本文選題：RIP2啟動子 + Sp1結(jié)合位點�。� 參考：《暨南大學》2010年碩士論文

【摘要】： 目的 生物信息學研究發(fā)現(xiàn)人RIP2基因啟動子中含有Spl結(jié)合位點,本研究構(gòu)建了含有Spl結(jié)合位點的人RIP2基因啟動子驅(qū)動的綠色熒光蛋白表達載體和缺失Spl結(jié)合位點的人RIP2基因啟動子驅(qū)動的綠色熒光蛋白表達載體,觀察其在真核細胞中表達情況；并進一步構(gòu)建人RIP2基因啟動子(含有Spl結(jié)合位點或缺失Spl結(jié)合位點)驅(qū)動的EGFP-RIP2綠色熒光融合蛋白表達載體,觀察其在真核細胞中綠色熒光蛋白以及RIP2 mRNA表達情況。探討Spl結(jié)合位點是否在RIP2基因調(diào)控中發(fā)揮作用。 方法 以人基因組DNA為模板,PCR擴增含有Spl結(jié)合位點的2段不同長度的人RIP2基因啟動子序列,以切除CMV啟動子的pEGFP-C2作為框架結(jié)構(gòu),將這2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Spl結(jié)合位點的人RIP2基因啟動子驅(qū)動的綠色熒光蛋白載體pEGFP-C2-RIP2(750bp)wt和pEGFP-C2-RIP2(941bp)wt,將構(gòu)建的重組質(zhì)粒經(jīng)陽離子聚合物JetPeiTM介導瞬時轉(zhuǎn)染HEK293細胞,在倒置熒光顯微鏡下觀察其能否在RIP2基因啟動子的調(diào)控下表達報告基因增強型綠色熒光蛋白(enhanced green fluorescent proteins, EGFP)。用突變試劑盒將重組質(zhì)粒pEGFP-C2-RIP2(750bp)wt中的Spl結(jié)合位點缺失突變,將構(gòu)建的突變重組質(zhì)粒mpEGFP-C2-RIP2(740bp)瞬時轉(zhuǎn)染HEK293細胞,觀察綠色熒光蛋白的表達情況。再以人cDNA為模板,PCR擴增RIP2基因的CDS區(qū)域,定向克隆入表達載體pEGFP-C2的多克隆位點區(qū)域。構(gòu)建RIP2基因啟動子(含有Spl結(jié)合位點或缺失Spl結(jié)合位點)驅(qū)動的EGFP-RIP2綠色熒光融合蛋白表達載體,轉(zhuǎn)染HEK293細胞,觀察綠色熒光蛋白和RIP2基因表達情況。 結(jié)果 pEGFP-C2-RIP2(750bp)wt、pEGFP-C2-RIP2(941bp)wt、mpEGFP-C2-RIP2(740bp)、pEGFP-C2-RIP2(S)、pEGFP-C2-RIP2(750bp+S)wt、mpEGFP-C2-RIP2(740bp+S)經(jīng)酶切鑒定和序列測定均證實重組質(zhì)粒構(gòu)建成功,并且Spl結(jié)合位點突變成功。細胞轉(zhuǎn)染結(jié)果表明,構(gòu)建的重組質(zhì)粒轉(zhuǎn)染HEK293細胞株后,在倒置熒光顯微鏡下均能看到綠色熒光,含有Spl結(jié)合位點的不同長度的人RIP2啟動子片段驅(qū)動的綠色熒光蛋白的表達的強度不相同(P0.05),其中pEGFP-C2-RIP2(750bp)wt重組質(zhì)粒在HEK293細胞中綠色熒光表達明顯強于轉(zhuǎn)染組熒光強度高于pEGFP-C2-RIP2(941bp)wt;因此將pEGFP-C2-RIP2(750bp)wt重組質(zhì)粒中的Sp1結(jié)合位點缺失突變,缺失Sp1結(jié)合位點的RIP2啟動子驅(qū)動的綠色熒光蛋白與含Sp1結(jié)合位點的RIP2啟動子驅(qū)動的綠色熒光蛋白的熒光強度相同(P0.05)；RIP2基因啟動子(含有Sp1結(jié)合位點或缺失Spl結(jié)合位點)驅(qū)動的EGFP-RIP2綠色熒光融合蛋白表達載體轉(zhuǎn)染HEK293細胞后,RT-PCR檢測RIP2 mRNA表達水平無差異(P0.05)。上述熒光和RT-PCR的強弱用灰度值表示,均經(jīng)統(tǒng)計學方法分析,P0.05,差異有統(tǒng)計學意義。 結(jié)論 (1)成功構(gòu)建了含有Sp1結(jié)合位點的不同長度的人RIP2基因啟動子的重組質(zhì)粒和含有Spl結(jié)合位點缺失突變的重組質(zhì)粒； (2)成功構(gòu)建含Sp1結(jié)合位點RIP2基因啟動子和不含Spl結(jié)合位點啟動子驅(qū)動的EGFP-RIP2綠色熒光融合蛋白表達載體； (3)含有Sp1結(jié)合位點的不同長度的人RIP2啟動子片段驅(qū)動的綠色熒光蛋白的表達強度不同,說明含有Sp1結(jié)合位點的不同長度人RIP2啟動子效率不同； (4)Sp1結(jié)合位點缺失突變重組質(zhì)粒在HEK293細胞中綠色熒光表達無明顯變化,且EGFP-RIP2綠色熒光融合蛋白表達載體轉(zhuǎn)染HEK293細胞后RIP2 mRNA表達水平無差異,本研究尚未能證明Sp1結(jié)合位點在RIP2基因表達中發(fā)揮調(diào)節(jié)作用。
[Abstract]:objective
Bioinformatics studies found that the human RIP2 gene promoter contains Spl binding sites. This study constructs a green fluorescent protein expression vector driven by the Spl binding site of human RIP2 gene promoter and a green fluorescent protein expression vector driven by human RIP2 gene promoter with the deletion of the Spl binding site, and to observe its expression in the eukaryotic cells. And further construction of the EGFP-RIP2 green fluorescent fusion protein expression vector, which is driven by the human RIP2 gene promoter (including Spl binding site or missing Spl binding site), to observe the expression of green fluorescent protein and RIP2 mRNA in eukaryotic cells, and to explore whether the Spl binding site plays a role in the regulation of RIP2 gene.
Method
Using the human genome DNA as a template, PCR amplified 2 segments of human RIP2 promoter sequences containing Spl binding sites and removed pEGFP-C2 from CMV promoter as a frame structure. The 2 segments were cut and directed to the expression vector pEGFP-C2 to construct a human RIP2 promoter with Spl binding sites. The green fluorescent protein carrier pEGFP-C2-RIP2 (750bp) WT and pEGFP-C2-RIP2 (941bp) wt were used to transfect HEK293 cells via the cationic polymer JetPeiTM, and could be observed under the inverted fluorescence microscope to express the enhanced green fluorescent protein (enhanced green fluore) under the regulation of the RIP2 promoter. Scent proteins, EGFP). The mutation of the Spl binding site in the recombinant plasmid pEGFP-C2-RIP2 (750bp) wt was mutated by a mutant kit, and the recombinant plasmid mpEGFP-C2-RIP2 (740bp) was transiently transfected to HEK293 cells, and the expression of green fluorescent protein was observed. To express the polyclonal loci region of the carrier pEGFP-C2, construct the EGFP-RIP2 green fluorescent fusion protein expression vector, which is driven by the RIP2 gene promoter (including the Spl binding site or the missing Spl binding site), and transfect the HEK293 cells to observe the expression of green fluorescent protein and RIP2 gene.
Result
PEGFP-C2-RIP2 (750bp) WT, pEGFP-C2-RIP2 (941bp) WT, mpEGFP-C2-RIP2 (740bp), pEGFP-C2-RIP2 (S), pEGFP-C2-RIP2 (750bp+S) proved that the recombinant plasmid was successfully constructed and the binding site mutation was successful. Cell transfection results showed that the recombinant plasmid was transfected into the cell line. After the inverted fluorescence microscope, green fluorescence can be seen, and the intensity of the expression of green fluorescent protein driven by human RIP2 promoter fragment with different lengths of Spl binding site is different (P0.05), and the green fluorescent expression of pEGFP-C2-RIP2 (750bp) wt recombinant plasmid is stronger than that of the transfection group in the transfection group. -C2-RIP2 (941bp) wt; therefore, the Sp1 binding site deletion mutation in the pEGFP-C2-RIP2 (750bp) wt recombinant plasmid, the green fluorescent protein driven by the RIP2 promoter with the deletion of the Sp1 binding site and the fluorescent intensity of the RIP2 promoter driven by the Sp1 binding site are the same as the fluorescence intensity of the green fluorescent protein, which is driven by the RIP2 promoter of Sp1 binding site. After the transfection of EGFP-RIP2 green fluorescent fusion protein expressing vector to HEK293 cells without Spl binding site, there was no difference in the expression level of RIP2 mRNA by RT-PCR (P0.05). The above fluorescence and the strength of RT-PCR were expressed by the gray value, and all were statistically analyzed, and the difference was statistically significant.
conclusion
(1) we successfully constructed recombinant plasmids containing human Sp1 RIP2 promoter with different length of binding sites and recombinant plasmids containing deletion mutation of Spl binding sites.
(2) successfully construct the EGFP-RIP2 green fluorescent fusion protein expression vector driven by Sp1 binding site RIP2 gene promoter and Spl binding site promoter.
(3) the expression intensity of green fluorescent protein (GFP) driven by human RIP2 promoter fragment with different lengths of Sp1 binding site is different, indicating that the efficiency of RIP2 promoter with different lengths containing Sp1 binding sites is different.
(4) there was no obvious change in the expression of green fluorescence in HEK293 cells by the deletion mutation of Sp1 binding site, and there was no difference in the expression level of RIP2 mRNA after transfection of EGFP-RIP2 green fluorescent fusion protein expression vector to HEK293 cells. This study failed to prove that the Sp1 binding site could play a regulatory role in the expression of RIP2 gene.

【學位授予單位】：暨南大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R346

【引證文獻】

相關(guān)博士學位論文 前1條

1 李靜;RIP2基因單核苷酸多態(tài)性及血清水平與系統(tǒng)性紅斑狼瘡的相關(guān)性研究[D];安徽醫(yī)科大學;2012年

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本文編號：1892888