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目的 :重组表达结核分枝杆菌融合蛋白TB10.4-Hsp16.3,分析其诊断结核病的价值。方法 :重组PCR扩增TB10.4-Hsp16.3融合基因,克隆至pMD18-T载体,测序鉴定后,亚克隆至原核表达载体pET28a(+),PCR和双酶切鉴定阳性重组子,将重组DNA转化E.coli BL21,筛选阳性菌株,经IPTG诱导表达融合蛋白TB10.4-Hsp16.3,超声裂解菌体,对包涵体进行变性溶解和复性,金属螯合层析纯化,Western blot分析其免疫学活性,ELISA评价其诊断结核病的价值。结果 :成功获得约750bp的融合基因TB10.4-Hsp16.3,构建了融合蛋白的重组表达质粒pETTB10.4-Hsp16.3,表达、纯化获得分子量约29kDa的融合蛋白,能被结核病患者血清特异识别;其ELISA诊断结核病的灵敏度为89.3%,特异度为90.7%,阳性预测值为90.9%,阴性预测值为89.1%,诊断效率达90.0%。结论 :成功表达可用于结核病诊断的结核分枝杆菌融合蛋白TB10.4-Hsp16.3。
Objective: To express recombinant Mycobacterium tuberculosis fusion protein TB10.4-Hsp16.3 and analyze the diagnostic value of TB10.4-Hsp16.3. Methods: The fusion gene TB10.4-Hsp16.3 was amplified by PCR and cloned into pMD18-T vector. After sequencing, it was subcloned into the prokaryotic expression vector pET28a (+). The positive recombinant was identified by PCR and restriction enzyme digestion. Recombinant DNA was transformed into E.coli BL21, and the positive strains were screened. The fusion protein TB10.4-Hsp16.3 was induced by IPTG, and the inclusion body was denatured, lysed and refolded, and purified by metal chelate chromatography. Western blot analysis Its immunological activity, ELISA evaluation of the diagnosis of tuberculosis value. Results: The fusion gene TB10.4-Hsp16.3 of about 750bp was successfully obtained. The recombinant plasmid pETTB10.4-Hsp16.3 was constructed and purified. The fusion protein of about 29kDa molecular weight was obtained, which could be detected by the serum-specific The sensitivity, specificity and accuracy of ELISA for the diagnosis of tuberculosis were 89.3%, 90.7%, 90.9%, 89.1%, and 90.0% respectively. Conclusion: Mycobacterium tuberculosis fusion protein TB10.4-Hsp16.3, which can be used for diagnosis of tuberculosis, was successfully expressed.