P53結(jié)合位點在NOD8基因調(diào)控中的作用
發(fā)布時間：2018-09-02 09:47
【摘要】： 目的: 構(gòu)建含有P53結(jié)合位點的人N0D8基因啟動子驅(qū)動的綠色熒光蛋白表達載體和含有P53結(jié)合位點缺失突變的人N0D8基因啟動子驅(qū)動的綠色熒光蛋白表達載體,觀察其在真核細(xì)胞表達情況,探討P53結(jié)合位點在N0D8基因調(diào)控中的作用。 方法: 以人基因組DNA為模板,PCR擴增含有P53結(jié)合位點的兩段不同長度的人NOD8基因啟動子序列,以切除啟動子的pEGFP-C2作為框架結(jié)構(gòu),將這兩段序列片段進行酶切并定向克隆入表達載體pEGFP-C2中,構(gòu)建含有P53結(jié)合位點的人NOD8基因啟動子驅(qū)動的綠色熒光蛋白載體pEGFP-C2- NOD8(520bp)wt、pEGFP-C2-NOD8(760bp)wt,將構(gòu)建的重組質(zhì)粒經(jīng)脂質(zhì)體Lipofectamine~(TM)2000介導(dǎo)瞬時轉(zhuǎn)染HEK293細(xì)胞K562細(xì)胞及Hela細(xì)胞,在倒置熒光顯微鏡下觀察其能否在NOD8基因啟動子的調(diào)控下表達報告基因綠色熒光蛋白(green fluorescent proteins,GFP)。用突變試劑盒將重組質(zhì)粒pEGFP-C2-3NOD8(760 bp)wt中的P53結(jié)合位點缺失突變,將構(gòu)建的突變重組質(zhì)粒mpEGFP-C2-NOD8瞬時轉(zhuǎn)染HEK293細(xì)胞,觀察綠色熒光蛋白的表達情況。 結(jié)果: pEGFP-C2-NOD8(760bp)wt和mpEGFP-C2-NOD8經(jīng)酶切鑒定和序列測定證實重組質(zhì)粒構(gòu)建成功,并且P53結(jié)合位點突變成功。細(xì)胞轉(zhuǎn)染結(jié)果表明,構(gòu)建的重組質(zhì)粒轉(zhuǎn)染三種細(xì)胞后,在倒置熒光顯微鏡下均能看到綠色熒光,含有P53結(jié)合位點的不同長度的人NOD8啟動子片斷驅(qū)動的綠色熒光蛋白的表達的強度不相同(P＜0.05),其中重組質(zhì)粒pEGFP-C2-NOD8(760bp)轉(zhuǎn)染組熒光強度高于pEGFP-C2-NOD8(520bp)wt轉(zhuǎn)染組;各重組質(zhì)粒均在HEK293細(xì)胞中綠色熒光表達最強,而在HELA細(xì)胞中很弱。缺失P53結(jié)合位點的NOD8啟動子驅(qū)動的綠色熒光蛋白的熒光強度明顯弱于含P53結(jié)合位點的NOD8啟動子驅(qū)動的綠色熒光蛋白的熒光,上述熒光的強弱用灰度值表示,均經(jīng)統(tǒng)計學(xué)方法分析,P＜0.05,差異有統(tǒng)計學(xué)意義。 結(jié)論: (1)成功構(gòu)建了含有P53結(jié)合位點的不同長度的人NOD8基因啟動子的重組質(zhì)粒和含有P53結(jié)合位點缺失突變的重組質(zhì)粒;(2)含有P53結(jié)合位點的不同長度的人NOD8啟動子片斷驅(qū)動的綠色熒光蛋白的表達的強度不相同;說明在人NOD8基因不同長度的啟動子中包含了不同作用的調(diào)控位點;(3)P53結(jié)合位點突變重組質(zhì)粒在HEK293細(xì)胞中綠色熒光表達明顯減弱,說明P53結(jié)合位點在NOD9基因調(diào)控中發(fā)揮了正調(diào)節(jié)作用;為進一步研究NOD8基因表達及調(diào)控機制奠定了良好的基礎(chǔ)。
[Abstract]:Aim: to construct a green fluorescent protein expression vector driven by human N0D8 gene promoter with p53 binding site and a green fluorescent protein expression vector driven by human N0D8 gene promoter with p53 binding site deletion mutation. To observe the expression of p53 binding site in eukaryotic cells and to explore the role of p53 binding site in the regulation of N0D8 gene. Methods: human genomic DNA was used as template to amplify two sequences of human NOD8 gene promoter containing p53 binding site. The pEGFP-C2 of the excision promoter was used as the frame structure. The two fragments were digested and cloned into the expression vector pEGFP-C2. A green fluorescent protein vector pEGFP-C2- NOD8 (520bp) wt,pEGFP-C2-NOD8 (760bp) wt, with p53 binding site of human NOD8 gene promoter was constructed. The recombinant plasmid was transiently transfected into HEK293 cell line K562 and Hela cells via liposome Lipofectamine~ (TM) 2000 mediated transfection. The expression of reporter gene green fluorescent protein (green fluorescent proteins,GFP) was observed under inverted fluorescence microscope under the regulation of NOD8 promoter. Mutation kit was used to mutate p53 binding site of recombinant plasmid pEGFP-C2-3NOD8 (760 bp) wt). The recombinant plasmid mpEGFP-C2-NOD8 was transiently transfected into HEK293 cells to observe the expression of green fluorescent protein (GFP). Results: pEGFP-C2-NOD8 (760bp) wt and mpEGFP-C2-NOD8 were identified by restriction endonuclease digestion and sequencing. The recombinant plasmid was successfully constructed and p53 binding site mutation was successful. The results of cell transfection showed that green fluorescence could be observed under inverted fluorescence microscope after transfection of the constructed recombinant plasmid into three kinds of cells. The intensity of green fluorescent protein (GFP) driven by human NOD8 promoter fragment with p53 binding site was different (P < 0. 05). The fluorescence intensity of recombinant plasmid pEGFP-C2-NOD8 (760bp) group was higher than that of pEGFP-C2-NOD8 (520bp) wt transfection group. All the recombinant plasmids expressed the strongest green fluorescence in HEK293 cells, but weak in HELA cells. The fluorescence intensity of green fluorescent protein driven by NOD8 promoter without p53 binding site was significantly weaker than that of NOD8 promoter with p53 binding site. The difference was statistically significant (P < 0.05). Conclusion: (1) Recombinant plasmids with different lengths of human NOD8 gene promoter containing p53 binding sites and recombinant plasmids with deletion mutation of p53 binding sites were successfully constructed; (2) different p53 binding sites were found in the recombinant plasmids containing p53 binding sites. The intensity of the expression of green fluorescent protein driven by human NOD8 promoter fragment was different. The results showed that the promoter of human NOD8 gene had different regulatory sites, (3) the expression of green fluorescence in HEK293 cells was significantly decreased. The results suggest that p53 binding site plays a positive role in the regulation of NOD9 gene, which lays a good foundation for further study on the expression and regulation mechanism of NOD8 gene.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R346
本文編號：2218987
[Abstract]:Aim: to construct a green fluorescent protein expression vector driven by human N0D8 gene promoter with p53 binding site and a green fluorescent protein expression vector driven by human N0D8 gene promoter with p53 binding site deletion mutation. To observe the expression of p53 binding site in eukaryotic cells and to explore the role of p53 binding site in the regulation of N0D8 gene. Methods: human genomic DNA was used as template to amplify two sequences of human NOD8 gene promoter containing p53 binding site. The pEGFP-C2 of the excision promoter was used as the frame structure. The two fragments were digested and cloned into the expression vector pEGFP-C2. A green fluorescent protein vector pEGFP-C2- NOD8 (520bp) wt,pEGFP-C2-NOD8 (760bp) wt, with p53 binding site of human NOD8 gene promoter was constructed. The recombinant plasmid was transiently transfected into HEK293 cell line K562 and Hela cells via liposome Lipofectamine~ (TM) 2000 mediated transfection. The expression of reporter gene green fluorescent protein (green fluorescent proteins,GFP) was observed under inverted fluorescence microscope under the regulation of NOD8 promoter. Mutation kit was used to mutate p53 binding site of recombinant plasmid pEGFP-C2-3NOD8 (760 bp) wt). The recombinant plasmid mpEGFP-C2-NOD8 was transiently transfected into HEK293 cells to observe the expression of green fluorescent protein (GFP). Results: pEGFP-C2-NOD8 (760bp) wt and mpEGFP-C2-NOD8 were identified by restriction endonuclease digestion and sequencing. The recombinant plasmid was successfully constructed and p53 binding site mutation was successful. The results of cell transfection showed that green fluorescence could be observed under inverted fluorescence microscope after transfection of the constructed recombinant plasmid into three kinds of cells. The intensity of green fluorescent protein (GFP) driven by human NOD8 promoter fragment with p53 binding site was different (P < 0. 05). The fluorescence intensity of recombinant plasmid pEGFP-C2-NOD8 (760bp) group was higher than that of pEGFP-C2-NOD8 (520bp) wt transfection group. All the recombinant plasmids expressed the strongest green fluorescence in HEK293 cells, but weak in HELA cells. The fluorescence intensity of green fluorescent protein driven by NOD8 promoter without p53 binding site was significantly weaker than that of NOD8 promoter with p53 binding site. The difference was statistically significant (P < 0.05). Conclusion: (1) Recombinant plasmids with different lengths of human NOD8 gene promoter containing p53 binding sites and recombinant plasmids with deletion mutation of p53 binding sites were successfully constructed; (2) different p53 binding sites were found in the recombinant plasmids containing p53 binding sites. The intensity of the expression of green fluorescent protein driven by human NOD8 promoter fragment was different. The results showed that the promoter of human NOD8 gene had different regulatory sites, (3) the expression of green fluorescence in HEK293 cells was significantly decreased. The results suggest that p53 binding site plays a positive role in the regulation of NOD9 gene, which lays a good foundation for further study on the expression and regulation mechanism of NOD8 gene.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R346
【參考文獻】
相關(guān)期刊論文 前1條
1 胡巢鳳;參與免疫及炎癥反應(yīng)調(diào)控的胞漿蛋白NODs[J];中國病理生理雜志;2004年07期
，本文編號：2218987
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