结核分枝杆菌重组融合蛋白t不b10.4-hsp16.3纳米金生物传感器诊断结核病的研究.docxVIP

阴性预测值、诊断效率依次为89．3％、90．7％、90．9％、89．1％、90．O％。 阴性预测值、诊断效率依次为89．3％、90．7％、90．9％、89．1％、90．O％。 (3)构建了基于融合蛋白TBl0．4．Hspl6．3的纳米金生物传感器，其诊 断结核病的灵敏度、特异性、阳性预测值、阴性预测值、诊断效率分 别为83．9％、85．2％、85．5％、83．6％、84．5％。 结论：(1)采用重组DNA技术获得了结核分枝杆菌重组融合蛋 白TBl0．4．Hspl6．3。(2)融合蛋白TBl0．4．Hspl6．3具有良好的免疫 活性，可用于结核病的诊断。(3)基于融合蛋白TBl0．4．Hspl6．3的 纳米金生物传感器对结核病具有较好的诊断价值。 关键词：结核分枝杆菌；融合蛋白；纳米金生物传感器；诊断；结核 病 II 万方数据 ABSTRACTobjective：To ABSTRACT objective：To clone and express the recombinant fusion protein TB 1 0．4-Hsp l 6．3 of Mycobacteria tuberculosis(M tuberculosis)and analyze the value of diagnosis of tuberculosis，then establish the Gold Nano-Biosensor(AuNRs)based on recombinant fusion protein TBl0．4-Hspl6．3 to detect tuberculosis in order to provide new experimental basis and technical support for serological diagnosis of tuberculosis．Methods：(1)The primers of TB 1 0．4 and Hsp l 6．3 of M tuberculosis were designed and synthesized based on their sequences in GenBank；genes of TB 1 0．4 and Hsp l 6．3 were amplified by polymerase chain reaction(PCR)from M tuberculosis H37Rv genomic DNA respectively,the fusion gene TB 1 0．4一Hsp l 6．3 Was amplified by overlap PCR and Was cloned into pMD 1 8一T vector．Positive clones were identified by colony PCR and DNA sequencing．The fusion gene TB 1 0．4一Hsp l 6．3 Was subcloned into the prokaryotic expression vector pET-28a(+)and identified by PCR and double enzyme digestion．(2) Recombinant fusion protein TB 1 0．4一Hsp l 6．3 Was expressed in E．coli BL2 1 containing recombinant plasmid pET-TB 1 0．4-Hsp l 6．3 when induced by isopropyl p-D—thiogalactoside(IPTG)；Bacterial precipitation Was dissociated by ultrasonic lysis method，and the inclusion body Was dissolved in 8mol／L urea and the fusion protein TB 10．4-Hsp 16．3 Was renatured by low urea gradient and then purified by nickel chelate chrom— III 万方数据 atography．The atography．The immunological activity of TBl0．4一Hspl6．3 WaS analyzed by western-bolt and the diagnosis efficiency for tuberculosis WaS evaluated by enzyme-linked immunoso