黑色素瘤抗原-3原核重组质粒的构建及表达

Construction and Expression of MAGE-3 Target Gene Prokaryotic Recombinant Plasmid

目的构建MAGE-3目的基因原核重组表达质粒pGEX-4T-1-MAGE-3并检测其在大肠杆菌(E.coli)BL21(DE3)中的表达。方法RT-PCR法制备MAGE-3目的基因,连接入原核表达载体pGEX-4T-1,构建MAGE-3目的基因的原核重组表达质粒pGEX-4T-1-MAGE-3并测序,将该质粒转化E.coliBL21(DE3)菌株,经IPTG诱导,12%SDS-PAGE和W estern B lot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GenBank收录序列相一致;在E.coli BL21(DE3)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。

Objective To construct MAGE -3 target gene prokaryotic recombinant expression plasmid pGEX - 4T - 1 - MAGE - 3 and analysis of its expression in BI21 （ DE3 ） E. coll. Methods MAGE -3 target gene was obtained by RT- PCR. After sequencing, the target gene was cloned into the expression vector pGEX -4T - 1 to construct MAGE -3 prokaryotic recombinant expression plasmid pGEX -4T - 1 - MAGE -3. The E. coli BI21 （ DE3 ） containing the expression plasmid was induced by IVFG,identified by 12% SDS -PAGE electrophoresis and Western -blot. Results MAGE -3 target gene fragment （349bp） was amplified and the prokaryotic recombinant expression plasmid pGEX - 4T - 1 - MAGE - 3 was correctly constructed. DNA sequencing results show that the sequence of MAGE - 3 aim gene in position clones is the completely same as the sequence of the GenBank public. The 35kD fusion protein was observed in BI21 （DE3 ） E. coli and verified that it is the very aim protein. Conclusion Prokaryotic recombinant expression plasmid pGEX- 4T- 1 -MAGE -3 was successfully constructed and the fusion protein was expressed by induced. These set a foundation for providing antigen which will be used as peptide vaccine and specific diagnose reagent based on MAGE -3 target gene, and also provide experimental basis for further research.