结核分枝杆菌抗原优势肽段融合抗原38kD-ESAT6-CFP10的构建与抗原性初步检测

The cloning,expression and antigenicity of the fused dominant peptide antigen 38 kD-ESAT6-CFP10 of Mycobacterium tuberculosis

目的为了提高结核病诊断试剂的特异性和敏感性,构建了结核分枝杆菌抗原优势肽段融合抗原38 kD-ESAT6-CFP10,并且采用双抗原夹心ELISA方法初步分析了该融合抗原的抗原性。方法运用生物学软件分析抗原优势肽段并模拟其串联后的三维结构,以确保串联后仍保持良好的抗原性。通过PCR方法分别扩增38 kD、ESAT-6和CFP10抗原优势肽段基因并将其进行连接,然后将融合基因分别连接入pBVIL1和pGEX-4T-2表达载体,在大肠杆菌菌株DH5α中进行表达。将两种不同载体表达的38 kD-ESAT6-CFP10融合抗原分别作为包被抗原和标记抗原,以双抗原夹心ELISA方法初步评价其抗原性。结果获得了38 kD、ESAT-6和CFP10的抗原优势肽段融合抗原38 kD-ESAT6-CFP10,并在大肠杆菌中进行了高效表达,初步验证所获得的抗原具有良好的抗原性。结论该种抗原优势肽段融合抗原有望成为结核病血清学诊断的重要候选抗原,并且双抗原夹心ELISA方法有助于提高检测的特异性和敏感性。

Objective In order to improve the specificity and sensitivity of diagnostic reagent for the tuberculosis, the fused dominant peptide antigen 38 kD-ESAT6-CFP10 was cloned and expressed,and the antigenicity of which was evaluated by double antigen sandwich ELISA.Methods The dominant pepfides of three antigens （38 kD,ESAT-6 and CFP10） were analyzed using the BIOSUN software.After connection,the three dimensional structure of the fused dominant peptide antigen was modeled so as to maintain the antigenicity of the antigen. The gene sequence of each dominant pepfide was amplified and ligated together by PCR, and then cloned into expression vector pBVIL1 and pGEX-4T-2, respectively. The fused antigen was expressed highly in E coli DH.Sa. The antigenicity of the fused dominant peptide antigen 38 kD-ESAT6-CFP10 was evaluated by double antigen sandwich ELISA.Results The fused dominant peptide antigen 38 kD-ESAT6-CFP10 was expressed highly in E coli DH5α. The fused antigen presented more favourable antigenicity than that of each antigen. Conclusion The fused dominant peptide antigen 38 kD-ESAT6-CFP10 might serve as a serodiagnostic candidate antigen,and the double antigen sandwich FLISA using the fused antigen might be used to detect the tuberculosis and improve the specificity and sensitivity of diagnostic reagent.