肾癌相关抗原G250的原核表达、纯化及抗原活性检测

Prokaryotic expression,purification and antigenicity identification of human renal cell carcinoma-associated antigen G250

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 amplify human renal cell carcinoma （RCC）-associated antigen G250 gene and construct a recombinant plasmid pET-42a-hG250, express and purify human G250 protein and identify its antigenicity. Methods The gene of human G250 was amplified from pGEM-T-G250 by PCR. After sequencing, the PCR product （112-1242 bp） was cloned into pET-42a prokaryotic expression vector to construct the recombinant plasmid pET-42a-hG250. The plasmid was transformed into BL21 （DE3） and human G250 protein was expressed under the induction of IPTG. The fusion protein was purified and identified by SDS-PAGE, Western blotting and ELISA sequentially. Results The human G250 prokaryotic expression vector pET-42a-hG250 was successfully constructed as confirmed by enzyme digestion and DNA sequencing. After transformation into BL21 （ DE3）, the target protein was successfully induced to express and purified as expected. West- ern blotting and ELISA demonstrated that the purified human G250 protein had a desirable immunogenicity. Conclusion The recombinant prokaryotic expression vector pET-42a-hG250 has been constructed successfully. The purified human G250 pro- tein has a good antigenicity.