猪札幌病毒VP1基因的表达及ELISA抗体检测方法的建立

为了在大肠杆菌中表达猪札幌病毒VP1基因,并以纯化的重组蛋白为抗原建立猪札幌病毒血清抗体ELISA检测方法。采用RT-PCR技术扩增VP1基因,并将其克隆到pMD18-T载体中,再将所克隆的衣壳蛋白的编码序列亚克隆到表达载体pET-28a(+)中,将成功构建的原核表达质粒pETSAVCAP转化大肠杆菌Rosetta(DE3)进行诱导表达。用纯化的VP1蛋白作为抗原建立了猪札幌病毒血清抗体间接ELISA检测方法并初步用于临床样品的检测。结果显示,猪札幌病毒VP1基因可在大肠杆菌中稳定、高效地表达,Western-blot分析表明该重组蛋白具有良好的反应原性。经对反应条件进行优化,确定间接ELISA的抗原最佳包被浓度为0.5μg/mL,且不与其他常见猪病的阳性血清发生交叉反应,建立的ELISA方法具有较好的敏感性、特异性和重复性。通过对从多个省份收集的490份猪血清样品进行检测,阳性率为65.31%。表明,以大肠杆菌表达的猪札幌病毒VP1重组蛋白为抗原建立的间接ELISA方法可以用于猪札幌病毒抗体的检测。

Expression of VP1 gene of porcine sapovirus and development of an indirect ELISA for the detection of antibodies against porcine sapovirus

The aim of the present study was to establish an indirect ELISA method for the detection of porcine sapovirus antibody based on the expression of VP1 gene of porcine sapovirus in Escherichia coli.Using a pair of specific primers designed according to the relevant nucleotide sequence from GenBank,the VP1 gene of porcine sapovirus was amplified with RT-PCR method from stool samples of piglet suffered from diarrhea.The PCR product was then cloned into a pET-28a(+) vector to construct a prokaryotic recombinant plasmid pETSAVCAP.The pETSAVCAP was transformed into E.coli BL21(DE3) for expression.Then an indirect ELISA was established to detect antibody against porcine sapovirus with the VP1 protein,which was purified by SDS-PAGE,as coating antigen.The VP1 protein was stably and highly expressed and manifested good reactinogenicity as was confirmed by Western-blot.The optimum working concentration of antigen was 0.5 μg in 100 μL per well.By using this novel ELISA approach,490 pig serum samples which were collected from six provinces of China were tested with a positive rate of 65.31% of porcine sapovirus antibodies.The method was specific,sensitive and reliable.The results indicated that the ELISA method based on the purified recombinant protein could be used to detect antibodies against porcine sapovirus.