摘要
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.
出处
《中国动物传染病学报》
CAS
2014年第5期28-34,共7页
Chinese Journal of Animal Infectious Diseases