浅谈重组结核分枝杆菌Mr-38-000蛋白的表达、纯化和鉴定.docx

浅谈重组结核分枝杆菌Mr38000蛋白的表达、纯化和鉴定摘要从H37Rv基因组中扩增Mr38000蛋白基因并高效表达和纯化。用PCR技术从结核分枝杆菌H37Rv基因组中扩增Mr38000蛋白序列，将其与pGEM-T-Easy载体连接转化DH5α，构建重组克隆载体pGEM-T-Easy/Mr38000，测序正确后将目的基因片断克隆入pQE-80L原核表达载体并转化DH5α，IPTG诱导目的蛋白表达。经Western-blot鉴定目的基因与(His)6融合表达，将已表达的蛋白质通过Ni-NTA亲和色谱柱进行纯化。PCR得到结核分枝杆菌Mr38000蛋白基因，测序结果与GeBank中报道的完全一致。SDS-PAGE显示，在Mr为×103处有相应的蛋白质表达条带，Wesernblot鉴定为(His)6融合表达蛋白。经Ni-NTA亲和色谱柱进行纯化后可得到纯化的蛋白。成功克隆了铁调节蛋白基因Mr38000蛋白序列，并在DH5α高效表达，亲和层析后获得了纯化目的蛋白。关键词Mr38000蛋白;表达;纯化;结核分枝杆菌；Cloning,ExpressionandPurificationofMr38000proteinfromMycobacteriumtuberculosisZHANGMing，LIJin-cheng，LINHang(Departmentofinternalneurology,2.emergencydepartment,theFuZhougeneralhospital,FuZhou,350025,P.R.China)[Abstract]ToobtainMycobacteriumtuberculosisMr38000proteinfromH37Rv-DNA,expressefficientlyinE.coliandpurifytheMr38000proteins.Mr38000proteingenewasamplifiedbyPCRfromthegenomeofH37RvandclonedintopGEM-T-Easyvector.Aftersequenced,Mr38000proteingenewasrecombinatedexpressionvectorpQE-80Lbyrestrictionenzymedigestion,namedpQE-80L-Mr38000.TheplasmidpQE-80L-Mr38000wastransformedintoDH5αandinducedbyIPTG.Mr38000proteinexpressionwasanalyzedbySDS-PAGEandconfirmedbywesternblotwithmice-specificmAbagainst(His)6.Mr38000proteingenewasidenticalwithGeBankreported.ThepQE-80L-Mr38000vectorexpressedproteinwithrelativemoleculeweightaboutkD,whichcouldbecaughtby(His)6mAb.TheexpressedproteincouldbepurifiedbyNi-NTAsystemkitwithdenativecondition.Mr38000proteingenehadbeensuccessfullyclonedandefficientlyexpressedin,TheresultsestablishedthebasisforfurtherstudyofthefunctionofMr38000protein.[Keywords]Mr38000protein;expression;purification;Mycobacteriumtuberculosis(MTB)结核分枝杆菌是结核病(tuberculosis,TB)的病原体。其在患者体内主要为细胞内生长。诊断方法的欠缺是结核病流行的重要原因。目前常用的PPD试验，常使BCG疫苗接种者被误检为阳性[1]，且对后期结核患者敏感性较低，存在明显不足。近年来，有研究发现Mr38000蛋白具有强的免疫原性，而成为结核分枝杆菌抗原的研究热点[2,3]，可能成为结核病血清学诊断试剂和疫苗研究的候选抗原之一。为此，我们在原核表达载体中表达结核分枝杆菌Mr38000蛋白并对其进行了纯化，为进一步研究其抗原性和主要功能提供方便。１材料和方法材料MTB毒株H37Rv、DH5α由本室保存;pGEM-T-Easy及WizardPlusPurificationsystem购自Promega公司；X-gal，限制性内切酶BamHⅠ,EcoRⅠ及T4-DNA连接酶，TaqDNA聚合酶均为TaKa