猪链球菌2型胞外因子短片段的原核表达及其免疫保护力  

Prokaryotic expression of short fragment of extracellular protein factor gene of Streptococcus suis type 2 and immune protective potency of the expressed protein in rabbits

作　　者：王冬梅[1,2] 王凡[1,3] 殷宏[3] 曾巧英[1] 

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]西北民族大学实验中心,甘肃兰州730030 [3]中国农业科学院兰州兽医研究所甘肃省动物寄生虫病重点实验室,甘肃兰州730046

出　　处：《中国兽医科学》2009年第5期421-425,共5页Chinese Veterinary Science

基　　金：国家自然科学基金项目(30571389);甘肃省农业生物技术研究与应用开发项目(GNSW-2008-09);中国博士后科学基金项目(20080430788);甘肃省自然科学基金项目(3ZS051-A25-060)

摘　　要：以江苏分离株HA9801基因组DNA为模板,扩增猪链球菌2型(SS2)胞外因子ef基因的1423～2203bp片段,并将其亚克隆至表达载体pGEX-4T-1上,构建了重组质粒pGEX-4T-1-ef。将鉴定阳性的质粒转化大肠杆菌BL21(DE3),0.5mmol/LIPTG诱导8h,表达的融合蛋白rGST-sEF大小为56ku,以包涵体形式存在。Western-blotting显示,rGST-sEF能与全菌兔抗血清发生特异性反应。用自制rGST-sEF亚单位疫苗免疫新西兰兔,以最小致死量HA9801攻击,保护率为60%。结果表明,表达的融合蛋白rGST-sEF具有良好的免疫原性和免疫反应性。A fragment（sequence positions 1423-2203） of extracellular factor（EF） gene was amplified by PCR from the genomic DNA of Streptococcus suis type 2（SS2） strain HA9801. The recombinant plasmid pGEX-4T-1-ef was constructed by inserting this fragment into plasmid pGEX-4T-1. After identification by PCR, restriction endonuclease digestion and sequencing, the recombinant plasmid pGEX-4T-1-ef was transformed into Escherichia coli BL21 （DE3）. The fragment was expressed under induction with 0. 5 mmol/L IPTG for 8 h. It was confirmed that the fusion protein of 56 ku named as rGST-sEF was expressed in the form of inclusion bodies by SDS-PAGE, and the rGST-sEF could specifically react with antiserum against whole bacterial cells of SS2. The rabbits were immunized with rGST-sEF protein emulsified with Freund＇s complete adjuvant,and then challenged with the strain HA9801 at 1 MLD. 60% of the immunized rabbits survived. The results demonstrated that the expressed rGST-sEF had good antigenieity and reactogenicity in rabbits.

关 键 词：猪链球菌2型 胞外因子 原核表达 免疫保护力 

分 类 号：S852.611[农业科学—基础兽医学] Q786[农业科学—兽医学]