Sp1结合位点在RIP2基因调控中的作用研究

发布时间：2018-05-15 15:25

本文选题：RIP2启动子 + Sp1结合位点　；参考：《暨南大学》2010年硕士论文

【摘要】： 目的生物信息学研究发现人RIP2基因启动子中含有Spl结合位点,本研究构建了含有Spl结合位点的人RIP2基因启动子驱动的绿色荧光蛋白表达载体和缺失Spl结合位点的人RIP2基因启动子驱动的绿色荧光蛋白表达载体,观察其在真核细胞中表达情况；并进一步构建人RIP2基因启动子(含有Spl结合位点或缺失Spl结合位点)驱动的EGFP-RIP2绿色荧光融合蛋白表达载体,观察其在真核细胞中绿色荧光蛋白以及RIP2 mRNA表达情况。探讨Spl结合位点是否在RIP2基因调控中发挥作用。方法以人基因组DNA为模板,PCR扩增含有Spl结合位点的2段不同长度的人RIP2基因启动子序列,以切除CMV启动子的pEGFP-C2作为框架结构,将这2段序列片段进行酶切并定向克隆入表达载体pEGFP-C2中,构建含有Spl结合位点的人RIP2基因启动子驱动的绿色荧光蛋白载体pEGFP-C2-RIP2(750bp)wt和pEGFP-C2-RIP2(941bp)wt,将构建的重组质粒经阳离子聚合物JetPeiTM介导瞬时转染HEK293细胞,在倒置荧光显微镜下观察其能否在RIP2基因启动子的调控下表达报告基因增强型绿色荧光蛋白(enhanced green fluorescent proteins, EGFP)。用突变试剂盒将重组质粒pEGFP-C2-RIP2(750bp)wt中的Spl结合位点缺失突变,将构建的突变重组质粒mpEGFP-C2-RIP2(740bp)瞬时转染HEK293细胞,观察绿色荧光蛋白的表达情况。再以人cDNA为模板,PCR扩增RIP2基因的CDS区域,定向克隆入表达载体pEGFP-C2的多克隆位点区域。构建RIP2基因启动子(含有Spl结合位点或缺失Spl结合位点)驱动的EGFP-RIP2绿色荧光融合蛋白表达载体,转染HEK293细胞,观察绿色荧光蛋白和RIP2基因表达情况。结果 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)经酶切鉴定和序列测定均证实重组质粒构建成功,并且Spl结合位点突变成功。细胞转染结果表明,构建的重组质粒转染HEK293细胞株后,在倒置荧光显微镜下均能看到绿色荧光,含有Spl结合位点的不同长度的人RIP2启动子片段驱动的绿色荧光蛋白的表达的强度不相同(P0.05),其中pEGFP-C2-RIP2(750bp)wt重组质粒在HEK293细胞中绿色荧光表达明显强于转染组荧光强度高于pEGFP-C2-RIP2(941bp)wt;因此将pEGFP-C2-RIP2(750bp)wt重组质粒中的Sp1结合位点缺失突变,缺失Sp1结合位点的RIP2启动子驱动的绿色荧光蛋白与含Sp1结合位点的RIP2启动子驱动的绿色荧光蛋白的荧光强度相同(P0.05)；RIP2基因启动子(含有Sp1结合位点或缺失Spl结合位点)驱动的EGFP-RIP2绿色荧光融合蛋白表达载体转染HEK293细胞后,RT-PCR检测RIP2 mRNA表达水平无差异(P0.05)。上述荧光和RT-PCR的强弱用灰度值表示,均经统计学方法分析,P0.05,差异有统计学意义。结论 (1)成功构建了含有Sp1结合位点的不同长度的人RIP2基因启动子的重组质粒和含有Spl结合位点缺失突变的重组质粒； (2)成功构建含Sp1结合位点RIP2基因启动子和不含Spl结合位点启动子驱动的EGFP-RIP2绿色荧光融合蛋白表达载体； (3)含有Sp1结合位点的不同长度的人RIP2启动子片段驱动的绿色荧光蛋白的表达强度不同,说明含有Sp1结合位点的不同长度人RIP2启动子效率不同； (4)Sp1结合位点缺失突变重组质粒在HEK293细胞中绿色荧光表达无明显变化,且EGFP-RIP2绿色荧光融合蛋白表达载体转染HEK293细胞后RIP2 mRNA表达水平无差异,本研究尚未能证明Sp1结合位点在RIP2基因表达中发挥调节作用。
[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.

【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346

【引证文献】