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摘要：HEK293T细胞中的Ago2蛋白中包含多种miRNA（其中包括miR-210）。本课题旨在寻找能与Ago2蛋白中的miR-210结合的mRNA，此即为miR-210的靶序列。通过pIRESneo-FLAG-HA-Ago2质粒使得293T细胞过表达FLAG-HA-Ago2蛋白。将pIRESneo-FLAG-HA-Ago2质粒与miR-210、pIRESneo-FLAG-HA-Ago2质粒与对照RNA分别转染入293T细胞，转染完成后，通过Western blotting法测定细胞中的Ago2含量，通过RT-qPCR法测定细胞中的miR-210含量，证明Ago2和miR-210过表达。然后分离Ago2蛋白，提取其中的RNA进行测序，并利用电脑对测序结果进行分析，从而确定miR-210的靶序列，利于进一步探究miR-210的功能和作用原理。本实验找到一种条件能够在293T细胞中过表达FLAG-HA-Ago2蛋白和miR-210 RNA，并且能够分离提取FLAG-HA-Ago2和miR-210的复合物。今后的工作是研究这种复合物中mRNA的表达，从而鉴定miR-210的靶基因。26203
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毕业论文关键词：miR-210；靶基因；转染；Western blotting；RT-qPCR
Identification and analysis of the target gene of miR-210
Abstract: The Ago2 protein in HEK293T cells contains many kinds of miRNA(including miR-210). The project aims to search for mRNA which can combine with miR-210 in Ago2. That is the target sequence of miR-210. Making 293T cells overexpress FLAG-HA-Ago2 protein through pIRESneo-FLAG-HA-Ago2 plasmid. Transfecting pIRESneo-FLAG-HA-Ago2 plasmid with miR-210 and pIRESneo-FLAG-HA-Ago2 plasmid with control RNA into 293T cells respectively. After transfection completed, determining the content of Ago2 in cells through Western blotting and determining the content of miR-210 in cells through RT-qPCR to prove the overexpression of Ago2 and miR-210. Then isolating Ago2, extracting RNA from it to sequence and analyse the resultant sequence by computer to determine the target sequence of miR-210. That is beneficial to exploring the function and principle of miR-210 further. The experiment has found a condition in which can overexpress FLAG-HA-Ago2 protein and miR-210 RNA in 293T cells.Then isolating and extracting the complex substance of FLAG-HA-Ago2 and miR-210. The further task is to study the expression of mRNA in this complex substance and identify the target gene of miR-210.
Key words: miR-210；target gene；transfection；Western blotting；RT-qPCR
目 录
摘要1
关键词1
Abstract1
Key words1
引言1
1 材料与方法2
1.1 实验材料2
1.1.1 HEK293细胞和HEK293T细胞（生工）2
1.1.2 pIRESneo-FLAG-HA-Ago2质粒2
1.2 实验方法2
1.2.1 细胞传代2
1.2.2 细胞转染2
1.2.3 免疫共沉淀（Co-IP）2
1.2.4 Western blotting3
1.2.5 RNA提取3
1.2.6 RT-qPCR3
1.2.7 RNA测序数据分析3
2 结果与分析3
2.1 HEK293T细胞形态观察4
2.2 Western blotting4
2.3 RT-qPCR扩增cDNA的表达量5
3 讨论5
致谢7
参考文献8
图1 未贴壁时的细胞和贴壁后的细胞的形态4
图2 Western blotting结果图4
图3 RT-qPCR扩增cDNA的表达量5
miR-210的靶基因鉴定与分析
引言：
miRNA是一种长约为18~25nt的内源性小分子非编码RNA，其自身结构中没有开放阅读框，因而无法进行蛋白质的编码。miRNA是由一小段包含近似于发夹状结构、长约70~80nt的单链RNA前体经过Dicer酶的剪切加工作用后形成。在大多数动物体内，miRNA分子能够与目标靶基因mRNA分子的3'-UTR区域互补配对，阻遏mRNA分子的翻译过程或造成其降解，从而对靶基因翻译后的蛋白质表达水平表现为负调控[1]。而miRNA自身与目标靶基因的关系也并非是一一对应的，即某一miRNA分子可以调控多种靶基因的蛋白质表达水平，而某一靶基因的3'-UTR区域也可以与不同的miRNA分子配对结合，由此可以推断某一靶基因的蛋白质表达水平是多种miRNA分子共同发挥调控作用的总和。