GST融合蛋白包涵体纯化方法的探索

克隆结核分枝杆菌培养滤液蛋白CFP10基因，并在大肠杆菌中进行表达和纯化。用PCR方法从结核分枝杆菌H37Rv基因组扩增出CFP10基因片段，克隆至pMD18-T载体中，序列测定正确后，将其亚克隆到表达载体pGEX-4T-1并在大肠杆菌BL21中表达，表达蛋白经SDS-PAG及Western-blot分析后，亲和层析法纯化蛋白。成功克隆了CFP10基因，并对其在E.coli中进行了表达，SDS-PAGE及Western blot分析表明表达产物正确。通过GST纯化系统获得36kD纯化蛋白，与文献报道相符，该蛋白可与CFP10 mAb特异结合，并且同时与活动期结核病人血清发生反应。成功获得了纯化的CFP10蛋白，为进一步研究CFP10蛋白的致病机理提供了实验依据。

Study on Method of Purification of GST-tagged proteins from inclusion bodies

The aim was to study the method of purification of GST-tagged proteins from inclusion bodies.The vector PGEX-4T1-Rpfd expressed Rpfd-GST fusion protein was transformed into E.coli DH5a and protein expression was induced with IPTG.The effect of these factors is observed on the impact of protein purification by comparing lysis with guanidine hydrochloride and pretreatment with Novagen Protein Refolding Kit,and adjusting the different types of protein refolding solution.The active GST fusion protein can be efficiently purified,through removing soluble protein after ultrasonic treatment,and then using the reaction mixture containing 20 mmol Tris-HCl and 0.1 mmol DTT for rehabilitation.To remove soluble protein expression and change the formula of protein refolding solutions can effectively improve protein purification of GST-tagged proteins from inclusion bodies.