【摘要】 目的构建原核表达载体pGEX-4T-1-MAGE-3并检测其在大肠杆菌(E.coli)BL21中的表达,为制备以黑色素瘤抗原-3(MAGE-3)基因片段为基础的肽疫苗及特异诊断试剂提供抗原打下基础。方法通过逆转录-聚合酶链反应(RT-PCR)法制备MAGE-3目的基因,以pGEM-TEasy为克隆载体,pGEX-4T-1为原核表达载体,将MAGE-3目的基因克隆至pGEM-TEasy载体和亚克隆至pGEX-4T-1载体上。筛选、鉴定阳性克隆并将其转化E.coliBL21.经IPTG诱导,12%SDS-PAGE电泳分离和WesternBlot表达鉴定。结果正确构建了原核重组表达质粒pGEX-4T-1-MAGE-3;在E.coliBL21中检测到含该重组表达质粒的转化菌表达出分子量35kD的融合蛋白;基因测序结果表明,阳性克隆中的MAGE-3目的基因序列与GenBank公布的已知序列完全一致。结论成功构建的原核重组表达载体pGEX-4T-1-MAGE-3及其所表达的融合蛋白,为以MAGE-3目的基因为基础的肽疫苗及特异诊断试剂的研制提供抗原打下了基础;为后续实验提供了依据。更多还原

【Abstract】 [Objective] To construct prokaryotic expression plasmid pGEX-4T-1-MAGE-3 and analyze its expression in BL21 E.coli, so that to lay a foundation for providing antigen of producing peptide vaccine and specific diognose reagent based on MAGE-3 gene. [Methods] MAGE-3 gene was obtained by RT-PCR. pGEM-T Easy was used as cloning vector, pGEX-4T-1 was used as prokaryotic expression vector. MAGE-3 gene was cloned into pGEM-T Easy and subcloned into pGEX-4T-1. Position clones were selected, identified and transformed into BL21 E.coli. Afterwards they were induced with IPTG, identified by 12%SDS-PAGE electrophoresis and Western blotting. [Results] The prokaryotic recombinant expression plasmid pGEX-4T-1-MAGE-3 was correctly constructed and 35KDa fusion protein was observed in BL21 E.coli. DNA sequecing results showed that the sequence of MAGE-3 gene in position clones was as completely same as the reported in the GenBank. [Conclution] Prokaryotic recombinant expression plasmid pGEX-4T-1-MAGE-3 was successfully constrcted and the fusion protein was expressed by induced. These lay a foundation for providing antigen which might be used as peptide vaccine and specific diognose reagent based on MAGE-3 gene, and also provide experimental basis for further research.更多还原