重组结核分枝杆菌TB10.4的表达及免疫反应性分析  

Diagnosis of tuberculosis with recombinant TB10.4 of Mycobacterium tuberculosis

作　　者：任琪琪 谢琳[1] 袁仕善[1] 郭婧玮 蒋华科 谭云洪 

机构地区：[1]湖南师范大学医学院,湖南长沙410013 [2]湖南省结核病防治研究所检验科

出　　处：《中国病原生物学杂志》2017年第7期623-626,共4页Journal of Pathogen Biology

摘　　要：目的构建结核分枝杆菌TB10.4的原核重组表达体系并表达TB10.4,分析其反应原性。方法 GenBank报道的结核分枝杆菌标准株H37Rv TB10.4基因序列设计并合成引物,从H37Rv基因组DNA为模板PCR扩增TB10.4基因片段,克隆至pMD-18T载体后转化大肠埃希菌JM109,筛选和鉴定阳性克隆,并进行测序分析。将TB 10.4基因插入原核表达载体pET-28a后转化大肠埃希菌E.coli DH5ɑ,PCR和双酶切鉴定阳性的重组子转化大肠埃希菌BL21,IPTG诱导表达TB 10.4蛋白。冰浴超声裂解重组菌,His-bindTM柱层析纯化TB10.4蛋白,Western blot分析其反应原性,ELISA评价其诊断结核病的价值。结果 PCR扩增和克隆获得TB10.4基因片段,大小约300bp,与GenBank中报道的序列一致。将TB10.4基因片段插入原核表达载体,构建TB10.4蛋白重组表达体系,金属螯合层析获得分子质量单位约为10.4ku的TB10.4。Western blot证实TB10.4蛋白可被结核患者血清特异识别。以重组TB10.4为抗原采用ELISA诊断结核病的灵敏度为88.5%,特异度为97.3%,阳性预测值93.8%,阴性预测值94.8%,诊断效率94.5%。结论成功构建了TB10.4蛋白的重组表达体系,表达的TB10.4蛋白具有反应原性,可用于结核病的免疫诊断。Objectives To clone and express TB10.4 of Mycobacteriurn tuberculosis and to analyze its value in diagno- sing tuberculosis. Methods Primers for the TB10. 4 gene were designed and synthesized in accordance with the se- quence of the TB10.4 gene in GenBank. The TB10.4 gene was amplified from M. tuberculosis genomie DNA via a poly- merase chain reaction （PCR） and cloned into a pMD18-T vector. Positive clones were identified using colony PCR and DNA sequencing. The gene TB10.4 was subcloned into the prokaryotic expression vector pET-28a（＋） and identified u sing PCR and restrictive enzyme digestion with BamH I and Hind III. TB10.4 was expressed in E. coli BL21 containing the recombinant plasmid pET TB10. 4 when induced with IPTG, and TB10.4 was purified using affinity chromatography with an His bindTMcolumn. The immunological activity of TB10.4 was analyzed using Western blotting, and the efficien cy with which tuberculosis was diagnosed was evaluated using an enzyme-linked immunosorbent assay. Results Ampli- fication of M. tuberculosis genomie DNA with PCR and cloning yielded the TB10.4 gene, which was about 300 bp in length. This gene was cloned into a pMD18-T vector. The recombinant expression plasmid pET-TB10.4 was found to yield a fragment about 300 bp in length according to colony PCR and double restrictive enzyme digestion. The recombinant protein TB10.4 was expressed in E. coli BL21 and purified using affinity chromatography. The protein about 10.4 ku and it was specifically recognized by antibodies in sera from patients With tuberculosis. Using ELISA with TB10.4 protein as an antigen to detect tuberculosis had a sensitivity of 88.5 %, a specificity of 97.3 %, a positive predictive value of 93.8 %, a negative predictive value of 94.8 %, and a diagnostic efficiency of 94.5 %. Conclusion Conclusion A system for pro- karyotic expression of M. tuberculosis TB10.4 was successfully constructed, and this protein can be used to test for tu- berculosis.

关 键 词：结核分枝杆菌 TB10.4 表达 结核病 

分 类 号：R378.911[医药卫生—病原生物学]