人Cuedc2真核表达载体的构建及其在HEK293细胞中的表达

目的:构建人Cuedc2的真核表达载体,并进行体外验证。方法:提取人卵巢癌细胞总RNA,通过RT-PCR的方法其反转录为cDNA;以之为模板,利用PCR获得Cuedc2的编码区,纯化后克隆入pcDNA3.1myc-his(-),利用菌落PCR及DNA测序进行鉴定。最后,采用瞬时转染的方法,将所构建的重组CUEDC2真核表达载体通过脂质体转染HEK293细胞,48h后通过western blot检测Cuedc2蛋白的表达。结果:Cuedc2编码区cDNA正确地插入真核表达载体pcDNA3.1myc-his(-)中,western blot检测证实其在HEK293细胞中表达,而空载体转染的细胞为阴性,表明所构建的pcDNA3.1myc-his(-)-Cuedc2能够在体外有效表达。结论:本研究成功地克隆了人Cuedc2 cDNA,构建了重组真核表达载体,并在HEK293细胞中有效表达,为进一步研究人Cuedc2的功能及其与肿瘤的关系奠定了实验基础。

Construction of Human Cuedc2 eukaryotic Expression Vector and Expressionof Cuedc2 in HEK293 Cells

Objective： To construct the recombinant expression vector of Cuedc2 and detect its expression in HEK293 cells. Methods： Total RNA was isolated from ovarian cancer cells and RT-PCR was conducted to acquire the cDNA. A 952 bp fragment containing the coding region of Cuedc2 was amplified by PCR and the resulting product was purified. Then this fragment was used as new template and the coding region of Cuedc2 （864 bp）was amplified, purified and inserted into the BamHI and HindlHsites of the pcDNA3.1 myc-his （-）A expression vector. The sequence was confirmed by colony PCR and DNA sequencing. In the end, pcDNA3.1 myc-his（-）A-Cuedc2 was transiently transfected into HEK293 cells and the expression of this new construct was detected by western blot. Results： The full length coding region of Cuedc2 was obtained and confirmed, the expression of Cuedc2 was detected successfully in HEK293 cells. Conclusion： The eukaryotic expression vector of Cuedc2 had been successfully constructed, which would provide a useful tool for designing an in-depth investigation of the role of Cuedc2 in tumorigenesis.