CAJ | 학술논문

背景:碱性螺旋-环-螺旋转录因子少突胶质细胞转录因子2在脊髓的少突胶质细胞的分化过程中起着关键性的作用.目的:构建大鼠少突胶质细胞转录因子2基因重组真核表达载体,观察其在真核细胞内的表达.方法:从新生大鼠脊髓组织中提取总RNA,采用RT-PCR方法获得目的基因片段,克隆至pGEM-T载体中.经测序确证后将少突胶质细胞转录因子2 cDNA片段与pEGFP-N1载体定向连接.将鉴定正确的重组体pEGFP-N1-Olig2以脂质体法转染至COS-7细胞中,反转录PCR鉴定少突胶质细胞转录因子2 mRNA的表达,免疫印迹法检测少突胶质细胞转录因子2蛋白质表达水平.结果与结论:克隆了少突胶质细胞转录因子2的cDNA,并构建了其真核表达载体pEGFP-N1-Olig2,经限制性内切酶酶切鉴定及测序分析证实了其序列的正确性;转染72 h时免疫印迹分析显示,COS-7/pEGFP-N1-Olig2实验组COS-7细胞表达Olig2-GFP融合蛋白.提示实验成功构建pEGFP-N1-Olig2真核表达载体,并且在COS-7细胞中得到高效表达.
배경:감성라선-배-라선전록인자소돌효질세포전록인자2재척수적소돌효질세포적분화과정중기착관건성적작용.목적:구건대서소돌효질세포전록인자2기인중조진핵표체재체,관찰기재진핵세포내적표체.방법:종신생대서척수조직중제취총RNA,채용RT-PCR방법획득목적기인편단,극륭지pGEM-T재체중.경측서학증후장소돌효질세포전록인자2 cDNA편단여pEGFP-N1재체정향련접.장감정정학적중조체pEGFP-N1-Olig2이지질체법전염지COS-7세포중,반전록PCR감정소돌효질세포전록인자2 mRNA적표체,면역인적법검측소돌효질세포전록인자2단백질표체수평.결과여결론:극륭료소돌효질세포전록인자2적cDNA,병구건료기진핵표체재체pEGFP-N1-Olig2,경한제성내절매매절감정급측서분석증실료기서렬적정학성;전염72 h시면역인적분석현시,COS-7/pEGFP-N1-Olig2실험조COS-7세포표체Olig2-GFP융합단백.제시실험성공구건pEGFP-N1-Olig2진핵표체재체,병차재COS-7세포중득도고효표체.
BACKGROUND:The basic helix-loop-helix transcription factor oligodendrocytes transcription factor 2 is a key regulator for the differentiation of oligodendrocyte lineage cel s during development. OBJECTIVE:To construct the recombinant eukaryotic expression vector of rat oligodendrocyte transcription factor 2, and to investigate its expression in eukaryocytes. METHODS:Oligodendrocyte transcription factor 2 cDNA fragments were cloned by reverse transcription-PCR with the total RNA from neonatal rat spinal cord as the template, and subsequently cloned into pGEM-T vector. The oligodendrocyte transcription factor 2 fragment with its sequence confirmed was then cloned into pEGFP-N1 vector directional y. The right recombinant was transfected into COS-7 cel s by lipofectamine 2000. The expression of oligodendrocyte transcription factor 2 in COS-7 cel s was detected by reverse transcription-PCR and immunoblot analysis. RESULTS AND CONCLUSION:Oligodendrocyte transcription factor 2 cDNA was cloned, and the correct pEGFP-N1-Olig2 cloning was verified by restriction endonuclease digestion and sequencing. The Western blot analysis indicated that Olig2-GFP fusion protein was expressed in COS-7/pEGFP-N1-Olig2 cel s at 72 hours after transfection. pEGFP-N1-Olig2 was constructed successful y. Olig2-GFP fusion protein could be abundantly expressed in COS-7/pEGFP-N1-Olig2 cel s.