猪细小病毒NS1蛋白主要抗原表位区的原核表达及间接ELISA方法的建立

Establishment of an indirect ELISA with the major epitope domain of NS1 of PPV expressed in prokaryotical expression system

通过分子生物学软件对GenBank中发表的猪细小病毒(Porcine parvovirus,PPV)中国株NS1蛋白的氨基酸序列进行分析,确定了主要抗原表位区域,针对此区域设计了1对特异性引物。通过PCR方法扩增出长度为843bp的基因片段,将此片段克隆到原核表达载体pET30a(+)上,构建了原核表达载体pET30a-NS1,实现了PPVNS1蛋白主要抗原表位区在大肠杆菌中的高效表达。重组蛋白相对分子质量约为43 000,与预期相符。Western-blot试验表明获得的表达产物具有良好的反应原性。以纯化的该蛋白作为包被抗原,通过方阵试验确定了包被抗原的最佳包被量为2 mg/L,一抗的最佳稀释倍数为1∶100,阳性标准为:待检血清D450≥0.36,且待检血清D450/阴性血清D450>2,作为阴阳性临界值,初步建立了检测PPV抗体的间接ELISA方法。用该方法对临床血清样本进行检测,间接ELISA判定为阳性的60份血清,经Western-blot试验只有45份为阳性。随机抽取100份猪血清样品与商品化试剂盒检测结果对比,符合率为96%,表明所建立的间接ELISA方法具有良好的特异性和敏感性,为流行病学调查和疾病的鉴别诊断奠定了基础。

According to the amino acid sequence in GenBank of China strain porcine parvovirus, the major epitope domain in NS1 protein was discovered by the software of the molecular biology. A pair of specific primer was designed to amplify the major epitope domain of NS1. The PCR product of interest was size of 843 base pairs and was cloned into the pET30a （＋） vector. The prokaryotic expression plasmid pET30a-NS1 was successfully constructed and transformed into E. coli BL21 （DE3） pLysS competent cells and induced with IPTG. The size of recombinant protein is 43 000 according to being expected. The parameters including cultivation temperature and concentration of IPTG were optimized,then protein of interest was highly expressed, suggesting that the recombinant protein hold a favourable immunogenicity confirmed by Western-blot. Then an indirect ELISA method was established to detect antibody against PPV with the purified NS1 protein as a coating antigen. The result showed that the optimal concentration of coated antigen was 2 mg/L and the optimal dilution of serum was 1 ： 100. The positive criterion of the ELISA assay was D450≥0.36 and D450 positive serum/D450 negative serumS2. In 60 positive sera samples by the indirect ELISA, there are 45 positive sera by Western-blot. It was conclude that the indirect ELISA established in this experi- ment is more sensitive than western blot. Compared with commercial kits, the assay showed a correlation rate of 96 % based on the data obtained from 100 samples. The results indicated that the ELISA is sensitive and specific,and suitable for routine diagnostics and epidemiological surveys.