本文選題：NOD2啟動子 + NF-κB結(jié)合位點　； 參考：《暨南大學(xué)》2008年碩士論文

【摘要】： 目的 構(gòu)建含有NF-κB結(jié)合位點的人NOD2基因啟動子驅(qū)動的綠色熒光蛋白表達(dá)載體和缺失NF-κB結(jié)合位點的人NOD2基因啟動子驅(qū)動的綠色熒光蛋白表達(dá)載體,觀察其在真核細(xì)胞中表達(dá)情況,探討NF-κB結(jié)合位點在NOD2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NF-κB結(jié)合位點的四段不同長度的人NOD2基因啟動子序列,以切除啟動子的pEGFP-N3作為框架結(jié)構(gòu),將這四段序列片段進(jìn)行酶切并定向克隆入表達(dá)載體pEGFP-N3中,構(gòu)建含有NF-κB結(jié)合位點的人NOD2基因啟動子驅(qū)動的綠色熒光蛋白載體pEGFP-N3-NOD2(617bp)wt、pEGFP-N3-NOD2(747 bp)wt、pEGFP-N3-NOD2(1136 bp)wt、pEGFP-N3-NOD2(1387 bp)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、Hela細(xì)胞及ECV304細(xì)胞,在倒置熒光顯微鏡下觀察其能否在NOD2基因啟動子的調(diào)控下表達(dá)報告基因綠色熒光蛋白(greenfluorescent proteins,GFP)。用突變試劑盒將重組質(zhì)粒pEGFP-N3-NOD2(617 bp)wt中的NF-κB結(jié)合位點缺失突變,將構(gòu)建的突變重組質(zhì)粒mpEGFP-N3-NOD2瞬時轉(zhuǎn)染Hela細(xì)胞及HEK293細(xì)胞,觀察綠色熒光蛋白的表達(dá)情況。 結(jié)果 pEGFP-N3-NOD2wt和mpEGFP-N3-NOD2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NF-κB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HEK293、Hela及ECV304細(xì)胞株后,在倒置熒光顯微鏡下均能看到綠色熒光,含有NF-κB結(jié)合位點的不同長度的人NOD2啟動子片段驅(qū)動的綠色熒光蛋白的表達(dá)的強度相同(P＞0.05),其中構(gòu)建的pEGFP-N3-NOD2wt重組質(zhì)粒在Hela及HEK293細(xì)胞中綠色熒光表達(dá)明顯強于突變質(zhì)粒mpEGFP-N3-NOD2的表達(dá)(P＜0.05)。 結(jié)論 (1)成功構(gòu)建了含有NF-κB結(jié)合位點的不同長度的人NOD2基因啟動子的重組質(zhì)粒和含有NF-κB結(jié)合位點缺失突變的重組質(zhì)粒; (2)含有NF-κB結(jié)合位點的不同長度的人NOD2啟動子片段驅(qū)動的綠色熒光蛋白的表達(dá)強度相同,說明含有NF-κB結(jié)合位點的不同長度人NOD2啟動子效率相同; (3)NF-κB結(jié)合位點突變重組質(zhì)粒在Hela細(xì)胞及HEK293細(xì)胞中綠色熒光表達(dá)明顯減弱,說明NF-κB結(jié)合位點在NOD2基因調(diào)控中可能發(fā)揮了正調(diào)節(jié)作用;為進(jìn)一步研究NOD2基因表達(dá)及調(diào)控機制奠定了良好的基礎(chǔ)。
[Abstract]:Purpose The green fluorescent protein expression vector driven by human NOD2 gene promoter with NF- 魏 B binding site and the green fluorescent protein expression vector driven by human NOD2 gene promoter with missing NF- 魏 B binding site were constructed to observe its expression in eukaryotic cells. To investigate the role of NF- 魏 B binding site in the regulation of NOD2 gene. Method Human genomic DNA was used as template to amplify four fragments of human NOD2 gene promoter with NF- 魏 B binding site. The pEGFP-N3 of the excision promoter was used as the frame structure. The four fragments were digested by enzyme and cloned into the expression vector pEGFP-N3. To construct a green fluorescent protein vector pEGFP-N3-NOD2N 617bpwtTN 747bpN3-NOD2N 747 bEGFP-N3-NOD2N 1136 BEGFP-N3-NOD2N 1136 bpwtpEGFP-N3-NOD2N 1387 bpwtand, the constructed recombinant plasmid was transfected into HEK293 cells and ECV304 cells by liposome Lipofectamine~(TM)2000, and the recombinant plasmid pEGFP-N3-NOD2N was transfected into Hela cells of HEK293 cells and ECV304 cells by liposome Lipofectamine~(TM)2000, and the recombinant plasmid pEGFP-N3-NOD2N was transfected into Hela cells and ECV304 cells by liposome Lipofectamine~(TM)2000, and the recombinant plasmid pEGFP-N3-NOD2N was constructed. The expression of the reporter gene green fluorescent protein (GFP) was observed under inverted fluorescence microscope under the regulation of promoter of NOD2 gene. The deletion of NF- 魏 B binding site in recombinant plasmid pEGFP-N3-NOD2(617 bp)wt was detected by mutation kit. The recombinant plasmid mpEGFP-N3-NOD2 was transiently transfected into Hela cells and HEK293 cells to observe the expression of green fluorescent protein (GFP). Result PEGFP-N3-NOD2wt and mpEGFP-N3-NOD2 were identified by restriction endonuclease digestion and sequencing. The recombinant plasmid was successfully constructed and NF- 魏 B binding site mutation was successful. The results of cell transfection showed that the green fluorescence could be observed under inverted fluorescence microscope after transfection of the recombinant plasmid into HEK293 Hela and ECV304 cell lines. The expression intensity of the green fluorescent protein driven by human NOD2 promoter fragment with different length of NF- 魏 B binding site was the same as that of the mutant plasmid mpEGFP-N3-NOD2 (P > 0.05). The green fluorescence expression of the constructed pEGFP-N3-NOD2wt recombinant plasmid was significantly stronger than that of the mutant plasmid mpEGFP-N3-NOD2 in Hela and HEK293 cells (P < 0.05). Conclusion 1) Recombinant plasmids containing different lengths of human NOD2 gene promoter with NF- 魏 B binding sites and recombinant plasmids with deletion mutation of NF- 魏 B binding sites were successfully constructed. (2) the expression intensity of green fluorescent protein driven by human NOD2 promoter fragment with different length of NF- 魏 B binding site was the same, which indicated that the efficiency of human NOD2 promoter with different length of NF- 魏 B binding site was the same. The expression of green fluorescence in Hela cells and HEK293 cells decreased obviously, indicating that NF- 魏 B binding sites may play a positive role in the regulation of NOD2 gene. It lays a good foundation for further study of NOD2 gene expression and regulation mechanism.
【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】：2008
【分類號】：R346

【共引文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 馮珊珊;RIP3誘導(dǎo)腫瘤細(xì)胞凋亡的分子機制研究[D];中國科學(xué)技術(shù)大學(xué);2007年

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本文編號：1894475