FMDV非结构蛋白3A和3B的表达与反应活性分析

根据已测得的O/China/99口蹄疫病毒(FMDV)基因组序列,设计两对特异性表达引物,以带有O/Chi-na/99 3ABC基因片段的重组质粒pGEM-3ABC为模板,扩增得到了3A、3B基因,通过两端的BamHI和SalI酶切位点插入原核表达载体pET28a,将重组表达质粒转化宿主菌BL21,用IPTG诱导表达目的蛋白.表达产物经SDS-PAGE检测表明,3A和3B基因成功的在大肠埃希氏菌中得到了表达;通过Western blotting分析和斑点ELISA分析表明,纯化后的蛋白能与FMDV感染动物血清发生特异性反应.

Expression and activity analysis of 3A and 3B non-structural protein of FMDV in E.coli

Two pairs of primers were designed for amplification of 3A and 3B genes accoding to the genome sequence of O/China/99 foot-and-mouth disease virus(FMDV).The 3A and 3B genes were amplified from pGEM-3ABC by PCR,and then inserted into pET-28a vector by two restriction sites BamHⅠand SalⅠ.Two recombinant plasmids were transformed into the host strain of E.coli BL21(DE3)pLys and the target proteins were expressed by inducing with IPTG at 37 ℃.The expressed products of these 3A and 3B genes were identified by SDS-PAGE,Western blotting and Dot-ELISA.The results revealed that the target genes of 3A and 3B had been expressed successfully and could react with sera derived from FMDV infected cattle.These two products will provide an useful antigens for the development of immunoelectro-transfer blot(EITB) diagnostic method,which cpuld be used for the differentiation of the FMDV infected animals from the vaccinated animals.