布鲁菌二氢硫辛酰胺脱氢酶的原核表达

摘要

以羊种布鲁菌043株基因组为模板，利用 PCR 扩增二氢硫辛酰胺脱氢酶（BMEI0145）基因，连接 pMD18-T 载体后测序，将测序正确的基因片段经双酶切后克隆至 pET-28a 载体中，转化大肠埃希菌BL21（DE3），经 IPTG 诱导表达，通过 SDS-PAGE 和 Western blot 分析鉴定表达产物。结果显示，成功克隆了 BMEI0145基因，片段大小为1404 bp；SDS-PAGE 显示，BMEI0145蛋白分子质量约为62 ku；Western blot 分析显示，BMEI0145蛋白可与布鲁菌阳性血清发生特异性反应。表明成功获得了 BMEI 0145蛋白，该蛋白具有与布鲁菌自身蛋白相同的抗原性，为进一步研究布鲁菌基因工程疫苗和布鲁菌病的诊断奠定了基础。%The dihydrolipoamide dehydrogenase(BMEI0145)gene was cloned from Brucella melitensis ,and its prokaryotic expressing vector was constructed,the antigenicity of BMEI0145 protein was analyzed.The full length of BMEI 0145 gene was amplified from Brucella melitensis 043 genome.The amplified fragment was cloned into a pMD18-T vector,the gene fregment after sequencing was digested with double enzymes and cloned into pET-28a vector.The recombinant expressing vector was transformed into E.coli BL21 (DE3)competent cells.After induction with IPTG,the expressed products were identified by SDS-PAGE and Western blot.The results suggested that we successfully cloned BMEI0145 gene of 1 404 bp length. The results of SDS-PAGE and Western blot showed that BMEI0145 protein with 62 ku specificly reacted with Brucella positive serum.Collectively,our data showed that BMEI 0145 protein was successfully ex-pressed and had the same antigenicity with the proteins of Brucella,which laid the foundation for further investigating Brucella genetic engineering vaccines and diagnosing brucellosis.foundation.