重组原核表达载体pGEX-4T-1-MAGE-1的构建及表达

目的:构建人肿瘤相关抗原MAGE-1基因原核表达载体.方法:从人肝细胞性肝癌组织中提取总RNA,RT-PCR扩增出MAGE-1基因.酶切鉴定后,将其插入pGEM-T载体,再克隆至原核表达载体pGEX-4T-1中,构建重组表达质粒pGEX-4T-1-MAGE-1.将重组质粒转化大肠杆菌BL21(DE3),经1 mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)诱导4 h,SDS-PAGE及凝胶密度扫描分析目的蛋白的表达情况.结果:RT-PCR扩增出长度为927 bp的MAGE-1基因.重组表达质粒pGEX-4T-1-MAGE-1转化大肠杆菌BL21(DE3)后经诱导表达,目的蛋白约57 000,占菌体总蛋白的23%.结论:成功构建出重组原核表达载体pGEX-4T-1-MAGE-1,该载体在大肠杆菌BL21(DE3)中高效表达.

Establishment and expression of recombinant prokaryotic vector pGEX-4T-1-MAGE-1

Aim: To establish recombinant prokaryotic vector of MAGE-1 gene.Methods:The mRNA of MAGE 1 gene was isolated from HCC tissue, and the cDNA was amplified by RT PCR. After cloning with pGEM T vector and sequencing, the MAGE 1 gene was inserted into the prokaryotic express vector pGEX 4T 1. BL21(DE3) was transformed with the recombinant pGEX 4T 1 MAGE 1, and was induced by IPTG for 4 h,and then the fusion MAGE 1 protein was detected using SDS PAGE and Western blot method. Results: A 927 bp MAGE-1 gene was obtained by RT -PCR. SDS PAGE indicated that the expressed protein was about 57 000 and account for 23% of the total bacterial proteins.Conclusion: Recombinant prokaryotic vector pGEX-4T-1-MAGE -1 is established successfully and can be highly expressed in BL21(DE3).