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肿瘤抗原基因MAGE-A9的克隆及原核表达
引用本文:徐璐,朱进,仇镇宁,李玉华,冯振卿.肿瘤抗原基因MAGE-A9的克隆及原核表达[J].山西医药杂志,2007,36(8):694-696.
作者姓名:徐璐  朱进  仇镇宁  李玉华  冯振卿
作者单位:南京医科大学 210029
摘    要:目的扩增和克隆人黑色素瘤抗原MAGE-A9cDNA,构建原核表达载体,表达并纯化MAGE-A9蛋白。方法从人肝癌组织提取总RNA,用反转录-聚合酶链反应(RT-PCR)从中扩增出MAGE-A9 cDNA,插入载体pMD18-T中,测序正确后,构建重组表达载体pBAD/gⅢ-MAGE-A9,转化大肠杆菌TOP10株。以L-Arabi-nose进行诱导表达,并纯化。结果获得MAGE-A9cDNA,测序结果与GenBank一致。成功构建表达载体,表达可溶重组蛋白,十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析其相对分子质量为35 000。结论成功地构建了pBAD/gⅢ-MAGE-A9原核表达质粒,获得了MAGE-A9蛋白,为该蛋白应用于肿瘤免疫治疗奠定了基础。

关 键 词:黑色素瘤  逆转录-聚合酶链反应  MAGE-A9  基因克隆  基因表达
修稿时间:2007-05-21

Cloning and prokaryotic expression of tumor antigen MAGE-A9
XU Lu,ZHU Jin,QIU Zhen-ning,LI Yu-hua,FENG Zheng-qing.Cloning and prokaryotic expression of tumor antigen MAGE-A9[J].Shanxi Medical Journal,2007,36(8):694-696.
Authors:XU Lu  ZHU Jin  QIU Zhen-ning  LI Yu-hua  FENG Zheng-qing
Institution:Nanjing Medical University, Jiangsu 210029, China
Abstract:Objective To amplify MAGE-A9 gene and construct the recombinant prokaryotic plasmid to induce expression of MAGE-A9.Methods The cDNA encoding human MAGE-A9 gene was amplified by RT-PCR from human hepatocelluar carcinoma tissue before the MAGE-A9 gene was inserted into plasmids pMD18-T.After sequencing,the MAGE-A9 was cloned into the prokaryotic expression vector pBAD/gⅢ to construct the recombinant expression vector pBAD/gⅢMAGE-A9 and was transformed into E.coli TOP10.The recombinant MAGE-A9 protein was expressed under induction of L-Arabinose and was purified through Hitrap column.Results The sequence of MAGE-A9 was identical to the sequence from GenBank.The expression plasmid pBAD/gⅢ-MAGE-A9 was successfully constructed.By affinity column and SDS-PAGE,the purified MAGEA9 fusion protein displayed a band of Mr 35 000.Conclusion The expression plasmid pBAD/gⅢ-MAGE-A9 was successfully constructed.MAGE-A9 was expressed and purified successfully,which may lay foundation for application of the protein in tumor immunotherapy.
Keywords:MAGE-A9
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