CAJ | 학술논문

[目的]构建犬黑皮质素受体4(MC4R)突变体D90N真核表达载体,并在MDCK细胞中进行表达.[方法]以犬MC4R基因组DNA为模板,应用重叠PCR扩增MC4R突变体D90N目的基因,将扩增产物进行T-A克隆;酶切、测序鉴定成功后将目的基因亚克隆到真核表达载体pcDNA3.1-myc-His/A.重组体pcDNA3.1-myc-His/A-cMC4R-D90N经酶切和测序鉴定;采用FuGENE HD介导转染技术将重组体导入MDCK细胞;转染后继续培养72.0 h,提取细胞内总RNA,用RT-PCR鉴定目的基因的表达;提取细胞总蛋白,用Western Blot检测蛋白表达.[结果]构建带标签的pcDNA3.1-myc-His/A-cMC4R-D90N重组真核表达载体,测序结果显示含有D90N突变位点.RT-PCR和Western Blot检测到目的基因表达.[结论]成功构建犬真核表达载体pcDNA3.1-myc-His/A-cMC4R-D90N,重组体能在MDCK细胞中表达.
[목적]구건견흑피질소수체4(MC4R)돌변체D90N진핵표체재체,병재MDCK세포중진행표체.[방법]이견MC4R기인조DNA위모판,응용중첩PCR확증MC4R돌변체D90N목적기인,장확증산물진행T-A극륭;매절、측서감정성공후장목적기인아극륭도진핵표체재체pcDNA3.1-myc-His/A.중조체pcDNA3.1-myc-His/A-cMC4R-D90N경매절화측서감정;채용FuGENE HD개도전염기술장중조체도입MDCK세포;전염후계속배양72.0 h,제취세포내총RNA,용RT-PCR감정목적기인적표체;제취세포총단백,용Western Blot검측단백표체.[결과]구건대표첨적pcDNA3.1-myc-His/A-cMC4R-D90N중조진핵표체재체,측서결과현시함유D90N돌변위점.RT-PCR화Western Blot검측도목적기인표체.[결론]성공구건견진핵표체재체pcDNA3.1-myc-His/A-cMC4R-D90N,중조체능재MDCK세포중표체.