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.
出处
《中国兽医科学》
CAS
CSCD
北大核心
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)
关键词
猪链球菌2型
胞外因子
原核表达
免疫保护力
Streptococcus suis type 2
extracellular protein factor
prokaryotic expression
immune protective potency
