發(fā)布時間：2018-02-25 04:15

  本文關鍵詞： 脂聯(lián)素 轉染 RT-PCR 質粒　出處：《安徽醫(yī)科大學》2005年碩士論文　論文類型：學位論文

【摘要】：目的:構建重組人脂聯(lián)素(adiponectin)真核表達質粒,并穩(wěn)定轉染3T3-L1細胞,為進一步研究脂聯(lián)素(ADPN)的功能提供實驗基礎。方法:應用限制性內切酶雙酶切載有人脂聯(lián)素cDNA的重組克隆質粒pMD18-T和真核表達質粒pcDNA3.1~+,回收目的基因片段與線性化的真核表達質粒,經連接、轉化大腸桿菌JM109,篩選陽性克隆,經酶切和核苷酸序列測定鑒定。脂質體法轉染3T3-L1細胞,G418篩選4周,挑選陽性細胞克隆擴增,按Rneasy Mini Kit操作說明從細胞中提取總mRNA,凝膠電泳鑒定總RNA質量,紫外分光光度計法檢測總RNA的純度和濃度。根據報道的人脂聯(lián)素基因序列,用計算機軟件設計特異性引物,以總RNA為模板,通過RT-PCR的方法逆轉錄合成脂聯(lián)素基因片段。結果:重組真核表達質粒經HindⅢ、EcoR Ⅰ酶切后獲得5.4kb和800bp兩個片段,大小與理論值一致。并經核苷酸序列測定證實�？俁NA電泳清晰顯示28S、18S、5S三條帶,28/18S約2:1,A260/A280=1.9,表明提取的總RNA純度高,其濃度為1μg/ml,RT-PCR產物經凝膠電泳后于紫外分析儀下可見在250 bp前有一清晰條帶,和擴增目的基因片段長度234bp一致。結論:成功地構建了人重組脂聯(lián)素真核表達質粒并在3T3-L1細胞中穩(wěn)定表達。
[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，

本文編號：1532968