结核分枝杆菌蛋白Rv1813c原核表达、纯化及免疫活性研究

Prokaryotic expression,purification,and immune activity of the Rv1813c protein of Mycobacterium tuberculosis

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(P<0.05)。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。

Objectives To clone, express, and purify Rv1813c protein, which is related to latent infection by Mycobacterium tuberculosis, and to analyze its immunological activity. Methods The amino acid sequence of Rv1813c protein was analyzed bioinformatically. Primers were synthesized in accordance with the sequence of the M. tuberculosis H37Rv genome, rv1813c was amplified with PCR and cloned into a pGEM-T vector. The gene from the positive clone was se- quenced, and then cloned into a pET30a vector. Expression of the recombinant protein in E. coli BL21 was induced with IPTG, and the protein was then purified using metal chelate chromatography. The titer of antibodies against the recombi- nant Rvl813c protein was determined in sera from 39 healthy individuals, 19 patients with tuberculosis, and 10 patients with a non-tuberculous respiratory disease according to ELISA. Results RvlS13c protein contained transmembrane do- mains and a signal peptide portion before amino acid 34; the protein may be an extracellular. Gene sequencing indicated that the recombinant plasmid pET30a-Rv1813c had the same sequence as intended. The recombinant protein was ex- pressed in the form of inclusion bodies, and expression peaked 3-4 hours after it was induced with IPTG. The recombi- nant RvlS13c protein accounted for more than 40% of all bacterial proteins expressed. The protein was more than 95%pure. ELISA results indicated that the recombinant RvlS13c protein had a significantly higher level of reactivity to sera from patients with tuberculosis （0.50±0.34） than to sera from healthy individuals （0.23±0.18） and sera from patients with a non-tuberculous respiratory disease （0.30±0.27） （P〈0.05）. Conclusion The recombinant Rvl813c protein of M. tuberculosis was successfully prepared and had good immunological activity, laying the foundation for further study of the protein＇s function and its potential use.