發(fā)布時間：2018-02-25 04:15

  本文關(guān)鍵詞： 脂聯(lián)素 轉(zhuǎn)染 RT-PCR 質(zhì)粒　出處：《安徽醫(yī)科大學(xué)》2005年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:構(gòu)建重組人脂聯(lián)素(adiponectin)真核表達(dá)質(zhì)粒,并穩(wěn)定轉(zhuǎn)染3T3-L1細(xì)胞,為進(jìn)一步研究脂聯(lián)素(ADPN)的功能提供實(shí)驗(yàn)基礎(chǔ)。方法:應(yīng)用限制性內(nèi)切酶雙酶切載有人脂聯(lián)素cDNA的重組克隆質(zhì)粒pMD18-T和真核表達(dá)質(zhì)粒pcDNA3.1~+,回收目的基因片段與線性化的真核表達(dá)質(zhì)粒,經(jīng)連接、轉(zhuǎn)化大腸桿菌JM109,篩選陽性克隆,經(jīng)酶切和核苷酸序列測定鑒定。脂質(zhì)體法轉(zhuǎn)染3T3-L1細(xì)胞,G418篩選4周,挑選陽性細(xì)胞克隆擴(kuò)增,按Rneasy Mini Kit操作說明從細(xì)胞中提取總mRNA,凝膠電泳鑒定總RNA質(zhì)量,紫外分光光度計(jì)法檢測總RNA的純度和濃度。根據(jù)報(bào)道的人脂聯(lián)素基因序列,用計(jì)算機(jī)軟件設(shè)計(jì)特異性引物,以總RNA為模板,通過RT-PCR的方法逆轉(zhuǎn)錄合成脂聯(lián)素基因片段。結(jié)果:重組真核表達(dá)質(zhì)粒經(jīng)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(jīng)核苷酸序列測定證實(shí)。總RNA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(chǎn)物經(jīng)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(kuò)增目的基因片段長度234bp一致。結(jié)論:成功地構(gòu)建了人重組脂聯(lián)素真核表達(dá)質(zhì)粒并在3T3-L1細(xì)胞中穩(wěn)定表達(dá)。
[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