猪链球菌9型荚膜多糖cps9G基因的原核表达  

Prokaryotic expression of capsular polysaccharide cps9G gene of Streptococcus suis type 9

作　　者：刘春生[1] 徐耀辉 陈陆[1] 杨霞[1] 王新娟[1] 刘春明 王川庆[1] 

机构地区：[1]河南农业大学牧医工程学院,河南郑州450002 [2]郑州牧业高等专科学校,河南郑州450002

出　　处：《中国兽医学报》2011年第7期998-1002,共5页Chinese Journal of Veterinary Science

基　　金：河南省重点科技攻关项目(072102130009);国家科技部支撑计划(2006BAK02A21)

摘　　要：本研究旨在克隆表达猪链球菌9型荚膜多糖部分抗原表位。设计1对引物,采用PCR法以猪链球菌9型河南分离株PY-2的基因组DNA为模板扩增出cps9G全基因序列。用限制性核酸内切酶BamHⅠ、XhoⅠ进行双酶切后,将其定向克隆到pET32a(+)中,构建重组表达质粒pET32a-cps9G,并将其转化至大肠杆菌BL21(DE3)感受态细胞中进行诱导表达,并优化表达条件。结果表明,经1.2mmol/L IPTG诱导4h后SDS-PAGE分析表明,重组菌株表达出了约50 700的融合蛋白条带,与预期一致。Western-blotting分析表明,该融合蛋白具有特异的生物学活性。这为以后建立快速、简便、特异的免疫学检测方法及制备单克隆抗体提供了较好的抗原,同时为研制猪链球菌亚单位疫苗和诊断试剂盒奠定了基础。A pair of primers were designed, by using PCR assay, cps9G gene was amplified from genomic DNA of Streptococcus suis type 9 isolated in Henan province. The gene was digested with BamH Ⅰ and Xho Ⅰ, and cloned to pET32a（＋） vector, a recombinant plasmid was constructed. Then the plasmid was transformed to E. coli BI.21 （DE3） competent cell to expression, then the expression product was analyzed by SDS-PAGE and Western-blotting. The result showed a fusion protein of about 50 700 was successfully expressed by E. coli BL21 containing the recombinant plasmid induced by 1.2 mmol/L IPTG for 4 h,and the protein has particular biologic activity. It may provide good antigen for immunological test and producing specific monoclonal antibody（McAbs） and may lay the foundation for preparing subunit vaccine and test kits.

关 键 词：猪链球菌 血清9型 cps9G基因 克隆 原核表达 

分 类 号：S852.61[农业科学—基础兽医学]