摘要
利用RT PCR技术 ,用包容口蹄疫病毒完整VP1基因的引物从口蹄疫分离株 (China/99 2 )中扩增得到一约 75 0bp的DNA片段 ,克隆后 ,经对该片段的核苷酸序列进行测序和同源性比较 ,显示其与国外同源参考序列 (O1K/66)的同源性为 80 .44% ,与国内同型参考株 (HB/WH/99)的核苷酸序列的同源性达 99.0 6%。随后以重组质粒pGEM VP1为模板 ,用特异性表达引物扩增得到目的基因VP1 (65 0bp) ,对目的基因和表达载体 pGEX 4T 1分别以相同的限制性内切酶酶切后构建成重组表达载体 ,转化宿主菌BL2 1 (DE3 )后得到重组质粒pGEX VP1 ,经酶切及PCR鉴定筛选出阳性克隆 ,测序证明目的基因正确插入到表达载体。用IPTG诱导VP1基因的表达 ,收集不同时间的菌液进行SDS PAGE电泳、Western blotting分析检测。结果表明 ,结构蛋白VP1基因可在大肠埃希氏菌中表达 ,表达产物的分子量约为 5 3ku ,并能被口蹄疫阳性血清所识别。经薄层扫描分析 ,表达蛋白约占菌体蛋白总量的 2 0 .0 0 %。证明口蹄疫病毒结构蛋白VP1基因可在大肠埃希氏菌中高效表达 。
In this study, the viral RNA was extracted fromtongue epithelium of cattle infected with FMDV ,and then the fragment of VP1 wasamplified with a primer pair by RT PCR.The interest fragment was inserted into pGEM Teasy vector.The recombinant plasmid was identified by restriction analysis and PCR.It was proved by DNA sequencing that theacquired recombinant containscomplete VP1 gene. The homologies of the nucleotide sequence of VP1 gene were 80.44%and 99.06% respectivelycomparing with that of strain O1K/66 and HB/WH/99.Afterwards the complete VP1 gene from the identified recombinant was amplified with another primer pair containing EcoRⅠand BamHⅠsites by PCR and digested it with EcoRⅠand BamHⅠ.The expression vector pGEX 4T 1 were digested by EcoRⅠand BamHⅠrespectively. The target gene VP1 was subcloned into vector pGEX 4T 1.Positive clones named as pGEX VP1 with interest gene were identificated by restriction analysis , PCR and DNA sequencing.Then the recombinant was transformed into Escherichia coli BL21(DE 3 ) for VP1 expression. The interest gene was inducedto express in E.coli with IPTG.The bacteria containing pGEX VP1 were collected at different time and subsequently were examinedby SDS PAGE and western blotting.Results showed that the structural protein VP1 gene of FMDV can express successfully in E.coli. Molecular weight of the fusion protein was 53 ku, and the amount of target proteinis over 20.00% of the total bacteria protein.The fusion protein can be recognized by the positive serum of cattle that was infected with FMDV.
出处
《中国兽医科技》
CSCD
北大核心
2003年第3期3-8,共6页
Chinese Journal of Veterinary Science and Technology
基金
国家"973"项目子专题部分内容 (1 9990 1 190 3)
