大肠杆菌K_(99)-ST_1双价基因工程菌株的构建及其免疫原性  

Construction and immunogenicity of gene engineering strain with protective antigen K_(99)-ST_1 of enteotoxigenic Escherichia col

作　　者：李书光[1] 管宇[1] 肖跃强[1] 王金良[1] 沈志强[1] 

机构地区：[1]山东省滨州畜牧兽医研究院山东绿都生物科技有限公司,山东滨州256600

出　　处：《中国兽医学报》2009年第5期598-602,共5页Chinese Journal of Veterinary Science

基　　金：山东省科技攻关资助项目(031020101)

摘　　要：为了有效预防肠毒素性大肠杆菌引起的犊牛和羔羊腹泻,利用基因工程技术构建了融合基因K99-ST1及其原核表达载体,并对融合蛋白进行了初步免疫原性分析。结果显示,将PCR获得的K99菌毛抗原基因和人工合成的ST1基因片段借助pUCm-T载体,成功构建重组质粒pUCm-K99-ST1,从而获得融合基因K99-ST1;使用EcoRⅠ+SalⅠ双酶切将该融合基因定向插入表达载体pET30a中,成功构建表达载体pET-K99-ST1;将该表达载体转入表达菌株BL21(DE3)中,经IPTG诱导得到大小约31000的蛋白;Western blotting显示,融合蛋白可以特异的与K99阳性血清反应;将BL21(DE3,pET-K99-ST1)诱导表达后免疫兔,ELISA检测K99抗体水平较高,乳鼠灌胃试验显示该菌株免疫后产生ST1抗体。这表明本试验已成功构建大肠杆菌K99-ST1双价基因工程菌株BL21(DE3,pET-K99-ST1),该菌株具有良好的免疫原性,为大肠杆菌K99-ST1双价基因工程疫苗开发奠定了基础。To prevent calf and lamb diarrhoea caused by enteotoxigenic Escherichia coli, fusion gene K99-ST1 and its prokaryotic expression vector were constructed by genetic engineering. The K99 antigen gene obtained by PCR, the synthetic nucleic acid fragment of ST1 , and the easy vector pUCm-T were recombinated to recombinant plasmid pUCm-K99-ST1 ,and the fusion gene K99-ST1 was obtained. Then the fusion gene K99-ST1 was directedly inserted into expression vector pET30a by EcoR I ＋ Sal I and T4 DNA ligase, and the recombinant expression vector pET-K99- ST1 was obtained. The pET-K99-ST1 was transferred into expression strain BL21（DE3） and a 31 000 fusion protein was got from BL21（DE3 ,pET-K99-ST1 ） induced by IPTG. Western blotting analysis showed that the fusion protein could be specifically recognizied by the K99 positive sera. Rabbits were immunized with the strain BL21 （DE3, pET- K99-ST1 ） induced,and the K99 antibody and ST1 antibody were detected by ELISA and suckling mice intragastric ad- ministration, respectively.

关 键 词：大肠杆菌 K99-ST1融合基因 融合蛋白 免疫原性 

分 类 号：S855.12[农业科学—临床兽医学] R535[农业科学—兽医学]