结核分枝杆菌蛋白Rv1813c原核表达、纯化及免疫活性研究

发布时间：2018-06-05 23:21

  本文选题：结核分枝杆菌 + Rvc　； 参考：《中国病原生物学杂志》2017年04期

【摘要】：目的克隆、表达、纯化结核分枝杆菌潜伏感染相关蛋白Rv1813c,分析其免疫活性。方法应用生物信息学方法分析Rv1813c蛋白序列,根据H37Rv基因组序列合成引物,PCR扩增Rv1813c基因,克隆至pGEM-T载体;挑取阳性克隆测序,将正确编码基因克隆到pET30a载体上,在大肠埃希菌BL21菌株中以IPTG诱导表达重组蛋白。以金属螯合层析分离纯化重组蛋白,采用ELISA法分析其免疫反应性。结果 Rv1813c蛋白第34位氨基酸残基之前含有跨膜区及信号肽部分,可能是一个胞外蛋白。对重组质粒pET30a-Rv1813c测序,与设计的序列相同。重组蛋白在大肠埃希菌BL21(DE3)细胞内以包涵体形式表达,IPTG诱导3h-4h重组蛋白在大肠埃希菌中的表达量较高,表达量约占细菌总蛋白的40%以上。纯化的重组Rv1813c蛋白纯度95%。ELISA分析重组Rv1813c蛋白与结核患者血清有较强的反应性,A450值为0.50±0.34,显著高于健康组A450值0.23±0.18及非结核呼吸疾病组A450值0.30±0.27(P0.05)。结论成功构建的结核分枝杆菌重组质粒能高效表达Rv1813c蛋白,该蛋白具有良好的免疫反应性,为结核病的免疫机制研究奠定了基础。
[Abstract]:Objective to clone, express and purify Mycobacterium tuberculosis latent infection related protein RV 1813 c and analyze its immune activity. Methods the Rv1813c protein sequence was analyzed by bioinformatics, and the Rv1813c gene was amplified by PCR according to the H37Rv genomic sequence and cloned into the pGEM-T vector, and the correct coding gene was cloned into the pET30a vector. The recombinant protein was induced by IPTG in Escherichia coli BL21 strain. The recombinant protein was purified by metal chelate chromatography and its immunoreactivity was analyzed by ELISA method. Results the 34th amino acid residues of Rv1813c protein contained transmembrane region and signal peptide, which may be an extracellular protein. The recombinant plasmid pET30a-Rv1813c was sequenced in the same sequence as the designed one. The recombinant protein was expressed as inclusion body in Escherichia coli BL21DDE3) cells. The expression of recombinant 3h-4h protein in Escherichia coli was higher than that in Escherichia coli cells, accounting for more than 40% of the total bacterial protein. 95%.ELISA analysis of purified recombinant Rv1813c protein the reactivity of recombinant Rv1813c protein to serum of tuberculosis patients was 0.50 卤0.34, which was significantly higher than that of healthy group (0.23 卤0.18) and non-tuberculous respiratory disease group (0.30 卤0.27) (P 0.05). Conclusion the recombinant plasmid of Mycobacterium tuberculosis can express Rv1813c protein efficiently, and the protein has good immunoreactivity, which lays a foundation for the study of the immune mechanism of tuberculosis.
【作者单位】： 解放军第三0九医院全军结核病研究所全军结核病防治重点实验室结核病诊疗新技术北京市重点实验室;
【基金】：“十二五”国家重大科技专项(No.2013ZX10003003-005) 军队医学科技“十二五”重点项目(No.BWS11J050) 北京市科技创新基地培育与发展工程专项(No.Z141107004414021) 解放军第309医院重点课题(No.2015ZD-004)
【分类号】：R378.911
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