肝细胞癌患者组织中MAGE-1基因的原核表达及鉴定

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。

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

Objective: To study MAGE-1 gene cloning,expression and characterization in human hepatocellular carcinoma(HCC).Methods: RT-PCR technique was used to amplify MAGE-1fragment 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.