CAJ | 학술논문

目的：构建重组质粒pET32a-AKT1，利用原核表达体系表达AKT1蛋白。方法逆转录聚合酶链反应（RT-PCR）扩增AKT1编码区基因，并将其与pET32a质粒融合，并转化大肠埃希菌DH5α及原核菌株BL21（DE3），采用异丙基-D-硫代半乳糖苷（IPTG）诱导其表达，采用十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）和Western-blot进行蛋白鉴定。结果目的基因与质粒完整融合；重组质粒pET32a-AKT1成功转入菌株DE3；IPTG诱导后，DE3表达相对分子质量为70000的蛋白。结论成功构建重组质粒pET32a-AKT1，且AKT1蛋白在原核表达菌DE3中完整、高效表达。
목적：구건중조질립pET32a-AKT1，이용원핵표체체계표체AKT1단백。방법역전록취합매련반응（RT-PCR）확증AKT1편마구기인，병장기여pET32a질립융합，병전화대장애희균DH5α급원핵균주BL21（DE3），채용이병기-D-류대반유당감（IPTG）유도기표체，채용십이완기류산납취병희선알응효전영（SDS-PAGE）화Western-blot진행단백감정。결과목적기인여질립완정융합；중조질립pET32a-AKT1성공전입균주DE3；IPTG유도후，DE3표체상대분자질량위70000적단백。결론성공구건중조질립pET32a-AKT1，차AKT1단백재원핵표체균DE3중완정、고효표체。
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 .