结核分枝杆菌蛋白TB9.7间接ELISA方法的建立  

Establishment of an Indirect ELISA Based on TB 9.7 of Mycobacteriu tuberculosis

作　　者：王燕[1] 元振杰[1] 曹旭东[2] 陈创夫[1] 

机构地区：[1]石河子大学动物科技学院,新疆石河子832003 [2]石河子大学医学院,新疆石河子832002

出　　处：《家畜生态学报》2011年第3期65-70,共6页Journal of Domestic Animal Ecology

基　　金：国际科技合作项目基金(2006DFA33740)

摘　　要：为获得新的鉴别诊断结核杆菌感染的候选抗原,根据Gene Bank中登录的结核杆菌H37Rv的TB 9.7基因序列设计1对引物,以结核杆菌H37Rv基因组DNA为模板,PCR扩增得到TB 9.7基因DNA片段。将该扩增片段克隆于PMD18-T载体中,得到载体PMD18-T-TB9.7。分别将pET32a质粒和PMD18-T-TB 9.7质粒用限制性内切酶EcoRⅠ和XhoⅠ双酶切,并将纯化的TB 9.7基因亚克隆至pET32a中,获得重组表达质粒pET32a-TB 9.7。将其转化E.coli BL21 IPTG诱导后,经SDS-PAGE、Western blot分析鉴定,可见约30ku的外源蛋白带。表明TB9.7基因在大肠杆菌中得到了表达。用Ni-NTA Agarose试剂盒进行蛋白纯化,获得纯化的TB9.7融合蛋白,建立间接ELISA诊断方法,检测了30份结核病人的痰检阳性血清,120份健康人的血清,敏感性为56.6%,特异性为97%,与痰检结果的符合率为84%。To obtain the new antigen for differential diagnosis of Mycobacterium tuberculosis（MTB） infection.A pair of primers specific to the TB 9.7 gene of MTB was designed and synthesized according to the nucleotide sequences of MTB H37Rv strain published in Gene Bank.TB 9.7 gene was amplified by PCR from inactivated MTB.The PCR product of TB 9.7 gene was cloned into the pMD-T vector and verified by sequencing.The TB 9.7 gene was subcloned into pET32a to construct recombinant plasmid of pET32a-TB 9.7 for expression.The resultant pET32a-TB 9.7 was transformed into E.coli BL21 cells and induced by IPTG.The SDS-PAGE and Western blot analysis indicated that the recombinant protein was about 30 ku.The recombinant protein was purified with Ni-NTA Agaros.The preliminary indirect ELISA method was developed by using the purified recombinant protein of TB 9.7 as antigen,test of 30 samples from patients with mycobacteriu tuberculosis and 120 samples from healthy person detected 56.6% positive with a specificity of 97.0%.The assay showed a coincidence rate of 84%.

关 键 词：结核分枝杆菌 TB9.7 原核表达载体 ELISA 

分 类 号：S811.5[农业科学—畜牧学]