TNF-α、胰岛素和地塞米松对稳定表达人脂联素3T3-L1细胞PPAR-γ2mRNA表达的影响

发布时间：2018-02-25 04:15

  本文关键词： 脂联素 转染 RT-PCR 质粒　出处：《安徽医科大学》2005年硕士论文　论文类型：学位论文

【摘要】：目的:构建重组人脂联素(adiponectin)真核表达质粒,并稳定转染3T3-L1细胞,为进一步研究脂联素(ADPN)的功能提供实验基础。方法:应用限制性内切酶双酶切载有人脂联素cDNA的重组克隆质粒pMD18-T和真核表达质粒pcDNA3.1~+,回收目的基因片段与线性化的真核表达质粒,经连接、转化大肠杆菌JM109,筛选阳性克隆,经酶切和核苷酸序列测定鉴定。脂质体法转染3T3-L1细胞,G418筛选4周,挑选阳性细胞克隆扩增,按Rneasy Mini Kit操作说明从细胞中提取总mRNA,凝胶电泳鉴定总RNA质量,紫外分光光度计法检测总RNA的纯度和浓度。根据报道的人脂联素基因序列,用计算机软件设计特异性引物,以总RNA为模板,通过RT-PCR的方法逆转录合成脂联素基因片段。结果:重组真核表达质粒经HindⅢ、EcoR Ⅰ酶切后获得5.4kb和800bp两个片段,大小与理论值一致。并经核苷酸序列测定证实。总RNA电泳清晰显示28S、18S、5S三条带,28/18S约2:1,A260/A280=1.9,表明提取的总RNA纯度高,其浓度为1μg/ml,RT-PCR产物经凝胶电泳后于紫外分析仪下可见在250 bp前有一清晰条带,和扩增目的基因片段长度234bp一致。结论:成功地构建了人重组脂联素真核表达质粒并在3T3-L1细胞中稳定表达。
[Abstract]:Objective: to construct a recombinant human adiponectin (Hadiponectin) eukaryotic expression plasmid and transfect it stably into 3T3-L1 cells. Methods: the recombinant clone plasmid pMD18-T and eukaryotic expression plasmid pcDNA3.1 ~ were digested with restriction endonuclease (restriction endonuclease) to study the function of adiponectin (ADPN). The target gene fragment was recovered from the linearized eukaryotic expression plasmid and transformed into E. coli JM109 after ligation, positive clones were screened and identified by restriction endonuclease digestion and nucleotide sequencing. 3T3-L1 cells were transfected with liposome for 4 weeks. Positive cells were selected for cloning and amplification, total RNA was extracted from cells according to Rneasy Mini Kit operation, total RNA quality was identified by gel electrophoresis, purity and concentration of total RNA were detected by UV spectrophotometer. According to reported human adiponectin gene sequence, The adiponectin gene fragment was synthesized by reverse transcription with total RNA as template by computer software. Results: the recombinant eukaryotic expression plasmid was digested with Hind 鈪，

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