Mir-200家族慢病毒载体构建与表达检测及其靶基因验证

发布时间：2018-05-23 22:32

  本文选题：miRNA + 载体　； 参考：《复旦大学》2013年硕士论文

【摘要】：第一部分 miR-200b/c/429过表达和敲降载体构建及慢病毒包转 目的：通过文献检索和生物信息学分析发现可能调控棕色脂肪发育过程中关键的转录因子PRDM16的miRNA,为了验证niRNA通过PRDM16对于棕色脂肪发育分化的调控,首先构建miRNA的过表达质粒和敲降miRNA表达的质粒。并且通过慢病毒包转验证慢病毒载体的构建。 方法：1.克隆约500bp的包含miR的前体序列的片段,通过酶切、连接、转化和测序等获得重组的miR-200b/c/429过表达质粒。2.根据miR-200b/c/429的成熟序列设计并合成anti-miRNA序列,然后通过克隆的构建anti-miR-200b/c/429的重组质粒。3.通过三质粒体系包装miRNA的过表达的慢病毒和敲降的miRNA的慢病毒。 结果：经过PCR扩增后琼脂糖凝胶电泳验证目的片段大小约为500bp,经过酶切和连接后构建出重组质粒。阳性克隆测序结果显示目的片段正确克隆入载体中。三质粒体系构建的转染包装细胞系后48小时,细胞显示绿色荧光。 结论：本研究成功构建了niR-200b/c/429过表达载体和anti-miR-200b/c/429载体,并且慢病毒包装成功。 第二部分 mir-200b/c/429的表达及其对靶基因的调控 目的：观察miR-200b/c/429在高脂诱导老鼠的皮下白色脂肪和3T3-L1细胞系诱导成熟分化过程中的表达变化。构建包含PRDM16基因3'UTR的双荧光报告载体,通过双荧光素实验验证]miR-200b/c/429对于PRDM16基因表达的调控。通过通过Q-PCR验证慢病毒干扰敲降3T3-L1中的miR-200b/c/429后,其中的miR-200b/c/429的表达,并观察1miR-200b/c/429表达量改变后,在3T3-L1其对PRDM16表达的影响。 方法：1、用Q-PCR的方法观察miR-200b/c/429在高脂肪诱导的C57小鼠的皮下白色脂肪中的表达,以及1niR-200b/c/429在3T3-L1前脂肪细胞系中诱导分化成熟过程中的表达的变化。4、通过构建PRDM16的基因的双荧光报告载体,并与miR-200b/c/429过表达质粒共转染到293FT细胞系中,观察荧光表达的变化。5、通过慢病毒包转后侵染3T3-L1细胞系,经过8天的诱导分化,观察PRDM16基因的表达的变化。 结果：1.成功构建了PRDM16基因的3'UTR,并且通过与miR-200b/c/429的共转染,观察到miR-200b/c下调了荧光素的表达,证明miR-200b/c可以调控PRDM16基因的3'UTR。2.在高脂肪诱导的C57BL/6小鼠的皮下脂肪中,miR-200b/c的表达量显著地下调,在3T3-L1诱导分化成熟过程中]miR-200b/c/429的表达量显著性上调。3.通过慢病毒侵染anti-miR-200b/c/429干扰miR-200b/c/429的表达后,经过诱导分化发现PRDM16的表达量上调。 结论：1.双荧光素酶实验证明miR-200b/c下调PRDM16, PRDM16的表达受到miR-200b/c/429的调控。2. miR-200b/c/429在3T3-L1的诱导分化过程中表达量上调,在高脂肪诱导的老鼠的皮下白色脂肪中miR-200b/c表达量下调。
[Abstract]:Part one Construction of miR-200b/c/429 overexpression and knock down Vector and Lentivirus Envelope Transformation Objective: to find out the possible regulation of PRDM16, a key transcription factor in brown fat development, by literature retrieval and bioinformatics analysis, in order to verify the regulation of niRNA on brown adipose development and differentiation by PRDM16. Firstly, the overexpression plasmid of miRNA and the plasmid of knockdown miRNA expression were constructed. The construction of lentivirus vector was verified by lentivirus envelope transfer. Method 1: 1. The fragment containing the precursor sequence of miR was cloned from about 500bp, and the recombinant miR-200b/c/429 overexpression plasmid. 2 was obtained by enzyme digestion, ligation, transformation and sequencing. According to the mature sequence of miR-200b/c/429, the anti-miRNA sequence was designed and synthesized, and then the recombinant plasmid. 3. 3 of anti-miR-200b/c/429 was cloned and constructed. Three plasmids were used to package lentivirus and lentivirus of miRNA and miRNA. Results: the size of the target fragment was about 500 BP by agarose gel electrophoresis after PCR amplification. The recombinant plasmid was constructed after digestion and ligation. The results of positive cloning and sequencing showed that the target fragment was correctly cloned into the vector. The three plasmids showed green fluorescence at 48 hours after transfection. Conclusion: niR-200b/c/429 overexpression vector and anti-miR-200b/c/429 vector were successfully constructed and lentivirus packaging was successful. Part two Expression of mir-200b/c/429 and its regulation on target gene Aim: to observe the expression of miR-200b/c/429 in hyperlipidemic mice induced by subcutaneous white fat and 3T3-L1 cell line induced maturation. The double fluorescent report vector containing PRDM16 gene 3'UTR was constructed and the regulation of PRDM16 gene expression by miR-200b/c/429 was verified by double fluorescein experiment. The expression of miR-200b/c/429 in lentivirus interference knockdown 3T3-L1 was detected by Q-PCR, and the effect of 3T3-L1 on PRDM16 expression was observed after the change of 1miR-200b/c/429 expression. Methods Q-PCR was used to observe the expression of miR-200b/c/429 in subcutaneous white fat of C57 mice induced by high fat. And the change of 1niR-200b/c/429 expression during differentiation and maturation in 3T3-L1 preadipoid cell line. The double fluorescent report vector of PRDM16 gene was constructed and co-transfected with miR-200b/c/429 overexpression plasmid into 293FT cell line. The changes of fluorescence expression were observed. The changes of PRDM16 gene expression were observed after infection of 3T3-L1 cell line with lentivirus envelope for 8 days. The result is 1: 1. The PRDM16 gene was successfully constructed, and by co-transfection with miR-200b/c/429, it was observed that miR-200b/c down-regulated the expression of fluorescein, which proved that miR-200b/c could regulate 3UTR.2of PRDM16 gene. The expression of miR-200b / c was significantly down-regulated in the subcutaneous fat of C57BL/6 mice induced by high fat, and the expression of miR-200b/c/429 was significantly up-regulated during the differentiation and maturation induced by 3T3-L1. After lentivirus infecting anti-miR-200b/c/429 to interfere with the expression of miR-200b/c/429, it was found that the expression of PRDM16 was up-regulated after induced differentiation. Conclusion 1. Double luciferase assay showed that miR-200b/c down-regulated PRDM16, and the expression of PRDM16 was regulated by miR-200b/c/429. 2.The expression of miR-200b/c/429 was up-regulated during the differentiation induced by 3T3-L1, and the expression of miR-200b/c was down-regulated in the subcutaneous white fat of high-fat induced mice.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R3416
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本文编号：1926634