【摘要】 目的构建原核重组表达载体pGEX-4T-1-MAGE-3并检测其在大肠杆菌(E.coli)BL21中的表达。方法RT-PCR法制备MAGE-3目的基因,采用DNA重组技术将其克隆至pGEM-TEasy和亚克隆至pGEX-4T-1载体,转化E.coliBL21株,经IPTG诱导,12%SDS-PAGE电泳分离和Western Blot表达鉴定。结果扩增出349bp的MAGE-3目的基因并构建了原核重组表达载体pGEX-4T-1-MAGE-3;测序结果与Gen Bank收录序列相一致;在E.coli BL21中检测到含该重组表达载体的转化菌表达出分子量约35kD的融合蛋白并证实其为目的蛋白。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 recombinant expression plasmid pGEX-4T-1-MAGE-3 and analysis of its expression in BL21 E.coli.Methods MAGE-3 aim gene was obtained by RT-PCR.The target gene was orientating cloned into pGEM-T Easy and subclone into pGEX-4T-1 vector by DNA recombinant technical.Position clones were transformed into BL21.And then they were induced with IPTG,identified by 12% SDS-PAGE electrophoresis and Western-blot.Results MAGE-3 fragment(349bp)was amplified and the prokaryotic recombinant expression plasmid pGEX-4T-1-MAGE-3 was correctly constructed.DNA sequecing results showed that the sequence of MAGE-3 gene fragment in position clones is the completely same as the sequence of the GenBank public.The 35kD fusion protein was observed in BL21 E.coli and verfied that it is the target protein.Conclusion Prokaryotic recombinant expression plasmid pGEX-4T-1-MAGE-3 was successfully constructed and the fusion protein was expressed by induced.These lay a foundation for providing antigen which will be used as peptide vaccine and specific diagnosis reagent based on MAGE-3 aim gene,and also provide an experimental basis for further research.更多还原