MAGE-D4a融合蛋白的原核表达、纯化及鉴定  

Prokaryotic expression,purification and identification of MAGE-D4a

作　　者：贺菽嘉[1] 肖绍文[2] 罗彬[1] 蓝秀万[3] 林文珍[3] 林永达[1] 谢小薰[1] 

机构地区：[1]广西医科大学组织学与胚胎学教研室,南宁530021 [2]广西医科大学第一附属医院神经外科,南宁530021 [3]广西医科大学生物化学与分子生物学教研室,南宁530021

出　　处：《中国免疫学杂志》2010年第5期432-435,440,共5页Chinese Journal of Immunology

基　　金：国家自然科学基金(30760055);广西省自然科学基金(桂科自0728148及0832144);广西高发疾病研究创新性团队基金(No.桂教人[2007-59]号);广西大型仪器协作共用网(499-2007-078及666-2008-079)资助

摘　　要：目的:构建肿瘤相关抗原MAGE-D4a的原核重组体系,表达、纯化融合蛋白。方法:PCR法从胶质瘤组织cDNA中扩增MAGE-D4a基因全长编码区,连接入原核表达载体pMAL-c2,筛选、鉴定阳性克隆并测序。将重组质粒转化至大肠杆菌Rosetta(DE3)诱导表达,表达产物经AmyloseResin亲和层析分离纯化,并且对纯化的融合蛋白进行质谱鉴定。结果:成功扩增了MAGE-D4a基因;重组质粒在Rosetta大肠杆菌中诱导表达出MBP/MAGE-D4a融合蛋白;优化了MAGE-D4a原核表达体系的最适条件;蛋白质谱分析结果显示表达的基因重组蛋白与目的蛋白相符。结论:获得了高效表达、可溶性的MAGE-D4a重组蛋白,为抗体的制备及血清学分析奠定了基础。Objective：To construct tumor associated antigen MAGE-D4a prokaryotic recombination system for expression and purification of the fusion protein.Methods：PCR technique was used to amplify MAGE-D4a of the full-length encoding sequence from human glioma sample,which was cloned into prokaryotic expression plasmid pMAL-c2.Positive clones were selected,identified,sequenced,and then transformed into E.coli Roseetta.After induced by IPTG,expressing products were purified with Amylose Resin affinitive chromatography and identified by mass spectrum.Results：The MAGE-D4a gene was obtained successfully.E.coli Roseetta strains which harbored the recombinant plasmid could express MBP/MAGE-D4a fusion protein.The inducing conditions of prokaryotic expression were optimized.The expressing protein was really identical to MAGE-D4a protein by mass spectrometry analysis.Conclusion：The highly efficient expression and soluble protein was manufactured,which could provide essential materials for further research of MAGE-D4a.

关 键 词：黑色素瘤相关抗原-D4a 原核表达 纯化 融合蛋白 

分 类 号：R739.4[医药卫生—肿瘤]