结核分枝杆菌CFP10和ESAT6优势肽段融合蛋白的表达及其免疫学研究

Expression of CFP10 and multi-epitope peptide of ESAT6 fusion protein from Mycobacterium tuberculosis and its immunological analysis

目的:构建结核分枝杆菌分泌蛋白的重组质粒及工程菌,获得纯化的CFP10-ESAT6P蛋白抗原,制备初步用于检测结核病患者的特异性抗体。方法:根据编码结核分枝杆菌基因序列设计引物,克隆表达结核CFP10-ESAT6重组蛋白,表达产物免疫新西兰大白兔,其中80%可产生高价的抗体。利用产物建立IgG间接ELISA方法鉴定其抗原性及实用性。结果:应用ELISA法,通过检测113例结核病患者、82例非结核病患者及健康献血员,CFP10-ESAT6P和对照PPD对结核病患者血清检测的敏感性分别为89.4%和79.6%,特异性分别为96.3%和93.9%。结论:高效表达纯化的CFP10-ESAT6蛋白抗原性强,可用于结核病患者抗体的检测,用于结核病的辅助诊断。

Objective： To construct the recombinant plasmid and engineering bacteria for expression of Mycobacterium tuberculosis secretion protein CFP10-ESAT6P and produce antibody for testing human tuberculosis antisera. Methods： DNA encoding protein CFP10 and ESAT6-peptide were amplified from Mycobacterium tuberculosis H37Rv chromosomal by PCR using primers which were designed in accordance with the reported gene sequence. The purified protein was injected into New Zealand rabbits and 80% showed high valence. Then antibody was analyzed by ELISA depended on IgG for test- ing the antigenicity and practicality. Results：By testing 113 cases of TB patients, 82 cases of none TB patients and healthy candidates by using ELISA, the sensitivity and specificity of CFP10-ESAT6P and control PPD to the antisera were com- pared, the sensitivity was 89.4% and 79.6% respectively, and the specificity was 96.3% and 93.9% respectively. Conclu- sion： The results show high titer polyclonal antibody against CFP10-ESAT6 fusion protein was obtained. It can be used for human tuberculosis antisera testing and aided diagnosis by IgG depended ELISA.