人粘膜血管定居因子/GST融合基因表达载体的构建与表达  

Construction and expression of recombinant hMAdCAM-1/GST fusion gene expression vector

作　　者：赖燕来[1] 陈志华[1] 孔祥英[1] 高杰英[1] 

机构地区：[1]军事医学科学院微生物流行病研究所免疫研究室,北京100850

出　　处：《免疫学杂志》2001年第4期251-253,264,共4页Immunological Journal

基　　金：国家自然科学基金资助项目 ( 3 97890 10 )

摘　　要：目的构建 h MAd CAM- 1/ GST融合基因表达载体并进行表达。方法采用 PCR技术扩增 h MAd CAM- 1c DNA5 '端 615 bp的基因片段 ,将其插入 p GEM- T质粒中 ,经全自动序列分析仪测序证实后 ,再亚克隆至表达载体 p GEX- 2 T,转化大肠杆菌 DH 5 a表达。结果 SDS- PAGE检测显示 ,表达出相对分子量 5 2 0 0 0 u的融合蛋白 ,占菌体总蛋白的 3 1%左右。West-ern blot分析表明 ,表达蛋白能与抗 h MAd CAM- 1多抗特异性结合。结论 h MAd CAM- 1/ GST融合基因表达载体的构建和表达 ,为深入研究 MAd CAM- 1提供了材料。ObjectiveTo construct and express a recombinant expression vector bearing hMAdCAM-1/GST fusion gene. MethodshMAdCAM-1 cDNA 5'-end 615 bp fragment encoding hMAdCAM-1 Ig-like domains was amplified by PCR from plasmid pUC21/hMAdCAM-1 and then inserted into pGEM-T plasmid. Determined by auto-sequencing, the target gene fragment was cloned into prokaryotic expression vector pGEX-2T in fusion form and transformed into E.coli DH5a. ResultsSDS-PAGE analysis showed that a new protein band with molecule weight of 52 000 u appeared as the expected size, representing 31% of the total bacterial protein. Western-blot indicate that the expressed fusion protein could bind with antiserum against hMAdCAM-1 specifically. ConclusionThe successful construction and expression of recombinant vector pGEX-2T-MAd might provide material for the further research about hMAdCAM-1.

关 键 词：MAdAM-1 原核表达 融合蛋白 GST 基因表达载体 

分 类 号：R392.11[医药卫生—免疫学] Q786[医药卫生—基础医学]