摘要
[目的]构建小鼠p300-HAT的真核表达质粒。[方法]利用PCR技术扩增p300的HAT域,然后构建p300的HAT域的真核表达质粒(p300-HAT-EGFP),并通过PCR验证和酶切验证重组质粒的正确性;提取不含内毒素的质粒,转染HepG2细胞,通过观测绿色荧光检测转染效率。[结果]PCR产物的电泳结果显示,成功扩增了小鼠p300基因HAT结构域,构建了小鼠p300基因HAT结构域的真核表达质粒,将其命名为m-p300-HAT-pEGFP-C1。重组质粒可以成功表达融合绿色荧光蛋白目的基因蛋白。[结论]p300-HAT-EGFP重组质粒的成功构建为后续开展巨噬细胞NF-kB信号通路的研究奠定了基础。
[ Objective] To construct mice p300-HAT eukaryotie expression plasmid. [ Method ] The study amplifieates, the HAT domain of p300 with PCR, and then constructs the p300 HAT domain of eukaryotic expression plasmid （p300-HAT-EGFP）, and the recombinant plas- mid is validated by enzyme digestion and PCR to verify the validity. Finally, we extracts the recombinant plasmid without endotoxin, and then transfect it to HepG2 cells. We can detect the transfection efficiency by observing the green fluorescence. [ Result ] The SDS-PAGE result showed that mice p300 gene HAT structural domain was amplified successfully, mice p300-HAT eukaryotic expression plasmid was constructed, namely m-p300-HAT-pEGFP-C1. [ Conclusion ] The success of reorganization of the recombinant plasmid （P300-HAT-EGFP） can make great contribution to the subsequent research which is important to understand the mechanism of the NF-kB signaling pathway in macrophages.
出处
《安徽农业科学》
CAS
2013年第27期10914-10916,10962,共4页
Journal of Anhui Agricultural Sciences
基金
合肥工业大学博士学位人员专项资助基金(2011HGB21280)
合肥工业大学青年教师创新项目(2012HGQC0019)
中国博士后科学基金资助项目(2013M541817)
