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

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(Western blot)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6 h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,Western blot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2500 u/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.

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

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 BL21 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 anion- exchange chromatography and RP-HPLC and identified with Western blot. Its N-terminal amino acid sequence and IL-2 bio- activity 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,anion-exchange 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 bioactivity 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.