结核分枝杆菌Mtb81蛋白的克隆、表达及抗原性分析

Cloning,expression,purification and antigenicity assay of Mtb81 protein of Mycobacterium tuberculosis.

目的对结核分枝杆菌Mtb81抗原基因进行克隆、表达及纯化,并评价其抗原性。方法应用聚合酶链反应技术扩增结核分枝杆菌H37Rv Mtb81 DNA序列;将目的基因构建大肠杆菌高效表达株;亲和层析柱纯化重组蛋白;经SDS-PAGE电泳鉴定;通用ELISA法进行重组蛋白的抗原性检测分析。结果获得了结核分枝杆菌抗原Mtb81基因,在大肠杆菌BL21中高效表达,Western印迹结果证实该重组蛋白能与抗结核分支杆菌多克隆抗体发生特异免疫结合反应。ELISA分析中,该纯化的重组抗原敏感性、特异性和准确性分别为:35.8%、98.3%和56.7%,具有良好的抗原性和较高的特异性。结论结核分枝杆菌Mtb81重组蛋白抗原具备良好的抗原性与特异性,为结核病诊断、重组疫苗应用和免疫效应检测以及抗原、抗体的大规模制备打下基础。

Objective To Clone,express and purify the Mtb81 gene of Mycobacterium tuberculosis and estimate its antigenicity. Methods The gene sequence of Mtb81 of M.tubereulosis strain H37Rv was cloned by PCR. The gene was induced to express in E cob. Expressed products were confirmed by SDS-PAGE. The antigenicity of recombinant protein was detected by indirect ELISA. Results The gene sequence of Mtb81 was obstainned and the antigen was expressed in E. coli BL21. Western blot analysis showed that the recombinant antigen could inununobind to the polyclonal antibody of Mycobacterium tuberculosis specifically. The sensitivity,specificity and accuracy of the purified recombinant antigen were： 35.8%,98.3% and 56.7% by ELISA. Conclusion The Mth81 recombinant antigen presented favourable antigenicity and specificity. It will be useful for the detection of immunity effectiveness and preparation of the antigen and antibody of Mtb81 protein in large quantity.