pIRES2-MLAA34-EGFP穿梭载体的构建及表达

Construction of p IRES2-MLAA34-EGFP vector and its expression

目的构建p IRES2-MLAA34-EGFP重组质粒,并对其进行原核诱导表达。方法用RT-PCR的方法从U937细胞中提取MLAA-34基因,利用酶切、T4连接等分子生物学技术将MLAA-34目的基因克隆至p IRES2-EGFP表达载体中,经PCR、酶切等技术对构建的重组表达载体p IRES2-MLAA34-EGFP进行鉴定后,转化大肠杆菌BL21并进行诱导表达。结果扩增出MLAA-34全长基因1 014 bp,构建了p IRES2-MLAA34-EGFP重组质粒,经PCR和双酶切鉴定,与预期大小一致。重组质粒在大肠杆菌中经IPTG诱导和SDS-PAGE检测,所表达的融合蛋白与预期蛋白相符合。结论成功构建了p IRES2-MLAA34-EGFP重组质粒,并使其在大肠杆菌中高效表达。

Objective To construct the pIRES2-MLAA34-EGFP recombinant vector and express the MLAA34-EGFP recombinant proteins in E.coli.Methods The MLAA34 gene were extracted from U937 cells by RT-PCR and insert into the pIRES2-EGFP vector.Results We amplified full-length sequence of MLAA-34 gene successfully and iden-tified the correctness of pIRES2-MLAA34-EGFP recombinant vector by PCR and BamHⅠ/EcoRⅠdouble digestion. Moreover,the MLAA34-EGFP recombinant proteins expressed in E.coli were consistent with the expected protein. Conclusion We construct the pIRES2-MLAA34-EGFP recombinant vector successfully and the MLAA34-EGFP re-combinant proteins was successfully induced to express by IPTG.