刚地弓形虫抗原基因SAG2的克隆、表达和纯化

CLONING、EXPRESSION AND PURIFICATION OF SAG2 GENE OF TOXOPLASMA GONDII

目的克隆刚地弓形虫抗原基因SAG2,并在大肠杆菌中进行高效表达及纯化。方法设计引物从弓形虫基因组DNA中扩增SAG2基因序列,构建pGEX-4T-SAG2重组质粒,双酶切鉴定后,在大肠杆菌中进行表达,并对其表达条件进行优化,所表达蛋白用GST亲和层析柱进行纯化。结果经SDS-PAGE检测,所表达的蛋白约为41KD。Western-blotting表明该蛋白能与兔抗弓形虫血清特异性结合。培养基2×YTA,IPTG浓度0.1 mmol/L,诱导时间3 h,培养温度30℃,摇瓶转速220 r/min是GST-SAG2融合蛋白表达的最佳条件,且得到的是可溶性融合蛋白GST-SAG2。结论得到高效表达的SAG2抗原蛋白,为建立弓形虫病快速免疫诊断试剂盒打下基础。

Objective In order to clone the SAG2 gene of Toxoplasma gondii and highly express it in E. coli, and then purify it. Methods The SAG2 gene fragment was amplified by PCR, and then the gene fragment was ligated to an expression vector pGEX-4T-2. After digestion with BamH I and Sal I , screening positive recombinants sequenced were induced for expression. The condition of experiment were optimized. It was purified by affinity chromatography on glutathione-sepharose system. Results SDS-PAGE indicated that the target protein was around 41KD. Western-blotting showed it could react with rabbit-anti Toxoplasma gondii serum. The higher yield could be gotten by 2× YTA, 0. 1 mmol/L IPTG, 220 r/min, till 3 hours at 30℃. Conclusion The soluble protein of SAG2 was highly expressed in E. coli, and the research foundation was laid for developing the new genetic engineering diagnostic reagents.