结核分枝杆菌c-di-AMP合成酶的表达和纯化以及小鼠多抗血清的制备  

Expression and purification of Mycobacterium tuberculosis c-di-AMP synthetase to prepare polyclonal antibodies in mice

作　　者：曹田宇 吉思雨 褚阳光 王丽梅[2] 康健[2] 王立飞[2] 徐志凯[2] 柏银兰[2] 

机构地区：[1]第四军医大学学员旅,陕西西安710032 [2]第四军医大学微生物学教研室,陕西西安710032

出　　处：《中国病原生物学杂志》2015年第8期681-684,688,共5页Journal of Pathogen Biology

基　　金：国家"十二五"科技重大专项课题(No.2012ZX10003008-007);国家自然科学面上项目(No.81371774);陕西省科研基金课题(No.2012D100);第四军医大学本科生导师(No.2013012;2015007)

摘　　要：目的表达和纯化结核分枝杆菌c-di-AMP合成酶,并制备多克隆抗体,为研究c-di-AMP在MTB生理过程中的作用奠定基础。方法从MTB基因组中PCR扩增c-di-AMP合成酶Rv3586基因,构建原核表达载体,序列鉴定后,IPTG诱导目的基因表达,采用Western-blot鉴定目的蛋白,并用离子亲和层析法纯化重组蛋白;用纯化蛋白经背部皮下免疫小鼠,收集小鼠血清,用免疫学方法测定抗体滴度及其特异性。结果 PCR成功扩增Rv3586基因,限制性酶切分析和序列测定结果表明成功构建原核表达载体;表达的重组c-di-AMP合成酶蛋白分子质量大小约43ku,该蛋白可被抗His标签抗体和感染小鼠血清识别。用纯化蛋白免疫小鼠,初次免疫后抗体滴度可达1︰12 800,且该多克隆抗体可识别结核分枝杆菌和卡介苗c-di-AMP合成酶。结论在E.coli中成功表达c-di-AMP合成酶,并制备了多克隆抗体,可用于结核分枝杆菌c-di-AMP生物学功能的进一步研究。Objective The aim of this study was to express and purify the c-di-AMP synthetase of Mycobacterium tuberculosis and prepare polyclonal antibodies for further research into the role of c-di-AMP in the physiological processes of bacteria. Methods Rv3586,the gene encoding c-di-AMP synthetase,was amplified from genomic DNA of M.tuberculosis using with PCR.The DNA fragments were cloned into a prokaryotic expression vector.After sequencing of the vector,isopropyl-β-thiogalactoside was used to induce expression of the recombinant protein.The protein was identified with Western blotting.The recombinant protein was purified with ion affinity chromatography and subcutaneously injected into the backs of mice three times.Sera were collected from mice.The antibody titer was detected with ELISA and the specificity of the antibodies was analyzed with Western blotting. Results The Rv3586 gene from the genome of M.tuberculosis was amplified with PCR.A prokaryotic expression vector with the target gene was verified using restriction endonuclease analysis and sequencing.The expression of a recombinant protein about 43 kDa in size was induced by isopropyl-β-thiogalactoside.This protein was recognized by anti-His antibody and serum from mice infected with M.tuberculosis according to Western blot analysis.The recombinant protein was purified with affinity chromatography and used to immunize mice.The titer of antibodies to c-di-AMP synthetase in sera rose to 1：12,800 after initial immunization.The polyclonal antibody recognized c-di-AMP synthetase from both M.tuberculosis and the Bacillus Calmette Guerin（BCG）vaccine strain. Conclusion c-di-AMP synthetase of M.tuberculosis was successfully expressed in E.coli via a prokaryotic expression vector.The recombinant protein induced a high antibody titer in mice.The prepared polyclonal antibodies can be used for further study of the biological function of c-di-AMP in M.tuberculosis.

关 键 词：结核病 结核分枝杆菌 c-di-AMP 原核表达 抗体 

分 类 号：R378.911[医药卫生—病原生物学]