紫藤脉花叶病毒cp基因在大肠杆菌中的表达及抗血清的制备

Expression of the cp gene and Antiserum Preparation of Wisteria Vein Mosaic Virus

采用RT-PCR方法自紫藤脉花叶病毒北京分离物(WVMV-BJ)的基因组中分离出其CP基因,连接到原核表达载体pET22b(+)上。获得的重组子pET-WVMVCP转化大肠杆菌BL21(DE3)后,用IPTG进行诱导表达。SDS-PAGE和Westernblot分析表明,cp基因在大肠杆菌中获得了高效表达,融合蛋白分子量约为34.4kDa。将融合蛋白纯化后免疫兔子,获得了特异性较高的抗血清。微量免疫沉淀法测定该抗血清的效价为1/1024,酶联法(enzyme-linkedimmunosorbantassay,ELISA)测定的效价为1/8192。

The coat protein (CP) gene of the Beijing isolate of Wisteria vein mosaic virus (WVMV-BJ) was amplified by RT-PCR, and ligated to the expression vector pET22b(+). The recombinant plasmid pET-WVMVCP was transformed into E. coli BL21(DE3) and then induced by IPTG. It was shon that the CP gene was highly expressed by SDS-PAGE and Western blot analysis. The molecular weight of the recombinant protein was about 34.4kDa. Antiserum with high specificity was produced after the rabbit was immunized with purified recombinant protein, and the titer was determined to be 1/1024 by micro-precipitation, or 1/8192 by antigen coating plate- ELISA(ACP-ELISA).