重组结核分枝杆菌TB10.4的表达及免疫反应性分析

发布时间：2017-12-27 22:12

本文关键词：重组结核分枝杆菌TB10.4的表达及免疫反应性分析　出处：《中国病原生物学杂志》2017年07期 　论文类型：期刊论文

【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。
[Abstract]:Objective to construct a Recombinant Prokaryotic Recombinant Expression System of Mycobacterium tuberculosis TB10.4 and to express TB10.4, and to analyze its reactivity. The method reported by GenBank standard strain of Mycobacterium tuberculosis H37Rv TB10.4 gene sequences were designed and synthesized primers, the TB10.4 gene fragment was amplified by PCR from H37Rv genome DNA as template, transformed into Escherichia coli JM109 was cloned into the pMD-18T vector, screening and identification of positive clones were sequenced and analyzed. The TB 10.4 gene was inserted into prokaryotic expression vector pET-28a and transformed into Escherichia coli E.coli DH5 alpha, PCR and double enzyme digestion of the recombinant plasmid was transformed into Escherichia coli BL21, IPTG induced the expression of TB 10.4 protein. The recombinant bacteria were cleavage by ice bath, TB10.4 protein was purified by His-bindTM column chromatography, Western blot was used to analyze its reactive progenicity, and ELISA was used to evaluate the diagnostic value of TB. Results the TB10.4 gene fragment was obtained by PCR amplification and cloning, and the size was about 300bp, which was consistent with the sequence reported in GenBank. The TB10.4 gene fragment was inserted into the prokaryotic expression vector, and the recombinant expression system of TB10.4 protein was constructed. The metal chelation chromatography was used to obtain the TB10.4 of the molecular mass of about 10.4ku. Western blot confirmed that the TB10.4 protein could be identified by the serum of patients with tuberculosis. Using recombinant TB10.4 as antigen, the sensitivity of TB diagnosis by ELISA is 88.5%, the specificity is 97.3%, the positive predictive value is 93.8%, the negative predictive value is 94.8%, and the diagnostic efficiency is 94.5%. Conclusion the recombinant expression system of TB10.4 protein has been successfully constructed, and the expressed TB10.4 protein has reactivity and can be used in the diagnosis of tuberculosis.
【作者单位】：湖南师范大学医学院;湖南省结核病防治研究所检验科;
【分类号】：R378.911
【正文快照】： 结核病(Tubeculosis,TB)是由结核分枝杆菌(Mycobacterium tuberculosis,Mtb)所致的慢性传染病。2015年,全世界约有1 040万例新发结核病例,约有180万例结核病患者死亡[1]。广泛耐药性肺结核具有使Mtb感染恶化的趋势[2],结核病的早期诊断和治疗是有效控制这种趋势蔓延的关键。

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