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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。
Objective To construct prokaryotic recombinant expression system of Mycobacterium tuberculosis TB10.4 and express TB10.4 and analyze its reactivity. Methods Genomic DNA of Mycobacterium tuberculosis strain H37Rv TB10.4 was sequenced and sequenced. Genomic DNA of H37Rv was used as a template to amplify the TB10.4 gene fragment. After cloned into pMD-18T vector, the recombinant plasmid was transformed into Escherichia coli JM109. Screening and identification of positive clones, and sequencing analysis. The TB 10.4 gene was inserted into the prokaryotic expression vector pET-28a and transformed into Escherichia coli DH5ɑ. The recombinant plasmid was identified by restriction endonuclease digestion and double restriction enzyme digestion. TB10 protein was induced by IPTG. The TB10.4 protein was purified by column chromatography on His-bindTM in ice bath and the antigenicity was determined by Western blot. ELISA was used to evaluate the diagnostic value of TB10.4. Results The fragment of TB10.4 gene was amplified by PCR and cloned. The size of the fragment was about 300 bp, which was consistent with the sequence reported in GenBank. The TB10.4 gene fragment was inserted into the prokaryotic expression vector to construct a recombinant expression system of TB10.4 protein, and the metal chelate chromatography was used to obtain TB10.4 with a molecular mass unit of about 10.4ku. Western blot confirmed that TB10.4 protein was specifically recognized by the serum of tuberculosis patients. The diagnostic sensitivity of TB10.4 was 88.5%, the specificity was 97.3%, the positive predictive value was 93.8%, the negative predictive value was 94.8% and the diagnostic efficiency was 94.5%. Conclusion The recombinant expression system of TB10.4 protein was constructed successfully. The expressed TB10.4 protein has the antigenicity and can be used for immunodiagnosis of tuberculosis.