重组人IL2-MUC1融合蛋白在大肠杆菌中的表达及鉴定  

Expression and identification of recombinant human IL-2 and MUC1 fusion protein in E. coli BL21

作　　者：周静[1] 马吉春[1] 赵小霞[1] 方芳[1] 宋献美[1] 柳忠辉[1] 台桂香[1] 

机构地区：[1]吉林大学基础医学院黏免疫学教研室,吉林长春130021

出　　处：《吉林大学学报（医学版）》2008年第5期763-766,共4页Journal of Jilin University：Medicine Edition

基　　金：教育部留学人员启动基金资助课题(2002);吉林大学创新基金资助课题(2004)

摘　　要：目的:构建表达人IL2-MUC1融合蛋白的工程菌,为制备新型MUC1疫苗提供实验依据。方法:通过PCR方法钓取人IL-2基因片段,将其克隆入pBS-SK-MUC1重组载体中,再将人IL2-MUC1串联基因片段插入原核表达载体pGEX-4T-1。将重组的pGEX-IL2-MUC1转化大肠杆菌BL-21,通过IPTG诱导表达,采用SDS-PAGE和Western blotting鉴定表达蛋白。结果:经酶切及基因测序证实获得的IL-2基因片段402bp及IL2-MUC1基因片段852bp基因序列正确;构建的pGEX-IL2-MUC1表达载体在BL21大肠杆菌中成功表达GST-IL2-MUC1蛋白,经SDS-PAGE证实其相对分子质量为64000,与理论预期值相符。Western blotting分析表明该表达产物与抗人MUC1多克隆抗体及抗人IL-2单克隆抗体均具有特异性结合能力。结论:成功构建表达人IL2-MUC1融合蛋白的工程菌,为进一步研究以人MUC1为靶点的新型抗肿瘤疫苗奠定基础。Objective To establish E. coli strain expressing human IL-2 and MUC1 fusion protein to provide experimental fundation for a novel MUC1 cancer vaccine. Methods Human IL-2 gene fragments were obtained by PCR and they were inserted into recombinant vector pBS-SK-MUC1. After digested, human IL2-MUC1 gene fragments were cloned into prokaryotic expression vector pGEX-4T-1 and E. coli BL21 was transformed by constructing pGEX-IL2-MUC1. IL2-MLTC1 fusion protein expression was induced by IPTG and analyzed by SDS-PAGE and Western blotting. Results Acquired gene fragments of human IL-2 （402 bp） and tandem IL2-MUC1 （852 bp） were identified by digestion and DNA sequencing. The recombinant vector of pGEX-IL2- MUC1 was constructed successfully. SDS-PAGE result proved that GST-IL2-MUC1 fusion protein with a relative molecular mass of about 64 000 was expressed. Western blotting result showed that the expressed products had specific reaction with both rabbit anti-human MUC1 polyclonal antibody and mouse anti-human IL-2 monoclonal antibody. Conclusion The engineering E. coli strain expressing human IL2-MUC1 fusion protein is successfully established. It is a basis to further study on novel cancer vaccine with MUC1 as target.

关 键 词：黏蛋白1 白细胞介素2 基因克隆 原核表达 

分 类 号：Q78[生物学—分子生物学]