粪肠球菌介导结核分枝杆菌重组Efs(pGEX-ESAT-6)疫苗的构建、鉴定及表达  

Construction,identification and expression of recombinant Efs(pGEX-ESAT-6)vaccine of Mycobacteria tuberculosis mediated by Enterococcus faecalis

作　　者：李文桂[1] 欧兴坤 何爱琳 LI Wengui;OU Xingkun;HE Ailin(Institute of Infectious and Parasitic Diseases,the First Affiliated Hospital,Chongqing Medical University,Chongqing 400016,China)

机构地区：[1]重庆医科大学附属第一医院传染病寄生虫病研究所,重庆400016

出　　处：《中国病原生物学杂志》2023年第10期1137-1140,共4页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.30801052,30671835,30500423,30200239)。

摘　　要：目的 构建粪肠球菌(Efs)介导的结核分枝杆菌重组Efs(pGEX-ESAT-6)疫苗,对表达的融合蛋白进行检测分析。方法 采用电穿孔方法将重组质粒pGEX-ESAT-6转化粪肠球菌ATCC47077株,构建rEfs(pGEX-ESAT-6)疫苗,抽提质粒进行PCR鉴定;经IPTG诱导表达后用SDS-PAGE和Western blot对表达产物进行分析和鉴定。结果 以从rEfs抽提的质粒为模板进行PCR,扩增出约288 bp的ESAT-6基因片段,SDS-PAGE显示表达产物为32 ku的融合蛋白,约占菌体总蛋白的20%;Western blot显示该融合蛋白能被结核病患者血清识别。结论 成功构建了结核分枝杆菌rEfs(pGEX-ESAT-6)疫苗,表达的融合蛋白具有反应原性。Objective To construct an Enterococcus faecalis_mediated recombinant Efs(pGEX-ESAT-6)vaccine of Mycobacteria tuberculosis and observe its expression efficiency.Methods The recombinant plasmid pGEX-ESAT-6was electroporated into Enterococcus faecalis(Efs)ATCC47077strain to construct rEfs(pGEX-ESAT-6)vaccine,the plasmid was extracted from rEfs for PCR identification.The rEfs vaccine was expressed through IPTG induction,and the recombi-nant protein was verified by SDS-PAGE and Western blotting.Results PCR showed that 288bp ESAT-6gene was am-plified when the extracted plasmid from rEfs as template;the relative molecular mass(Mr)of the expressed ESAT-6-GST fusion protein was approximately 32ku by SDS-PAGE,the amount of the expressed protein was 20%of the total bacterial proteins;Western blotting demonstrated that the expressed protein could be recognized by sera from patients with tuber-culosis.Conclusion The rEfs(pGEX-ESAT-6)vaccine was constructed in success,and the expressed fusion protein was provided with special antigenicity.

关 键 词：粪肠球菌 结核分枝杆菌 ESAT-6 疫苗 

分 类 号：R378.1[医药卫生—病原生物学] R378.91[医药卫生—基础医学]