结核分枝杆菌Rv2660c蛋白原核重组表达及其免疫活性研究

目的利用原核表达系统表达、纯化结核分枝杆菌潜伏感染相关蛋白Rv2660c,评价其在血清学方面用于结核病诊断的价值。方法合成Rv2660c全基因核酸序列,构建pET28a-Rv2660c融合表达载体,在原核系统内进行重组表达,亲和层析纯化重组Rv2660c蛋白,采用Western-Blot、ELISA实验分析重组蛋白的免疫反应。结果pET28a-Rv2660c在大肠杆菌内成功表达重组Rv2660c蛋白,SDS-PAGE电泳鉴定重组蛋白分子量为10.2 KD,与预期一致,Western-Blot结果表明重组蛋白Rv2660c只能与活动性结核患者血清反应,出现一条杂交条带;ELISA结果发现重组Rv2660c蛋白与结核患者血清能产生较强的反应,结核病菌阳患者组、菌阴患者组、潜伏感染组以及健康对照组A450平均值分别为0.52、0.48、0.28和0.25。结论重组结核分枝杆菌Rv2660c蛋白具有一定的免疫原性,对活动性结核病的诊断具有一定的价值,但对结核分枝杆菌潜伏感染的诊断没有价值。

Prokaryotic recombinant expression and immune activity research of Rv2660c protein of Mycobacterium tuberculosis

Objective To obtain Rv2660c protein related to latent infection by Mycobacterium tuberculosis expression,purification in prokaryotic system,and to evaluate its serological value in the diagnosis of tuberculosis.Methods Rv2660c nucleic acid sequence was synthesized and cloned into pET28a vector.After expression and purification using metal chelate chromatography from E.coli BL21(DE3),the feasibility and antibody titer of fusion protein was evaluated with clinical sera sample by Western Blot and ELISA assay.Results pET28a-Rv2660c was successfully constructed and Recombinant Rv2660c protein had a molecular weight of about 10.2 kD according to the results of SDS-PAGE as correct.Rv2660c protein could be recognized by serum antibody of active TB with Western Blot only.ELISA results indicated that recombinant Rv2660c protein had a higher level of reactivity to sera from TB or patients with bacterial negative pulmonary tuberculosis than to sera from LTBI or healthy controls,and A450 values were 0.52,0.48,0.28 and 0.25 respectively.Conclusion The recombinant Rv2660c protein of tuberculosis had a high immunogenicity and it is valuable for the diagnosis of active tuberculosis,but not for the latent infection of Mycobacterium tuberculosis.