结核分枝杆菌Ag85B蛋白的原核表达、纯化及其促PBMC增殖活性的测定

Prokaryotic Expression and Purification of Mycobacterium Tuberculosis Ag85B Protein and Evaluating of Its Activity on Promoting Human PBMC Proliferation

【目的】在大肠杆菌中表达结核分枝杆菌Ag85B蛋白,并对其进行纯化和测定其促人PBMC增殖活 性。【方法】将构建的pGEX Ag85B/BL21活化后接种于LB培养基中,异丙基硫代半乳糖苷(IPTG)诱导重组蛋 白的表达,包涵体变性后经梯度浓度尿素复性,GST Sepharose亲和层析、凝血酶切除GST并再次亲和层析纯 化;MTT法测定重组Ag85B对人PBMC增殖的促进作用。【结果】成功在大肠杆菌中高效表达出GST/Ag85B 融合蛋白,占菌体总蛋白的30%,主要以包涵体形式表达;包涵体复性后经GST Sepharose亲和层析、凝血酶切 除GST并再次亲和层析纯化获得纯度为96%的重组Ag85B蛋白;该蛋白能够促进人PBMC的增殖,增殖幅度 随浓度增加而增加,刺激72h时达增殖最高峰。【结论】高效表达并纯化了结核分枝杆菌Ag85B蛋白,该蛋白具 有促进人PBMC增殖的活性,为进一步研究其在膀胱肿瘤免疫治疗中的作用奠定了基础。

ObjectiveTo express the Mycobacterium tuberculosis Ag85B protein in E.coli and purify it and research its activity of promoting human PBMC proliferation .Induced by IPTG , GST/ Ag85B fusion protein was expressed in E.coli BL21. After the denaturalized inclusion body was renatured by grade concentration urea, Ag85B recombinant was purified by GST-sepharose affinity chromatography and digested by thrombin and purified by second affinity chromatography . The proliferative response of human PBMC to Ag85B was measured in MTT assay.A novel fusion protein GST/Ag85B was expressed in E.coli in a way of inclusion bodies ,and its amount was 30% in total lysate protein of bacteria. After being purified by GST-sepharose affinity chromatography and digested by thrombin and purified by second affinity chromatography,the purity of final Ag85B protein was 96%.The protein could promote the proliferation of human PBMC and its activity had a positive correlation to the concentration of Ag85B protein.[Conclusion]Mycobacterium tuberculosis Ag85B protein was successfully expressed in prokaryotic cell and purified, and it may maintain the activity of promoting human PBMC proliferation. This result establish a groundwork for the further research of Ag85B in the bladder tumor immune therapy.