小鼠GATA4基因真核表达载体的构建及其在P19细胞中的表达

The construction of eukaryotic expression vector of mouse GATA4 and its expression in P19 cells

目的:构建pCDNA3.1A-GATA4真核表达载体,检测其在P19细胞中的表达。方法:由于GATA4基因的全长编码区的GC含量很高,因此本实验采用分段克隆后PCR拼接的方法获得GATA4基因的全长编码序列,连接入pMD19-T载体,经鉴定正确后亚克隆入pCDNA3.1A,构建pCDNA3.1A-GATA4真核表达载体,测序验证无误后,采用脂质体介导法转染至P19细胞,Western blot检测其在P19细胞中的表达。结果:成功扩增小鼠GATA4基因全长编码区,测序证明重组真核表达载体pCDNA3.1A-GATA4构建成功,Westernblot确认目的基因在P19细胞中过表达。结论:pCDNA3.1A-GATA4真核表达载体构建成功,并在P19细胞内过表达,将为研究其在心肌分化、心脏发育中的功能奠定基础。

Objective：To construct the eukaryotic expression vector of pCDNA3.1A-GATA4 and detect its expression in P19 cells. Methods： Because the GC level of the coding sequence of mouse GATA4 was too high, a two step PCR was adopted to clone the fulllength coding regions of GATA4,and then the fight PCR product was inserted into PMD19-T vector. After DNA sequence analysis, GATA4 was subcloned into pCDNA3.1A vector. Finally,a eukaryotic expression vector of pCDNA 3.1A-GATA4 was obtained. The pCDNA3.1A-GATA4 expression vector was transfected into P19 cells by liposome mediation and the expression was determined by Western blot. Results：The coding sequence of mouse GATA4 gene was successfully amplified and the analysis of DNA sequence approved that the recombinant eukaryotic expression vector contained GATA4 eDNA. Western blot showed that P19 cells transfeeted with pCDNA3.1A-GATA4 could overexpress the mouse GATA4. Conclusion ： The pCDNA3. IA-GATA4 expression vector was constructed successfully and the transfected P19 cells could overexpress mouse GATA4 gene,which pave the way for further studies on the function of GATA4 in the differentiation myocardium and development of heart.