重组原核表达载体pGEX-4T-1-MAGE-1的构建及表达

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

目的:构建人肿瘤相关抗原MAGE1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MAGE1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pGEX4T-1中,构建重组表达质粒pGEX4T-1MAGE-1。将重组质粒转化大肠杆菌BL21(DE3),经1mmol/L异丙基βD硫代半乳糖苷(IPTG)诱导4h,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927bp的MAGE1基因。重组表达质粒pGEX4T1MAGE1转化大肠杆菌BL21(DE3)后经诱导表达,目的蛋白约57000,占菌体总蛋白的23%。结论:成功构建出重组原核表达载体pGEX4T-1MAGE-1,该载体在大肠杆菌BL21(DE3)中高效表达。

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）.