减蛋综合征病毒五邻体基因的原核表达及其表达蛋白的部分活性分析

Prokaryotic expression of Penton gene of egg drop syndrome virus and activity analysis of the expressed protein

通过PCR方法扩增得到减蛋综合征病毒(EDSV)结构蛋白五邻体(Penton)基因,将其克隆到原核表达载体pET-30a(+)的多克隆位点,构建了原核表达载体pET-30a-Penton,将其转化到感受态细胞Rosetta(DE3)pLysS中,经IPTG诱导,SDS-PAGE分析,结果获得了54.0ku的融合蛋白。用纯化的融合蛋白免疫家兔制备抗血清,通过血凝试验、Western-blot、血凝抑制试验、细胞中和试验进行分析,表明该融合蛋白不能引起鸡红细胞凝集,无血凝活性;可与EDSV阳性血清发生特异性反应,能够诱导机体产生中和抗体,具有良好的抗原性。

The Penton gene of egg drop syndrome virus（EDSV）was amplified by PCR,and then cloned into the polycloning sites of pET-30a vector.The recombinant prokaryotic expression plasmid was constructed,and named pET-30a-Penton.The recombinant plasmid was transformed into competent cells Rosetta（DE3）pLysS for expression and induced with IPTG.The fusion protein of 54ku was expressed.Rabbits were immunized with the purified fusion protein to prepare polyclonal anti-EDSV serum.The hemagglutination test showed that protein could not agglutinate chicken erythrocytes.Western-blot test,hemagglutination inhibition test and neutralization test indicated that the expressed protein had good antigenicity.