Construction and expression of recombinant plasmids pET32a-AKT1 / 国际检验医学杂志

ABSTRACT

Objective To construct the recombinant plasmid pET32a-AKT1 and express human AKT1 protein using prokaryotic expression system .Methods Reverse transcriptase-polymerase chain reaction(RT-PCR) was employed to amplify the gene AKT1 in coding region and integrated it with pET 32a plasmid ,following by transforming it into Escherichia coli DH5α and prokaryotic strains BL21(DE3) .Isopropyl-beta-D-thiogalactopyranoside(IPTG) was adopted to induce its expression .Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE ) and Western-blot were used for protein identification .Results Complete fusion of target gene and plasmid was observed .The recombinant plasmid pET32a-AKT1 was successfully transferred into the strain DE3 . After IPTG induction ,protein with relative molecular mass 70 000 was expressed by DE3 .Conclusion The recombinant plasmid pET32a-AKT1 is constructed successfully and AKT 1 protein is completely and efficiently expressed by prokaryotic strain DE 3 .