胃癌相关基因GCRG213在大肠杆菌中的表达及重组蛋白纯化  

Expression of gastric cancer related gene GCRG213 in E.coli and purification of recombinant protein

作　　者：伍银桥[1] 王刚石[1] 王孟薇[1] 吴本俨[1] 尤纬缔[1] 王卫华[1] 

机构地区：[1]解放军总医院,北京100853

出　　处：《解放军医学杂志》2004年第5期406-408,共3页Medical Journal of Chinese People's Liberation Army

基　　金：军队"十五"重点科研基金项目资助课题 (编号 0 1Z0 35)

摘　　要：目的　利用硫氧还蛋白融合表达系统表达胃癌相关基因GCRG2 13,制备高纯度的GCRG2 13蛋白。方法　采用PCR技术从pGEM T质粒上扩增出含完整ORF的GCRG2 13cDNA序列 ,将其克隆至硫氧还蛋白融合表达载体 pET10 2 /D TOPO中 ,转化大肠杆菌BL2 1,经IPTG诱导表达融合蛋白 ,凝胶回收目的蛋白。结果　SDS PAGE证实在大肠杆菌中高效表达出相对分子量约 2 9 4kD的Thioredoxin/GCRG2 13融合蛋白。薄层凝胶扫描显示 ,其表达量占菌体总蛋白的 2 8 7%。经凝胶回收法得到纯度近 10 0 %的蛋白产品。结论　在大肠杆菌中成功表达了Thioredoxin/GCRG2 13融合蛋白 ,并制备出高纯度蛋白产品 。Objective To express gastric cancer related gene GCRG213 by using thioredoxin fusion expression system, and to prepare human GCRG213 fusion protein. Methods GCRG213 cDNA with complete open reading frame was amplified by PCR from plasmid pGEM-T, and then was cloned into thioredoxin fusion expression vector pET102/D-TOPO. The recombinant plasmid was further transformed into E.coli BL21 strain. After induction with IPTG, the thioredoxin/GCRG213 fusion protein was expressed in E.coli. The product was obtained by means of direct purification from a denaturing polyacrylamide gel. Results SDS-PAGE analysis showed the thioredoxin/GCRG213 fusion protein with relative molecule mass of 29.4kDa was highly expressed. The thin layer gel scanning analysis showed that the yield of GCRG213 fusion protein was 28.7% of the total bacterial protein. The product was obtained with a purity of about 100% by means of direct purification from a denaturing polyacrylamide gel. Conclusion The thioredoxin/GCRG213 fusion protein was successfully expressed in E.coli and the product with high purity was obtained, which laid the foundation for the function research and antibody preparation hereafter.

关 键 词：胃肿瘤 基因 GCRG213 融合蛋白 原核表达 纯化 

分 类 号：R392.7[医药卫生—免疫学]