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目的:探索结核分枝杆菌Rv2461c基因编码的clpP蛋白作为结核检测抗原的可行性。方法:克隆结核分枝杆菌clpP蛋白的编码基因Rv2461c,构建pET30a-Rv2461c重组质粒,并将其成功的转化到大肠杆菌JF1125克隆载体中,测序鉴定结果正确。将pET30a-Rv2461 c重组质粒转化到大肠杆菌BL21中,表达纯化目的蛋白进行质谱分析.制备clpP蛋白的多克隆抗体,通过亚细胞分离及Western检测分析蛋白的亚细胞定位,纯化的clpP蛋白通过间接ELISA实验进行抗原性的初步检测。结果:结核分枝杆菌clpP蛋白大量存在于细胞质中,少量存在于细胞壁和细胞膜中。重组抗原在检测肺结核病人中的检出率为38.3%(23/60),检测敏感性为38.3%,特异性为90%。结论:为后续深入研究clpP蛋白的生物功能、clpP作为诊断靶标、药靶候选蛋白的可行性提供了基础。
Objective: To explore the feasibility of tuberculosis clpP protein encoded by Mycobacterium tuberculosis Rv2461c gene. Methods: Clone of the clpP gene of M. tuberculosis Rv2461c was cloned into pET30a-Rv2461c recombinant plasmid and successfully transformed into E. coli JF1125 cloning vector. The sequencing result was correct. The recombinant plasmid pET30a-Rv2461c was transformed into E.coli BL21, and the purified protein was expressed and analyzed by mass spectrometry.The polyclonal antibody against clpP protein was prepared, and subcellular localization of the protein was analyzed by subcellular isolation and Western analysis. ELISA test antigenicity of the initial test. Results: Mycobacterium tuberculosis clpP protein is abundant in the cytoplasm, a small amount in the cell wall and cell membrane. The detection rate of recombinant antigen in detecting tuberculosis patients was 38.3% (23/60), the detection sensitivity was 38.3% and the specificity was 90%. Conclusion: In order to further investigate the biological function of clpP protein, clpP serves as a basis for the diagnosis of the target protein and the potential of target protein candidates.