猪流行性腹泻病毒CH/ZJ分离株S基因的克隆、原核表达和免疫原性分析

Prokaryotic expression and immunogenicity analysis of S gene of Porcine epidemic diarrhea virus CH/ZJ strain

为了获得具有免疫活性的猪流行性病毒S蛋白,试验参照GenBank中所收录的猪流行性腹泻病毒(PEDV)CV777株S基因序列设计1对特异性引物,去除信号肽,以PEDVCH/ZJ株为模板,通过RT-PCR扩增出目的基因,克隆入原核表达载体pET28a(+),转化到Top10感受态细胞中,测序并分析。结果表明:目的基因含有1个长966bp、编码322个氨基酸残基组成的多肽;与CV777株相比较,同源性为96.0%,但在398bp处缺失编码1个异亮氨酸的密码子,将重组质粒命名为pET-P-SP1;将pET-P-SP1转化到大肠杆菌E.coliBL21-DL3(Rosetta)中,在IPTG诱导下表达;通过SDS-PAGE表达出与预期大小相符的约35.6ku的重组蛋白,重组蛋白以包涵体形式存在;表达产物占菌体蛋白的80%;表达蛋白能与抗猪流行性腹泻病毒高免血清反应,说明该重组蛋白具有免疫活性。

In order to obtain Porcine epidemic diarrhea virus S gene that has immune activity.The specific primers were designed by sequence of Porcine epidemic diarrhea virus（PEDV）CV777 strain S gene reported in GenBank excised signal peptide.The purpose gene of porcine epidemic diarrhea virus CH/ZJ strain was amplified by RT-PCR and then cloned into prokaryotic expression vector pET28a（＋）.The plasmid was transformed into E.coli Top10,and sequence was compared with PEDV CV777 strain.The gene comprised 966 bp encoding a polypeptide of 322 amino acids residues and the homology shared 96.0% nucleotide sequence with CV777,but deletion 3 bp at 398 bp position which encoding a isoleucine.The recombinant plasmid was transformed into E.coli BL21-DL3（Rosetta）and induced with IPTG.The expression product accounted for 80% of the total bacterial proteins by thin layer scanning.The recombinant protein was purified and then detected by SDS-PAGE and western-blot.The recombinant protein could react with the anti-PEDV hyperimmune serum.