黄瓜绿斑驳花叶病毒cp基因的原核表达及抗血清制备  

Expression of cp gene and antiserum preparation of Cucumber green mottle mosaic virus

作　　者：陈红运[1] 赵文军[1] 白静[1] 李桂芬[1] 朱水芳[1] 

机构地区：[1]中国检验检疫科学研究院动植物检疫研究所,北京100029

出　　处：《植物病理学报》2007年第5期467-471,共5页Acta Phytopathologica Sinica

基　　金：国家质检总局资助项目(2007IK253)

摘　　要：采用RT-PCR方法克隆了黄瓜绿斑驳花叶病毒辽宁分离物(Cucumber green mottle mosaic virus Liaoning isolate,CGMMV-LN)的cp基因并连接到原核表达载体pGEX-4T-3和pET-22b(+)上,将获得的重组子pGEX-4T-3-CGMMV CP和pET-22b(+)-CGMMV CP转化大肠杆菌BL21后用IPTG进行诱导表达。SDS-PAGE和W estern blot分析表明,cp基因在大肠杆菌中获得了高效表达,融合蛋白分子量分别为43.8 kDa和17.3 kDa。将17.3 kDa融合蛋白纯化后免疫家兔,制备了CGMMV特异性抗血清,抗原包被间接ELISA法(ACP-ELISA)测定抗血清的效价为1/20 000。The coat protein （CP） gene of Cucumber green mottle mosaic virus Liaoning isolate （CGMMV- LN） was amplified by RT-PCR, and ligated to the expression vectors pGEX-4T-3 and pET-22b（ ＋ ）. The recombinant plasmids pGEX-4T-3-CGMMV CP and pET-22b（ ＋ ）-CGMMV CP were transformed into E. coli BL21 respectively and then induced by IPTG. It was shown that the cp gene was highly expressed by SDS- PAGE and Western blot analysis. The molecular weights of recombinant proteins were 43.8 kDa and 17.3 kDa in pGEX-4T-3 and pET-22b（ ＋ ） vectors. Antiserum with high specificity was produced after the rabbit was immunized with 17.3 kDa purified recombinant protein, and the titer was 1/20 000 detected by antigen coating plate ELISA （ACP-ELISA）.

关 键 词：黄瓜绿斑驳花叶病毒 外壳蛋白 原核表达 抗血清 ACP-ELISA 

分 类 号：S435.72[农业科学—农业昆虫与害虫防治]