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1、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M138000蛋白基因并高效表达和纯化。用PCR技术从结核分枝杆菌IT37Rv基因组中扩增壯38000蛋白序列,将其与pGEM-T-Easy载体连接转化DH5a,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DH5a,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Mr380
2、00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X103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DH5a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,ExpressionandPurificationofMr3800OproteinfromMycobacteriumtuberculosisZHANGM
3、ing,LIJin-cheng,LINHang(Departmentofinternalneurology,2.emergencydepartmerit,theFuZhougeneralhospital,FuZhou,350025,P.R.China)[Abstract]ToobtainMycobacteriumtuberculosisMr38000protentlyinE・cOOproteinsdbyPCRfromEasyeinfromH37Rv-DNA,expressefficioliandpurifytheMr380・Mr3800
4、0proteingenewasamplifiethegenomeofH37RvandclonedintopGEM-T-vector.Aftersequenced,Mr38000proteingenewasrecombirestrictioMr38000.ThsformedintroteinexprconfirmedbAbagainst(nticalwithOOOvectoreeculeweighnatedexprenenzymedigeplasmidpQoDH5aandinessionwasaywesternblHis)6.Mr38Ge
5、BankrepoxpressedprtaboutkD,wssionvectoestion,namE-80L-Mr38rpQE-80LbyedpQE~80L-OOOwastranducedbylPTnalyzedbySotwithmiceOOOproteinrted.ThepQG.Mr38000pDS-PAGEand-specificmgenewasideE-80L-Mr38oteinwithrhichcouldbelativemolecaughtby(His)6mAb・Theexpressedproteincouldbepurified
6、byNi-NTAsystemkitwithdenativecondition.Mr38000proteingenehadbeensuccessfullyclonedandefficientlyexpressedin,TheresultsestablishedthebasisforfurtheTstudyofthefunctionofMr38000protein.[Keywords]Mr38000protein;expressionjpurification;Mycobacteriumtuberculosis(MTB)结核分枝杆菌(Myc
7、obacteriumtuberculosis,MTB)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
8、方便。1材料和方法材料MTB毒株H37Rv、DH5a由本室保存;pGEM-T-Easy及WizardPlusPurificationsystem购自Promega公司;X-gal,限制性内切酶BamH1,E