目的构建原核表达质粒pET-42a-hG250,表达并纯化肾癌相关抗原G250融合蛋白,并检测G250融合蛋白的抗原活性。方法利用PCR从pGEM-T-G250质粒中扩增G250基因片段(112～1 242 bp),测序正确后将其克隆至原核表达载体pET-42a中构建重组载体pET-42a-hG250。将其转化至大肠杆菌BL21(DE3)中,通过IPTG诱导G250蛋白的原核表达,随后将表达的融合蛋白进行纯化。经SDS-PAGE分析后,Western blot法检测纯化的蛋白,将纯化蛋白进一步包板后用ELISA对其抗原活性进行评价。结果酶切和测序结果证实pET-42a-hG250原核表达载体构建成功;转化后可以成功诱导并纯化出大小与预期一致的G250融合蛋白;Western blot法和ELISA检测证实纯化的蛋白能与特异性的抗体发生反应,显示纯化后的G250融合蛋白具有良好的免疫原性。结论成功构建了肾癌相关抗原G250基因的原核表达载体,纯化获得了G250融合蛋白,该蛋白具有良好的抗原活性。 Objective To construct the prokaryotic expression plasmid pET-42a-hG250, express and purify the G250 fusion protein of renal cell carcinoma and detect the antigenic activity of G250 fusion protein. Methods G250 gene fragment (112-142 bp) was amplified by PCR from pGEM-T-G250 plasmid and cloned into prokaryotic expression vector pET-42a to construct recombinant vector pET-42a-hG250. This was transformed into E. coli BL21 (DE3), prokaryotic expression of G250 protein was induced by IPTG, and then the expressed fusion protein was purified. After SDS-PAGE analysis, the purified protein was detected by Western blot, and the purified protein was further coated with ELISA kit to evaluate its antigenic activity. Results The recombinant plasmid pET-42a-hG250 was constructed successfully. The G250 fusion protein of the same size as expected was successfully induced and purified. Western blot and ELISA confirmed that the purified protein could bind to specific The antibody reacted, showing that the purified G250 fusion protein has good immunogenicity. Conclusion The prokaryotic expression vector of G250 gene of renal cell carcinoma was successfully constructed and the G250 fusion protein was purified. The protein has good antigen activity.