结核分枝杆菌Ag85B/IL-2融合蛋白的原核表达、纯化、鉴定及活性初步测定

Prokaryotic expression,purification and identification of Mycobacterium tuberculosis Ag85B/IL-2 fusion protein and detection of its biological activity

目的在大肠杆菌中表达结核分枝杆菌Ag85B/IL-2融合蛋白,并对其进行纯化、鉴定和活性初步测定.方法将构建的pGEX-Ag85B/IL-2重组菌BL21扩增后接种于LB培养基中,异丙基硫代半乳糖苷(IPTG)诱导重组融合蛋白的表达,筛选最适诱导剂浓度和最适诱导时间;梯度浓度尿素复性包涵体,经GST-Sepharose亲和层析、凝血酶切、阴离子交换层析和反相-高效液相(RP-HPLC)层析纯化Ag85B/IL-2融合蛋白,免疫印迹(Westernblot)鉴定,并测定其N-末端氨基酸序列和IL-2比活性.结果成功在大肠杆菌中高效表达带有GST的Ag85B/IL-2融合蛋白,占菌体总蛋白的30%,主要以包涵体形式表达,于诱导后6h表达量达最高峰.对包涵体复性,纯化获得纯度为98.32%的Ag85B/IL-2融合蛋白,Westernblot鉴定阳性,N-末端氨基酸测序与理论预期完全一致,IL-2比活性为2500u/mg.结论高效表达并纯化了Ag85B/IL-2融合蛋白,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.

AIM： To express Mycobacterium tuberculosis Ag85B/ IL-2 fusion protein in E. coli, to purify and identify it, and to detect its biological activity. METHODS： By using genetic engineering techniques, we constructed a recombinant expression plasmid pGEX-Ag85B/IL-2, and expressed Ag85B/ IL-2-GST fusion protein in E. coli BI21 under the induction of IPTG. Moreover, we also studied the optimal expression conditions concerning IPTG concentration and induction time. After renatured by urea in a concentration gradient, and purified by GST-Sepharose affinity chromatography and digested by thrombin, the Ag85B/IL-2 fusion protein was further purified by anionexchange chromatography and RP-HPLC and identified with Western blot. Its N-terminal amino acid sequence and IL-2 bioactivity were measured as well. RESULTS： A novel fusion protein Ag85B/IL-2-GST was expressed in E. coli in a way of inclusion body,accounting for 30% of total lysate protein of bacteria. After purified by GST-Sepharose affinity chromatography, anionexchange chromatography and RP-HPLC, the desired Ag85B/IL-2 fusion protein with purification degree of 98.32% was acquired and confirmed by Western blot. Its N-terminal amino acid sequence was identical to the anticipation, and its specific IL-2 bioaetivity was 2500 u/mg. CONCLUSION： Ag85B/IL-2 fusion protein was successfully expressed in E. coli and purified. This results established a groundwork for the further researches of Ag85B/IL-2 fusion protein in the immunotherapy of bladder tumor.