棉铃虫单核衣壳核多角体病毒3个bro基因的原核表达与抗体制备  

Prokaryotic Expression of Three bro Genes from Helicoverpa armigera Singlenucleocapsid Nucleopolyhedrovirus and Generation of Antibodies

作　　者：陈明明[1,2] 吴东[1,2] 袁丽[1,2] 陈新文[1,2] 胡志红[1,2] 

机构地区：[1]中国科学院武汉病毒研究所无脊椎动物病毒学联合开放实验室,湖北武汉430071 [2]中国科学院武汉病毒研究所分子病毒学重点实验室,湖北武汉430071

出　　处：《中国病毒学》2003年第6期597-602,共6页Virologica Sinica

基　　金：国家杰出青年基金(3002503);中国科学院创新工程重要方向性项目(kscx2-1-02;kscx2-sw-301-09);863项目(2001AA214031:101-06-10-01);国家973计划(2003CB114202)

摘　　要：根据棉铃虫单核衣壳核多角体病毒(HaSNPV)基因组全序列,设计引物,用PCR的方法扩增得到bro-a、bro-b和bro-c三个全长基因。将这三个基因的片段分别克隆至原核表达载体pProExHTb,经IPTG诱导,在E.coli DH50菌株中得到了目的基因的高效表达。表达的目的蛋白大小分别为32kDa、64kDa和58kDa,经SDS-PAGE分离纯化,免疫新西兰大白兔制备了多克隆抗体。抗体经1∶2500倍稀释后用于WestemBlot分析,获得特异性显色信号,所制备的三种抗体适合用作bro基因的功能的进一步研究。Three baculovirus repeated open reading frame(bro) genes were amplified by PCR from the genome DNA of HaSNPV. The PCR products were cloned into pBluescript KS(+) plasmid. The genes were introduced into the expression vector pProExHTb. After IPTG induction, the E. coli DHSa strain containing each of the recombinant plasmids expressed proteins with molecular weights of 32 KDa、 64 kDa and 58 kDa, respectively, which were in agreement with the prospectation. The purified recombinant proteins were used to immune the rabbits separately. Western blot analysis using the multiclonal antibodies derived from the rabbits indicated that these antibodies could react specifically with the target proteins and were suitable to be used for further functional analysis of the bro genes.

关 键 词：棉铃虫单核衣壳核多角体病毒 克隆 表达 抗体 bro基因 

分 类 号：S476[农业科学—农业昆虫与害虫防治]