high specificity. Conclusion The high titer polyclonal antibody of VP4 protein of GCRV strain HZ08 was successfully prepared for the further study on the function of VP4 protein, epitope analysis, establishment of a diagnostic method for grass carp hemorrhage and the development of related genetic engineering vaccines.