Ago2基因miRNA RNAi慢病毒表达载体与pEGFP-C1-Ago2表达载体构建及其对血睾屏障相关基因表达的影响

发布时间：2018-03-09 19:07

  本文选题：Argonaute　切入点：2　出处：《重庆医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的: 我们在前期研究中通过sertoli细胞与生精细胞共培养发现Argonaute 2基因可能与血睾屏障相关基因claudin-11表达有关。为了深入研究相关机制,本课题设计构建Ago2基因miRNA RNAi慢病毒表达载体与pEGFP-C1-Ago2表达载体,为研究其对血睾屏障相关基因claudin-11表达的影响奠定基础。 方法: 提取小鼠睾丸组织中总RNA,采用RT-PCR的方法扩增Ago2基因编码区全长,扩增产物克隆入pEGFP-C1载体EcoRI、Sa1I位点,获得重组表达质粒,并对重组质粒进行PCR、酶切及测序鉴定。应用LipofectamineTM 2000将重组质粒转染15P-1细胞,并通过RT-PCR及Western blotting法分别检测Ago2基因及血睾屏障相关基因claudin-11基因和蛋白表达水平。以Ago2为靶向基因,利用www.rnaidesigner.invitrogen.com/rna1iexpress在线设计网站设计2对Ago2 miR RNAi序列,并将其克隆至pcDNA?6.2-GW/EmGFP-miR载体中,经测序验证后,用Lipofectamine? 2000瞬时转染15P-1细胞,通过RT-PCR法检测Ago2表达情况,选择干扰效果较强的质粒克隆至BLOCK-iTTM HiPerformTM Lentiviral PolⅡmiR RNAi Expression System,经过氨苄青霉素和氯霉素筛选后,转染293FT细胞中包装慢病毒,转染15P-1支持细胞,杀稻瘟菌素筛选获得稳定转染后,通过RT-PCR及Western blotting法分别检测Ago2及血睾屏障相关基因claudin-11基因和蛋白表达水平。 结果: 1.成功构建了pEGFP-C1-Ago2表达载体,可有效上调15P-1 sertoli细胞Ago2基因表达;成功构建了Ago2基因miRNA RNAi慢病毒表达载体,通过杀稻瘟菌素筛选,获得15P-1 Ago2-miRNA-RNAi稳定株,可显著下调Ago2基因表达。 2.RT-PCR与western blotting结果显示,转染pEGFP-C1-Ago2表达载体上调claudin-11基因表达。相反,转染Ago2-miRNA RNAi慢病毒下调claudin-11基因表达。 结论: 1.成功构建了Ago2基因pEGFP-C1-Ago2表达载体与miRNA RNAi慢病毒表达载体,为进一步研究Ago2基因功能奠定了基础。 2.Ago2基因正性调节claudin-11表达。
[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，

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