肝细胞癌患者组织中MAGE-1基因的原核表达及鉴定  

Expression and characterization of MAGE-1 gene from human hepatocellular carcinoma cells

作　　者：徐凛峰[1] 朱进[2] 焦永军[2] 张建平[1] 

机构地区：[1]南京医科大学第二附属医院普外科,江苏南京210011 [2]南京医科大学医学分子生物学研究所,江苏南京210029

出　　处：《南京医科大学学报（自然科学版）》2006年第7期534-537,共4页Journal of Nanjing Medical University(Natural Sciences)

基　　金：江苏省卫生厅基金资助(H200103)

摘　　要：目的:克隆MAGE-1基因并对其进行原核表达及鉴定。方法:通过逆转录多聚酶链式反应(RT-PCR)从原发性肝癌的细胞中扩增MAGE-1基因片段,将该片段克隆在原核表达载体PET-32a中,重组克隆转化大肠杆菌BL21(DE3),表达的蛋白以His亲和柱层析纯化,并用Dot-blot和Western-blot对其进行鉴定。结果:经RT-PCR扩增出930bp基因片段,经测序分析后的DNA序列与Genbank中的报道序列同源程度达98%,构建含重组表达载体PET32-M1的工程菌,经IPTG诱导表达、纯化,MAGE-1蛋白浓度为0.48mg/ml,经Dot-blot和Western-blot检测,纯化蛋白可以与商业化的驴抗MAGE-1的多抗结合。结论:成功扩增了肝细胞肝癌中的MAGE-1基因全长cDNA并对其进行了原核表达与鉴定,为进一步研究MAGE-1基因的功能和研制有临床价值的全人抗MAGE-1抗体奠定了基础。Objective： To study MAGE-1 gene cloning, expression and characterization in human hepatocellular carcinoma （HCC）. Methods： RT-PCR technique was used to amplify MAGE-lfragment from primary HCC samples, which was cloned to the expression vector pET-32a, and transformed into E.coli. BL21 （DE3） and the proteins were purified and identified by Dot-blot and Western-blot. Results： 930 bp-size MAGE-1 fragment was amplified and its sequence had 98% homology with that in Genebank. The expressed protein had a concentration of 0.48 mg/ml after purified process, and could react with commercial anit-MAGE-1 polyclonal antibody. Conclusion： MAGE-1 gene has been successfully cloned, expressed and identified, which will be used for exploring MAGE-1 human antibodies.

关 键 词：人肝细胞癌(HCC) MAGE-1 原核表达 

分 类 号：R511.1[医药卫生—内科学] Q34[医药卫生—临床医学]