摘要
根据GenBank登录的猪细小病毒NADL-2株和China株NS1基因序列设计1对特异性引物,对贵州大学动物生物技术实验中心分离的猪细小病毒GZ株接种ST细胞传代培养后,提取病毒DNA,进行PCR扩增PPV NS1基因主要抗原表位区,克隆后测序,随后将该基因亚克隆至pET-32a(+)载体,构建pET-32a-NS1重组质粒,转化得到重组菌,经IPTG诱导表达,通过亲和柱纯化并复性,制备重组蛋白。Western-blot检测发现,经诱导表达的NS1基因主要抗原表位区蛋白约为64 ku,与预测的大小一致,而且,重组蛋白在纯化复性后能被PPV阳性血清所识别,具有较好的反应原性。
According to the NS1 gene sequences of porcine parvovirus （PPV） NADL-2 and China strains published in GenBank,a pair of specific primers was designed.The PPV GZ strain was isolated and inoculated into ST cells for proliferation.The viral DNA of GZ strain was extracted and used to clone the major antigen regions of NS1 gene.Subsequently,NS1 gene fragment was subcloned into pET-32a （＋） vector to construct pET-32a-NS1 recombinant plasmid.The recombinant plasmid was induced by IPTG,and the recombinant protein was purified by affinity column chromatography.Western-blot analysis indicated that the molecular weight of the recombinant protein was the same as predicated size of approximately 64 ku.Further,the purified and refolded recombinant protein showing good reactionogenicity,could be identified by PPV positive serum.
出处
《山地农业生物学报》
2013年第5期397-402,共6页
Journal of Mountain Agriculture and Biology
基金
贵州大学研究生创新基金项目"猪细小病毒NS1蛋白主要抗原区间接ELISA方法的建立"(研农2013020)
贵州省2011年农业攻关项目"贵州省规模化猪场主要病毒性疫病的调查及综合防控对策的研究与示范"[黔科合NY字(2011)3103号]
