摘要
以猪胸膜肺炎放线杆菌血清7型25＿4株基因组DNA为模板,PCR方法扩增外膜蛋白(OMP)5′末端保守区基因片段(OMPc),酶切及核苷酸序列分析鉴定后,与原核表达载体质粒pGEX＿6P＿1进行连接,构建成重组表达载体pGEX＿OM Pc,转入大肠杆菌BL21中,以IPTG进行诱导,SDS＿PAGE电泳分析发现,转化了重组质粒的菌株所表达的融合蛋白相对分子量为34kD,与实际预测相符,命名为GST＿OMPc。GST亲和层析柱进行纯化,ELISA方法对纯化蛋白进行检测。结果表明:纯化蛋白GST＿OMPc能够与兔抗猪胸膜肺炎放线杆菌血清7型的阳性血清反应。OMPc蛋白的成功表达为其功能的研究打下基础。
The conservative region fragment of outer membrane protein (OMPc) from actinobacillus pleuropneumoniae (APP) 25_4 strain chromosomal DNA was amplified by PCR technique.PCR product was cloned into pGEX_6P_1 expressing vector.Plasmid DNA (pGEX_OMPc)was extracted and digested with enzymes and sequenced to confirm its rightness.The right recmbinant plasmids pGEX_OMPc were transformed into E.Coli BL21 strain.Bacterial lysates prepared from 1?mmol/L IPTG induced cultures were loaded directly for SDS_PAGE.The result shows the recombinant pGEX_OMPc produced a fusion protein (GST_OMPc) with an apparent MW of 36 KDa.The fusion protein reacted with rabbit anti_pig positive sera in ELISA.In conclusion,we obtained the recombinant pGEX_OMPc expressing vector which contains OMPc gene and can be expressed successfully.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2004年第6期409-412,共4页
Chinese Journal of Preventive Veterinary Medicine
