【作者】 吕自兰；

【导师】 谢怡；

【作者基本信息】 重庆医科大学 ， 遗传学， 2011， 硕士

【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。更多还原

【Abstract】 Objective:In previous study,we found that Argonaute 2(Ago2) may be involved in the regulation of claudin-11 expression.Then to further investigate the cellular and molecular mechanism by which Ago2 regulate the expression of claudin-11,we constructed the miRNA RNAi vector and the eukaryotic expression vector of Ago2 and investigated the biological role of Ago2 in claudin-11 expression.Methods:To consturct the eukaryotic expression vector of Ago2,the coding region of Ago2 amplified by RT-PCR from mouse testis RNA was cloned into the pEGFP-C1 vector to form the recombinant pEGFP-C1-Ago2 plasmid.Then the recombinant plasmid was transfected into 15P-1 sertoli cells.Additionaly,BLOCK-iT? HiPerform? Lentiviral Pol II miR RNAi Expression System with EmGFP was adopted to construct lentiviral expression vector targeting for mouse Ago2, subsequently, Ago2 RNAi lentiviruses were transfected into 15P-1 sertoli cells. The expression of Ago2 and claudin-11 in 15P-1 sertoli cells were analyzed by RT-PCR and western blotting,respectively.Results:Ago2 coding region was successfully cloned into the eukaryotic vector pEGFP-C1.The recombinant vector was transfected into 15P-1 sertoli cells,results of RT-PCR and Western blotting showed that the expression of Ago2 was significantly up-regulated in 15P-1 sertoli cells. Ago2 RNAi lentiviruses were transfected into 15P-1,and a stable 15P-1 sertoli cell line bearing Ago2-miRNA RNAi lentivirus was successfully constructed by Blasticidin screening. Furthermore, Ago2 silenced induced a significant decrease in claudin-11 expression,which indicated that Ago2 is a positive regulator of claudin-11.Additionally,15P-1 sertoli cell with pEGFP-C1-Ago2 transfected exhibited high level of claudin-11 strengthen the evidence for claudin-11 is regulated by Ago2.Conclusion1. Ago2 RNAi lentivirus is successfully prepared,which could effectively inhibit Ago2 expression in 15P-1 cells.Also,We have constructed a recombinant vector expressing Ago2 and eGFP fusion protein, which is in favour of the further study on the gene function of Ago2.2. Ago2 is a positive regulator of claudin-11 expression.更多还原