β-hCG蛋白在大肠杆菌系统的表达、纯化及活性鉴定  

The expression,purification and characterization of β-hCG protein in E.coli

作　　者：王伟[1,2] 殷小涛[1,2] 李云奇[1,2] 田仁礼[1,2] 吴昊[2] 阎瑾琦[2] 高江平[1] 于继云[2] 

机构地区：[1]解放军总医院泌尿外科,北京100853 [2]军事医学科学院基础医学研究所,北京100850

出　　处：《细胞与分子免疫学杂志》2013年第5期481-484,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(30840094);国家高技术研究发展计划(863)项目(2007AA02Z451)

摘　　要：目的获得β-人绒毛膜促性腺激素(β-hCG)融合蛋白β-hCG/GST并验证其抗原活性。方法利用PCR法扩增β-hCG全长基因,将其克隆至原核表达载体pET-42a中,构建重组质粒pET-42a-β-hCG,将该重组质粒转化至大肠杆菌BL21(DE3)中IPTG诱导表达,经SDS-PAGE分析,亲和层析法纯化融合蛋白,目的蛋白用Western blot法鉴定,ELISA检测纯化得到的融合蛋白的抗原活性。结果测序结果证实成功扩增出目的基因β-hCG;酶切和测序结果证实pET-42a-β-hCG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蛋白;Western blot法及ELISA检测证实纯化后的β-hCG/GST融合蛋白具有良好的免疫原性。结论成功获得β-hCG/GST融合蛋白,该蛋白具有良好的免疫原性,为以β-hCG为靶位的抗肿瘤主动免疫治疗研究奠定了基础。Objective To obtain IS-chain human chorionic gonadotropin （[5-hCG） fusion protein （[3-hCG/GST） and identify its antigenicity. Methods The full-length gene of IS-hCG was amplified by PCR. The PCR product was cloned into pET-42a prokaryotic expression vector to construct the recombinant plasmid pET-42a-IS-hCG, and then it was transformed into B1_21 （DE3） for 15-hCG/GST fusion protein expression under IPTG induction. After SDS-PAGE assay, the fusion protein was purified by affinity chromatography and identified by Western blotting. The antigenicity of the purified fusion protein was char- acterized by ELISA. Results The IS-hCG gene we obtained had an identical sequence to that retrieved in GenBank. The prekaryotic expression vector pET-42a-IS-hCG was successfully constructed as confirmed by enzyme digestion and DNA sequencing. Both Western blotting and ELISA demonstrated that the purified IS-hCG fusion protein had satisfactory antigenicity. Conclusion The purified β-hCG/GST fusion protein with satisfactory antigenicity has been obtained, which will facilitate further study on active anti-tumor immunotherapy targeting 15-hCG.

关 键 词：Β-HCG 原核表达 蛋白纯化 

分 类 号：R392.12[医药卫生—免疫学]