猪圆环病毒2型多表位串联体诱导表达及其免疫活性

为了获得体外诱导表达的具有良好免疫原性的PCV2多表位串联体。本试验筛选PCV2主要抗原表位,顺序组成多表位串联体,人工合成其cDNA序列,通过BamHⅠ和SalⅠ定向克隆至原核表达载体pEGX-4T-1多克隆位点,转化BL21感受态细胞,筛选阳性克隆,进行IPTG诱导表达。使用SDS-PAGE对目的基因表达情况进行分析,提取与纯化融合蛋白多肽,Western blot鉴定表达多表位串联蛋白免疫活性。融合蛋白多肽免疫BALB/c小鼠,ELISA测定其抗体,评价其免疫原性。结果显示,成功构建了含7个PCV2抗原表位串联体的表达重组质粒pEGX-4T-1-ep,SDS-PAGE分析显示,融合蛋白多肽在大肠杆菌中获得了有效表达,其分子量约35ku,主要以可溶性形式存在。Western blot结果显示,提取与纯化融合蛋白多肽与PCV2阳性血清具有良好的反应原性。ELISA检测结果显示,纯化的融合蛋白多肽可有效刺激机体产生PCV2特异性抗体,具有良好的免疫原性。结果表明,构建的PCV2多表位串联体表达重组体具有良好的体外表达特性,表达蛋白具有良好的免疫原性。该研究为PCV2表位筛选,功能鉴定及多表位疫苗研究奠定了一定基础。

Induced expression of multi-epitope of porcine circovirus type 2 and its immune activity

In this study,in order to get good expression in vitro and good immunogenicity multiepitope of porcine circovirus type 2. The cDNA sequence encoding multi-epitope of PCV2 major antigen was sequentially installed and artificially synthesized that was cloned into pEGX-4T-1 plasmid by BamH I and sal I The resultant plasmid was transformed into E. coli BL21 in which a fusion protein was expressed with IPTG. The epitope gene expression was analysed by SDS- PAGE method and the recombinant protein polypeptide was extracted and purified by GST affinity chromatography that the tandem of multiTepitope was identified by Western blot. Tandem con- taining 7 epitopes of PCV2 was set up and the recombinant plasmid p EGX-4T-I-ep containing the epitope tandem was constructed by gene engineering. The SDS-PAGE analysis showed that the fusion protein was 35ku,with high efficient expression in E. coli. The expression of polypeptide protein was mainly in soluble form. Western blot and ELISA detection indicated that the polypep- tide protein reacted to positive PCV2 antibody. In this study,the expression recombinant plasmid of PCV2 polypeptide was successfully constructed in vitro and the fusion protein has good immu- nogenicity against to positive PCV2 antibody, which laid a certain foundation for the screening and identification of PCV2 epitope and research of muti-epitope vaccine.