人野生型和突变型CITED2真核表达质粒的构建和表达

发布时间：2018-03-27 12:29

本文选题：CITED2　切入点：突变　出处：《重庆医科大学》2012年硕士论文

【摘要】：目的构建人转录辅助因子CITED2突变型(c.573-578de16)及野生型真核表达质粒,将人转录辅助因子CITED2突变型(c.573-578del6)及野生型真核表达质粒转染至HEK293细胞,并检测两组重组质粒pEGFP-C1-mtCITED2和pEGFP-C1-wtCITED2中CITED2蛋白的表达情况。方法分别以健康儿童和CITED2基因杂合突变(c.573-578del6)的先天性心脏病患儿血细胞DNA为模板,PCR定向克隆扩增野生型CITED2和突变型CITED2基因的编码链,分别T/A克隆连接到PMD19-T simple质粒上,经质粒转化、蓝白斑筛选、菌液PCR、质粒抽提后酶切及测序鉴定,筛选出野生型CITED2和突变型CITED2(c.573-578del6)的T载体重组质粒。T载体重组质粒上的CITED2基因片段分别酶切后胶回收目的基因CITED2,将两组基因CITED2分别亚克隆入pEGFP-C1,构建pEGFP-C1-wtCITED2和pEGFP-C1-mtCITED2真核表达质粒。将空载体pEGFP-C1(2ug)、pEGFP-C1-wtCITED2(2ug)和pEGFP-C1-mtCITED2(2ug)分别转染至HEK293细胞,转染后24小时,以空载体pEGFP-C1转染的HEK293细胞组作为阳性对照,未转染的HEK293细胞组作为阴性对照,荧光显微镜下观察pEGFP-C1-wtCITED2和pEGFP-C1-mtCITED2真核表达质粒的转染情况；转染后48小时运用流式细胞仪检测其转染效率；Western blotting检测两组重组质粒中CITED2蛋白的表达。结果 1.成功构建了pMD19-T-wtCITED2和pMD19-T-mtCITED2质粒； 2.人野生型pEGFP-C1-wtCITED2和突变型pEGFP-C1-mtCITED2真核表达质粒构建成功； 3.将空载体pEGFP-C1、pEGFP-C1-wtCITED2和pEGFP-C1-mtCITED2分别成功转染至HEK293细胞； 4.培养HEK293细胞至转染后24小时,在荧光显微镜下可观察到各转染组EGFP的表达,阴性对照组的HEK293细胞中未观察到绿色荧光蛋白的表达； 5.转染后48小时,以空白组细胞作为参照,应用流式细胞仪检测转染空载体pEGFP-C1、pEGFP-C1-wtCITED2和pEGFP-C1-mtCITED2组细胞的转染效率为50%-60%;运用Western blotting检测到CITED2蛋白与EGFP的融合表达。结论 1.成功构建了人转录辅助因子CITED2突变型及野生型CITED2真核表达质粒。 2.空载体pEGFP-C1、pEGFP-C1-wtCITED2和pEGFP-C1-mtCITED2分别成功转染至HEK293细胞。 3.转染pEGFP-C1-wtCITED2组和pEGFP-C1-mtCITED2组HEK293细胞分别检测到CITED2蛋白的表达。
[Abstract]:Objective to construct the eukaryotic expression plasmids of human transcription cofactor CITED2 mutant c573-578de16 and wild-type eukaryotic expression plasmid, and to transfect the eukaryotic expression plasmid of human transcription cofactor CITED2 mutant and wild-type eukaryotic expression plasmid into HEK293 cells. The expression of CITED2 protein in two groups of recombinant plasmids pEGFP-C1-mtCITED2 and pEGFP-C1-wtCITED2 was detected. Methods the coding chains of wild-type CITED2 and mutant CITED2 gene were amplified by DNA from blood cells of healthy children and children with congenital heart disease (CITED2 gene heterozygous mutation, c.573-578del6), respectively, and ligated to PMD19-T simple plasmid by T / A cloning. After plasmid transformation, blue spot screening, bacterial liquid PCR, plasmids extraction, restriction endonuclease digestion and sequencing, T-vector recombinant plasmids. The CITED2 gene fragments on T vector recombinant plasmids were screened out from wild type CITED2 and mutant CITED2C. 573-578del6). The target gene CITED2 was recovered by restriction endonuclease digestion. The two groups of genes CITED2 were subcloned into pEGFP-C1 to construct the eukaryotic expression plasmids of pEGFP-C1-wtCITED2 and pEGFP-C1-mtCITED2. The empty vectors pEGFP-C1-wtCITED2ugand pEGFP-C1-mtCITED2ug) were transfected into HEK293 cells, respectively. 24 hours after transfection, HEK293 cells transfected with empty vector pEGFP-C1 were used as positive control and HEK293 cells without transfection as negative control. The transfection of pEGFP-C1-wtCITED2 and pEGFP-C1-mtCITED2 eukaryotic expression plasmids was observed under fluorescence microscope. At 48 hours after transfection, the transfection efficiency was detected by flow cytometry and the expression of CITED2 protein in two groups of recombinant plasmids was detected by Western blotting. Results. 1. PMD19-T-wtCITED2 and pMD19-T-mtCITED2 plasmids were constructed successfully. 2.The eukaryotic expression plasmids of human wild type pEGFP-C1-wtCITED2 and mutant type pEGFP-C1-mtCITED2 were successfully constructed. 3.The empty vector pEGFP-C1pEGFP-C1-wtCITED2 and pEGFP-C1-mtCITED2 were successfully transfected into HEK293 cells. 4. The expression of EGFP was observed under fluorescence microscope in HEK293 cells cultured for 24 hours after transfection, but no green fluorescent protein expression was observed in HEK293 cells in negative control group. 5. 48 hours after transfection, the transfection efficiency of pEGFP-C1-wtCITED2 and pEGFP-C1-mtCITED2 cells was 50 ~ 60 and 50 ~ 60% respectively, and the fusion expression of CITED2 protein and EGFP was detected by Western blotting. Conclusion. 1. The eukaryotic expression plasmids of human transcription cofactor CITED2 mutant and wild type CITED2 were successfully constructed. 2. The empty vector pEGFP-C1pEGFP-C1-wtCITED2 and pEGFP-C1-mtCITED2 were successfully transfected into HEK293 cells, respectively. 3. The expression of CITED2 protein was detected in HEK293 cells transfected with pEGFP-C1-wtCITED2 and pEGFP-C1-mtCITED2, respectively.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R725.4

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