摘要
应用RT PCR方法扩增了编码猪瘟病毒石门株 (CSFVshimenstrain)囊膜糖蛋白E2全基因 ,然后将其克隆到pMD 1 8T质粒中 ,获得重组质粒pMD E2。再以pMD E2为模板 ,另行设计两对引物 ,同时扩增其中一段适于在E .coli中表达且抗原反应性较好的基因片段 (E2蛋白A D抗原区基因序列 ) ,将扩增的两片段串联插入原核表达载体pET 32a中构建成重组质粒pET 2e。用酶切和序列分析鉴定插入目的基因的正确性。SDS PAGE和Western blot分析表明 ,经pET 2e转化、IPTG诱导的受体菌可表达目的蛋白 ,克隆在硫氧还蛋白 (thioredoxinprotein ,TrxA)基因下游的E2蛋白基因与TrxA基因获得了高效融合表达 ,并且具有免疫学反应活性 。
Full E2 gene of classical swine fever virus(CSFV) Shimen strain was isolated from CSFV genome by RT PCR method and cloned into pMD 18T vector to get the recombinant plasmid pMD E2. Then,another 2 pairs of primers were used to amplify the A/D domains of E2 gene with pMD E2 as template.The two subcloned sequences were cloned into prokaryotic expressing vector pET 32a tandemly and the recombinant plasmid named pET 2e was constructed .Then,the recombinant plasmid was sequenced and analyzed with computer software .The result showed that the homologies between the cloned gene and the gene published in GenBank reached 99 7%.Then the recombinant plasmid pET 2e was used to transform into E.coli BL21 (DE3) and induced by IPTG.The results of SDS PAGE and Western blot indicated that the gene cloned in downstream of thioredoxin protein (trxA) gene was expressed in high level and the recombinant fusion protein had immunologically reactive activity.The foundation for the development of the diagnosis methods in serology for CSFV were laid.
出处
《中国生物工程杂志》
CAS
CSCD
2004年第8期54-58,共5页
China Biotechnology
基金
国家"8 63"计划资助项目 ( 2 0 0 1AA2 490 12 )
关键词
猪瘟
E2基因
原核表达
免疫学反应
血清学诊断
Classical swine fever virus (CSFV) Glycoprotein E2 gene Prokaryotic expression
