發(fā)布時間：2018-03-09 19:07

  本文選題：Argonaute　切入點：2　出處：《重慶醫(yī)科大學》2011年碩士論文　論文類型：學位論文

【摘要】：目的: 我們在前期研究中通過sertoli細胞與生精細胞共培養(yǎng)發(fā)現(xiàn)Argonaute 2基因可能與血睪屏障相關基因claudin-11表達有關。為了深入研究相關機制,本課題設計構建Ago2基因miRNA RNAi慢病毒表達載體與pEGFP-C1-Ago2表達載體,為研究其對血睪屏障相關基因claudin-11表達的影響奠定基礎。 方法: 提取小鼠睪丸組織中總RNA,采用RT-PCR的方法擴增Ago2基因編碼區(qū)全長,擴增產物克隆入pEGFP-C1載體EcoRI、Sa1I位點,獲得重組表達質粒,并對重組質粒進行PCR、酶切及測序鑒定。應用LipofectamineTM 2000將重組質粒轉染15P-1細胞,并通過RT-PCR及Western blotting法分別檢測Ago2基因及血睪屏障相關基因claudin-11基因和蛋白表達水平。以Ago2為靶向基因,利用www.rnaidesigner.invitrogen.com/rna1iexpress在線設計網站設計2對Ago2 miR RNAi序列,并將其克隆至pcDNA?6.2-GW/EmGFP-miR載體中,經測序驗證后,用Lipofectamine? 2000瞬時轉染15P-1細胞,通過RT-PCR法檢測Ago2表達情況,選擇干擾效果較強的質�？寺≈罛LOCK-iTTM HiPerformTM Lentiviral PolⅡmiR RNAi Expression System,經過氨芐青霉素和氯霉素篩選后,轉染293FT細胞中包裝慢病毒,轉染15P-1支持細胞,殺稻瘟菌素篩選獲得穩(wěn)定轉染后,通過RT-PCR及Western blotting法分別檢測Ago2及血睪屏障相關基因claudin-11基因和蛋白表達水平。 結果: 1.成功構建了pEGFP-C1-Ago2表達載體,可有效上調15P-1 sertoli細胞Ago2基因表達;成功構建了Ago2基因miRNA RNAi慢病毒表達載體,通過殺稻瘟菌素篩選,獲得15P-1 Ago2-miRNA-RNAi穩(wěn)定株,可顯著下調Ago2基因表達。 2.RT-PCR與western blotting結果顯示,轉染pEGFP-C1-Ago2表達載體上調claudin-11基因表達。相反,轉染Ago2-miRNA RNAi慢病毒下調claudin-11基因表達。 結論: 1.成功構建了Ago2基因pEGFP-C1-Ago2表達載體與miRNA RNAi慢病毒表達載體,為進一步研究Ago2基因功能奠定了基礎。 2.Ago2基因正性調節(jié)claudin-11表達。
[Abstract]:Objective:. In our previous study, we found that the Argonaute 2 gene may be related to the expression of the blood-testis barrier gene claudin-11 through co-culture of sertoli cells and spermatogenic cells. In this study, we designed and constructed miRNA RNAi lentivirus expression vector and pEGFP-C1-Ago2 expression vector of Ago2 gene, which laid a foundation for the study of its effect on claudin-11 expression of blood-testis barrier related gene. Methods:. Total RNAs were extracted from mouse testis. The full length of Ago2 gene coding region was amplified by RT-PCR. The amplified product was cloned into the pEGFP-C1 vector Ecor RIN Sa1I site, and the recombinant expression plasmid was obtained. LipofectamineTM 2000 was used to transfect the recombinant plasmid into 15P-1 cells. The expression levels of Ago2 gene and claudin-11 gene and protein related to blood testis barrier were detected by RT-PCR and Western blotting, respectively. Ago2 was used as target gene. Design 2 pairs of Ago2 miR RNAi sequences using www.rnaidesigner.invitrogen.com/rna1iexpress online design site and clone them into pcDNAs? In 6.2-GW / EmGFP-miR vector, after sequencing, Lipofectamine? After transient transfection of 15P-1 cells in 2000, the expression of Ago2 was detected by RT-PCR method. The plasmid with strong interfering effect was cloned into BLOCK-iTTM HiPerformTM Lentiviral Pol 鈪，

本文編號：1589854