MAGE-3基因的表达、纯化及其抗血清的制备  

Prokaryotic Expression and Purification of MAGE-3 Gene and Preparation of Polyclonal Antibody against MAGE-3

作　　者：肖刚[1] 张文敏[1] 张萌[1] 谢丹[1] 郭爱林[1] 文剑明[1] 

机构地区：[1]中山大学中山医学院病理学教研室,广东广州510080

出　　处：《中山大学学报（医学科学版）》2005年第2期176-179,共4页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：广州市科技计划项同基金资助项目(2003J1-C0221)

摘　　要：【目的】构建MAGE-3的原核表达质粒,表达、纯化蛋白并制备多克隆抗体。【方法】利用RT- PCR方法扩增MAGE-3基因片段.连接入原核表达载体pGEX-4T-1,构建MAGE-3的原核表达质粒pGEX- MAGE-3并测序。将该质粒转化大肠杆菌BL21进行诱导表达,并通过谷胱甘肽巯基转移酶纯化系统进行分离纯化。用纯化的融合蛋白GST-MAGE-3免疫新西兰大白兔,ELISA法测定抗体效价,饱和硫酸铵沉淀法纯化抗体,Western-blot检测抗体活性。【结果】构建了pGEX-MAGE-3原核表达质粒,含有该质粒的大肠杆菌经IPTG诱导后表达-相对分子质量约为72×103的蛋白,经GST融合蛋白纯化系统纯化,得到了MAGE-3的重组蛋白GST-MAGE-3。制备了兔抗GST-MAGE-3抗体。Western blot证实该抗体可与GST-MAGE-3蛋白特异性结合。【结论】获得了肿瘤抗原MAGE-3的纯化重组蛋白及其特异的多克隆抗体,为进一步研究MAGE-3抗原在肿瘤免疫治疗中的作用提供了一件检测工具。[Objective]To construct MAGE-3 prokaryotic expression plasmid and express and purify protein for preparation of polyclonal antibody. [Methods]MAGE-3 gene was amplified by reverse transcription polymerase chain reaction (RT-PCR). After sequencing, the gene was cloned into the expression vector pGEX-4T-1 to construct MAGE-3 expression plasmid pGEX-MAGE-3. The E.coli 8L21 contained the expression plasmid was induced by IPTG and the protein was purified with GSTrap FF column. White rabbits were immunized with the immunogen GST-MAGE-3. The titer was measured by ELISA and the polyclonal antibody was obtained. [Results]The expression plasmid pGEX-MAGE-3 was constructed. The E.coli BL21 contained the plasmid expressed an 72×103 protein after being induced by IPTG and the 72 ku protein was purified by GSTrap FF column. The polyclonal anti-GST-MAGE-3 antibody could specifically recognize MAGE-3 protein in Western blot. [Conclusion]In this study, the protein of human MAGE-3 segment was expressed and purified successfully, polyclonal anti-GST-MAGE-3 antibody has been prepared, which can be used as the diagnostic tool for further research of the MAGE-3 protein as a promising candidate in tumor immunotherapy.

关 键 词：黑色素瘤抗原-3 原核表达 重组蛋白 纯化 抗体 

分 类 号：R730.3[医药卫生—肿瘤]