结核分支杆菌16000抗原基因在大肠杆菌中的高效表达及其产物纯化

High level expression of Mycobacterium tuberculosis 16 000 antigen in E coli

目的 获得高效表达结核分支杆菌１６ ０００ 抗原的大肠杆菌工程菌，将培养物纯化后得到重组１６ ０００ 蛋白抗原纯品，并检测其免疫学特性。方法 用聚合酶链反应（ＰＣＲ） 方法获得目的基因构建大肠杆菌高效表达株。聚丙烯酰胺凝胶电泳（ＳＤＳＰＡＧＥ） 分析重组蛋白抗原的相对分子质量大小及表达形式。ＤＥＡＥ及ＰＥＩ凝胶纯化重组蛋白。Ｗｅｓｔｅｒｎ 印迹及酶联免疫吸附测定法（ＥＬＩＳＡ） 分析重组蛋白的免疫原性。结果 构建了具有正确基因序列的重组表达载体，在大肠杆菌ＢＬ２１（ＤＥ３）中诱导表达，重组蛋白占菌体总蛋白的４０％ ，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，该纯化的重组抗原能区别抗结核抗体阳性患者血清及正常人血清。结论 实验中获得了能高效表达１６ ０００ 蛋白抗原的大肠杆菌工程菌，以可溶性形式存于胞浆内。该重组蛋白具有特异的免疫原性。

Objective To gain the recombinant protein antigen 16 000 of Mycobacterium tuberculosis highly expressed in E coli and study its immunological characteristics Methods DNA fragments code for the protein were obtained by PCR, then cloned into the pET plasmid vector to gain recombinant E coli Cells were cultured and induced to produce recombinant protein, whose molecular size and present form were analyzed by SDS PAGE,and its immunological characteristics were analyzed by Western blotting and ELISA technology Results The clone was analyzed at the nucleotide lever and shown the same DNA sequence coding for natural 16 000 protein Analyzed by SDS PAGE and Western blotting,it was found to produce immunoreactive proteins with mobilities very similar to those of the 16 000 protein antigen,and the recombinant protein amounted to 40% of total cell proteins ELISA results indicated that the purified recombinant protein could distinguish sera from tuberculosis patients with anti PPD antibody and those from tuberculin positive contacts Conclusions Recombinant 16 000 protein antigen highly expressed in the form of solution in E coli was gained and this antigen was located in cell plasma This recombinant protein showed specific immunogenicity