牛病毒性腹泻病毒Core蛋白的原核表达及多克隆抗体制备

为制备牛病毒性腹泻病毒(BVDV)的Core蛋白及其多克隆抗体,根据Core蛋白的编码基因序列,设计合成一对特异性引物,利用RT-PCR扩增BVDV Core基因并定向插入Pet30a载体中,构建重组质粒Pet30a-Core,经酶切鉴定后转化大肠埃希菌TOP 10感受态细胞,筛选获得阳性重组菌,以IPTG进行诱导后成功表达出分子质量为20 ku的重组Core蛋白。将诱导表达的蛋白产物经融合蛋白的Ni柱亲和法进行纯化,利用纯化的重组蛋白免疫新西兰白兔制备多克隆抗体,并利用间接ELISA法测出多克隆抗体效价达到1∶512 000。蛋白质印迹法(Western blot)和间接免疫荧光试验(IFA)证实,多克隆抗体可与细胞中的BVDV抗原发生反应,具有良好的免疫原性和特异性。本实验成功制备了BVDV重组Core蛋白的兔源多克隆抗体,为BVDV的检测及其Core蛋白功能研究奠定了基础。

Prokaryotic expression of Core protein of bovine viral diarrhoea viruses and preparation of polyclonal antibodies

In order to obtain the Core protein of bovine viral diarrhea virus (BVDV) and its polyclonal antibody, a pair of specific primers was designed and synthesized according to the coding sequence of the Core protein, and the BVDV Core gene was amplified by RT-PCR and inserted into the Pet30a vector. The recombinant plasmid Pet30a-Core was constructed and transformed into Escherichia coli TOP 10 competent cells by enzyme digestion. The positive recombinant bacteria were screened and the recombinant Core protein with molecular mass of 20 ku was induced to express with IPTG. The protein was purified by Ni column affinity method of the fusion protein, and the polyclonal antibody was prepared by immunizing New Zealand white rabbit with the purified recombinant protein, and the polyclonal antibody titer was determined by indirect ELISA to reach 1∶512 000. Western blot and indirect immunofluorescence assay (IFA) confirmed that polyclonal antibodies reacted with BVDV antigens in cells and had good immunogenicity and specificity. The rabbit polyclonal antibody against BVDV recombinant Core protein was successfully prepared, which laid a foundation for the detection of BVDV and the study of Core protein function.