本文選題：CITED2　切入點(diǎn)：突變　出處：《重慶醫(yī)科大學(xué)》2012年碩士論文

【摘要】：目的構(gòu)建人轉(zhuǎn)錄輔助因子CITED2突變型(c.573-578de16)及野生型真核表達(dá)質(zhì)粒,將人轉(zhuǎn)錄輔助因子CITED2突變型(c.573-578del6)及野生型真核表達(dá)質(zhì)粒轉(zhuǎn)染至HEK293細(xì)胞,并檢測(cè)兩組重組質(zhì)粒pEGFP-C1-mtCITED2和pEGFP-C1-wtCITED2中CITED2蛋白的表達(dá)情況。 方法分別以健康兒童和CITED2基因雜合突變(c.573-578del6)的先天性心臟病患兒血細(xì)胞DNA為模板,PCR定向克隆擴(kuò)增野生型CITED2和突變型CITED2基因的編碼鏈,分別T/A克隆連接到PMD19-T simple質(zhì)粒上,經(jīng)質(zhì)粒轉(zhuǎn)化、藍(lán)白斑篩選、菌液PCR、質(zhì)粒抽提后酶切及測(cè)序鑒定,篩選出野生型CITED2和突變型CITED2(c.573-578del6)的T載體重組質(zhì)粒。T載體重組質(zhì)粒上的CITED2基因片段分別酶切后膠回收目的基因CITED2,將兩組基因CITED2分別亞克隆入pEGFP-C1,構(gòu)建pEGFP-C1-wtCITED2和pEGFP-C1-mtCITED2真核表達(dá)質(zhì)粒。將空載體pEGFP-C1(2ug)、pEGFP-C1-wtCITED2(2ug)和pEGFP-C1-mtCITED2(2ug)分別轉(zhuǎn)染至HEK293細(xì)胞,轉(zhuǎn)染后24小時(shí),以空載體pEGFP-C1轉(zhuǎn)染的HEK293細(xì)胞組作為陽(yáng)性對(duì)照,未轉(zhuǎn)染的HEK293細(xì)胞組作為陰性對(duì)照,熒光顯微鏡下觀察pEGFP-C1-wtCITED2和pEGFP-C1-mtCITED2真核表達(dá)質(zhì)粒的轉(zhuǎn)染情況；轉(zhuǎn)染后48小時(shí)運(yùn)用流式細(xì)胞儀檢測(cè)其轉(zhuǎn)染效率；Western blotting檢測(cè)兩組重組質(zhì)粒中CITED2蛋白的表達(dá)。 結(jié)果 1.成功構(gòu)建了pMD19-T-wtCITED2和pMD19-T-mtCITED2質(zhì)粒； 2.人野生型pEGFP-C1-wtCITED2和突變型pEGFP-C1-mtCITED2真核表達(dá)質(zhì)粒構(gòu)建成功； 3.將空載體pEGFP-C1、pEGFP-C1-wtCITED2和pEGFP-C1-mtCITED2分別成功轉(zhuǎn)染至HEK293細(xì)胞； 4.培養(yǎng)HEK293細(xì)胞至轉(zhuǎn)染后24小時(shí),在熒光顯微鏡下可觀察到各轉(zhuǎn)染組EGFP的表達(dá),陰性對(duì)照組的HEK293細(xì)胞中未觀察到綠色熒光蛋白的表達(dá)； 5.轉(zhuǎn)染后48小時(shí),以空白組細(xì)胞作為參照,應(yīng)用流式細(xì)胞儀檢測(cè)轉(zhuǎn)染空載體pEGFP-C1、pEGFP-C1-wtCITED2和pEGFP-C1-mtCITED2組細(xì)胞的轉(zhuǎn)染效率為50%-60%;運(yùn)用Western blotting檢測(cè)到CITED2蛋白與EGFP的融合表達(dá)。 結(jié)論 1.成功構(gòu)建了人轉(zhuǎn)錄輔助因子CITED2突變型及野生型CITED2真核表達(dá)質(zhì)粒。 2.空載體pEGFP-C1、pEGFP-C1-wtCITED2和pEGFP-C1-mtCITED2分別成功轉(zhuǎn)染至HEK293細(xì)胞。 3.轉(zhuǎn)染pEGFP-C1-wtCITED2組和pEGFP-C1-mtCITED2組HEK293細(xì)胞分別檢測(cè)到CITED2蛋白的表達(dá)。
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R725.4

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 高燕;黃國(guó)英;;先天性心臟病病因及流行病學(xué)研究進(jìn)展[J];中國(guó)循證兒科雜志;2008年03期

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本文編號(hào)：1671532