MAGE-D4a融合蛋白的原核表达、纯化及鉴定

Prokaryotic expression,purification and identification of MAGE-D4a

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.