猪圆环病毒2型Cap蛋白表达及免疫原性鉴定

EXPRESSION AND IMMUNOGENICITY OF RECOMBINANT CAP PROTEIN OF PORCINE CIRCOVIRUS TYPE 2

以猪圆环病毒2型(Porcine circovirus type 2,PCV2)WH株基因组DNA为模板,扩增ORF2截短基因,经BamH I/NotI双酶切处理后与经相同酶切处理的pET32a(+)原核表达载体连接,获得重组质粒pET32a-Cap2。将重组质粒转化至BL21(DE3),经IPTG诱导,对表达产物进行SDS-PAGE和Western blot分析。纯化的重组蛋白免疫小鼠,并对获得的血清进行间接ELISA检测和中和活性测定。SDS-PAGE分析表明,ORF2截短基因在大肠杆菌中得到表达,蛋白分子质量大小为40 kDa,重组PCV2 Cap蛋白主要以上清的形式存在。Western blot证实重组蛋白能够识别抗PCV2阳性血清。经间接ELISA检测,鼠抗PCV2 Cap血清抗体效价能达到1:25 600,间接免疫荧光检测分析表明,鼠抗PCV2 Cap血清能特异性识别PCV2感染细胞中的Cap蛋白。病毒血清中和实验证实,抗PCV2 Cap血清抗体具有中和病毒的活性,中和效价为1:36。猪圆环病毒2型Cap蛋白的表达,为进一步研究该蛋白的功能及Cap蛋白亚单位疫苗和检测试剂盒的制备奠定了基础。

The truncated ORF2 of Porcine circovirus type 2（PCV2） WH strain was amplified and cloned into expression vector pET32a （＋）. The expression vector pET32a-Cap2 was then transformed into BL2 （DE3） for expression with induction of IPTG. The expressed pET32a-Cap products were analyzed in SDS-PAGE and Western blot. The recombinant Cap protein was a soluble protein with molecular weight of 40 kDa. The purified Cap protein was used to immunize mice to prepare antiserum. The resulting murine antiserum had ELISA titer at over 1：25 600 and reacted with PCV2 in infected PK-15 cells in indirect assay （IFA）. The murine antiserum also showed neutralizing titer at 1：36. The availability of the recombinant PCV2 Cap protein provides the basis for development of vaccine and diagnostic reagents.