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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。
Objective: To construct eukaryotic expression vector of human Cuedc2 and verify the expression in vitro. Methods: Total RNA was extracted from human ovarian cancer cells and reverse transcribed into cDNA by RT-PCR. The coding region of Cuedc2 was obtained by PCR and cloned into pcDNA3.1myc-his (-) after purification. Colony PCR and DNA sequencing were identified. Finally, HEK293 cells were transfected with the recombinant eukaryotic expression vector of CUEDC2 by transient transfection. The expression of Cuedc2 protein was detected by western blot after 48 hours. Results: The coding region of Cuedc2 was correctly inserted into the eukaryotic expression vector pcDNA3.1myc-his (-). The expression of Cuedc2 was confirmed by western blot in HEK293 cells, while the negative cells transfected with empty vector showed that pcDNA3 was constructed. 1myc-his (-) - Cuedc2 is efficiently expressed in vitro. CONCLUSION: The present study successfully cloned human Cuedc2 cDNA and constructed a recombinant eukaryotic expression vector. The recombinant eukaryotic expression vector was constructed and expressed in HEK293 cells. This study laid the foundation of further study on the function of human Cuedc2 and its relationship with tumor.