非洲猪瘟病毒PP62蛋白在昆虫细胞中的表达与鉴定

Expression and identification of PP62 of ASFV in the baculovirus expression system

为表达非洲猪瘟病毒PP62蛋白,将非洲猪瘟病毒pp62基因克隆至pFastBac-HTA载体,经转座、转染后获得重组杆状病毒rBac-pp62。将该重组病毒感染Sf9细胞后,采用间接免疫荧光(IFA)检测PP62蛋白的表达,通过亲和层析纯化重组蛋白PP62,以Western-blot和间接ELISA方法鉴定纯化的重组蛋白PP62的免疫反应活性。结果显示,重组杆状病毒rBac-pp62感染的Sf9细胞可以大量表达PP62蛋白,纯化的PP62重组蛋白能够与非洲猪瘟阳性血清发生特异性反应,以重组蛋白PP62为包被抗原建立的间接ELISA方法可以区分非洲猪瘟阳性血清和阴性血清。结果表明,表达的重组蛋白PP62具有良好的反应活性,可作为开发非洲猪瘟抗体检测试剂盒的抗原以及用于PP62蛋白的结构和生物学功能研究。

In order to express ASFV PP62,the ORF of ASFV PP62 was cloned into the p Fast Bac-HTA vector.By translocation and transfection,the recombinant baculovirus r Bac-pp62 was prepared.Recombinant PP62 protein was purified by Ni-NTA Fast Start Kit.The reactivity of PP62 protein was identified by IFA test,Western-blot and indirect-ELISA.It was found that the specific fluorescence appeared in the infected Sf9 cells in IFA test.The purified PP62 protein can specifically react with anti-His Mc Ab and ASFV positive serum detected by Western-blot.Expressed PP62 was used as the coating antigen to develop an indirect ELISA which could differentiate ASFV positive serum and negative serum.All these results showed that the purified PP62 protein had good reactivity.It laid a foundation for development of the diagnosis kit and research of the structure and biological functions of ASFV PP62.