Homer1基因真核表达重组质粒构建及HT22细胞转染鉴定

Construction of Homer1 gene eukaryotic expression recombinant plasmid and its identification in HT22 cells

目的构建Homer1基因真核表达重组质粒并在HT22细胞中转染和鉴定。方法提取APP/PS1双转基因小鼠海马组织总RNA,经逆转录、PCR扩增,获取Homer1基因编码序列,1%琼脂糖凝胶电泳分离后,将Homer1基因序列插入p3×FLAG-CMV-10载体的多克隆位点区域,获得p3×FLAG-CMV-10-Homer1基因真核表达重组质粒,双酶切后鉴定重组质粒并进行基因测序。取HT22细胞,随机分为p3×FLAG-CMV-10-Homer1重组质粒组、p3×FLAG-CMV-10空载体组、空白对照组,p3×FLAG-CMV-10-Homer1重组质粒组、p3×FLAG-CMV-10空载体组分别转染p3×FLAG-CMV-10-Homer1重组质粒、p3×FLAG-CMV-10空载体,空白对照组不予转染。采用Western blotting法检测各组转染后Homer1蛋白表达。结果成功构建了p3×FLAG-CMV-10-Homer1基因真核表达重组质粒,经双酶切鉴定和基因测序,该重组质粒序列与目的基因序列一致。p3×FLAG-CMV-10-Homer1重组质粒组Homer1蛋白相对表达量明显高于p3×FLAG-CMV-10空载体组、空白对照组(P均<0.05),而p3×FLAG-CMV-10空载体组与空白对照组Homer1蛋白相对表达量比较P>0.05。结论本研究成功构建了p3×FLAG-CMV-10-Homer1基因真核表达重组质粒,并成功转染HT22细胞,这为研究Homer1蛋白对脑内Aβ清除的影响及在阿尔茨海默病发病机制中的作用奠定了基础。

Objective To construct the eukaryotic expression plasmid of Homer1 gene and its transfection and identification in HT22 cells.Methods We extracted the whole RNA from APP/PS1 mouse hippocampus,and Homer1 gene coding sequence was obtained by reverse transcription.After being isolated and purified though 1%agarose gel electrophoresis,Homer1 gene coding sequence was inserted into the eukaryotic expression vector p3×FLAG-CMV-10 polyclonal site area,then the p3×FLAG-CMV-10-Homer1 eukaryotic expression vector was obtained.The Homer1 recombinant plasmid was identified by restriction enzyme digestion and sequencing.HT22 cells were randomly divided into three groups,and the first two groups were transfected with p3×FLAG-CMV-10-Homer1 recombinant plasmid and p3×FLAG-CMV-10 empty vector;the cells in the blank control group were not transfected.Western blotting was used to detect the expression of Homer1 protein.Results We successfully constructed the p3×FLAG-CMV-10-Homer1 gene eukaryotic expression recombinant plasmid.The sequencing results showed that the recombinant sequence was completely identical to the target gene.The expression of Homer1 protein in the p3×FLAG-CMV-10-Homer1 recombinant plasmid group was significantly higher than that in p3×FLAG-CMV-10 empty vector group and blank control group(P<0.05).Conclusion We constructe the p3×FLAG-CMV-10-Homer1 eukaryotic recombinant plasmid and transfect it into HT22 cells successfully,which lays a foundation for the study of the effect of Homer1 protein on Aβclearance in brain and its role in the pathogenesis of Alzheimer disease.