PCV2衣壳蛋白羧基端基因在T4噬菌体表面的展示及ELISA方法的建立

The establishment of ELISA assay with the purifild recombined T4 bacteriophage displaying of the carboxyl terminus fragment of capsid gene on porcine circovirus type 2

用PCR扩增猪圆环病毒Ⅱ型广东分离株的衣壳蛋白羧基端基因,将PCR产物连接到pR质粒,转化DH5α细胞,筛选阳性克隆进行PCR鉴定并测序后,转化E2菌,将重组E2菌与缺陷型噬菌体T4-Z1同源重组后,得到重组噬菌体,经SDS-PAGE和Western blotting分析,表明衣壳蛋白片段在噬菌体表面正确展示,表达的融合蛋白的分子质量约为25ku。用差速离心法纯化重组噬菌体,将纯化噬菌体作为包被抗原,建立了ELISA诊断方法,经对356份临床发病猪血清样品检测,结果表明仔猪的血清阳性率为34.8%,育肥猪的血清阳性率为57.8%,种猪的血清阳性率为67.9%。

Based on the gene sequence of Cap gene,a pair of primers was designed and,a 384 bp fraction of the carboxyl end of the capsid protein gene was obtained by PCR amplification,which was then cloned into plasmid pR to yield an integrative plasmid pR-Cap.The integrative recombinant plasmid was used to transform E.coli E2 host bacteria,and then E2 containing the recombinant plasmid was used for homologous recombination with lysozyme gene-deficient T4,thus yielding a recombinant bacteriophage T4-Cap.When purified T4-Cap was tested with SDS-PAGE and Western blotting,a 25 ku protein band was detected in polyacrylamide gel and nitrocellulose membrane,which could combine specifically with PCV2 antiserum,attesting to the successful display of SOC fusion protein on the T4 bacteriophage surface.With the purified recombined T4 bacteriophage as plate-coating antigen,a ELISA assay for PCV2 was preliminarily established.356 sera samples of sick pigs were examined with this ELISA.The positive incidence in piglets,hogs and sows was 34.8%,57.8% and 67.9%,respectively.