Klotho基因真核表达载体的构建及其在TCMK-1细胞中的稳定表达

Construction of eukaryotic expression vector of Klotho gene and its stable expression in TCMK-1 cell line

目的构建小鼠Klotho基因真核表达载体,进而获得稳定表达Klotho分子的小鼠肾上皮细胞株TCMK-1。方法应用PCR方法扩增带有FLAG标签的Klotho编码基因,并克隆至载体pCD-CXIN(CMVMCS-IRES-Neomycin)的相应位点;经酶切及测序鉴定重组质粒,脂质体转染TCMK-1细胞,通过G418筛选得到稳定表达Klotho分子的TCMK-1细胞株;Real-Time PCR检测Klotho mRNA水平,Western blotting检测Klotho蛋白的表达。结果将带有FLAG标签的Klotho基因重组表达载体转染至TCMK-1细胞后,经G418筛选获得TCMK-1细胞株。与对照组相比,Klotho mRNA和蛋白表达均显著升高。结论成功构建了Klotho基因的重组表达载体,并获得了稳定表达Klotho基因的TCMK-1细胞株,为进一步研究Klotho基因和蛋白在小鼠肾上皮细胞中的作用奠定了基础。

Objective To construct the eukaryotic expression vector of the mouse Klotho gene and to acquire mouse kidney epithelial cell line TCMK-1 with stable expresses Klotho. Methods The Klotho-FLAG gene was amplified by PCR and cloned to the corresponding site of eukaryotic expression vector pCD-CXIN （CMV- MCS-IRES-Neomycin）. Once confirmed by enzyme digestion and sequencing, the recombinant pGD-CXIN- Klotho plasmids were transfected to TCMK-1 cells by lipidosome and a cell line that stably expressed Klotho was obtained by G418 screening. The expression of Klotho mRNA was detected by Real-Time PCR, and the expression of Klotho protein was detected by Western blotting. Results The recombinant Klotho gene expression vector with FLAG tag was constructed, and the cell line obtained by transfection and CA18 screening presents higher expression of Klotho mRNA and protein compared with that of control groups. Conclusion The recombinant Klotho expression vector has been successfully constructed and the TCMK-1 cell line with stable expression of Klotho is obtained, which might be constructive for the research on the functions of Klotho protein in mouse kidney epithelial cells.