摘要
目的进一步研究端粒结合蛋白PinX1基因在乳腺癌细胞中对端粒酶活性的调控作用,构建PinX1基因的真核表达载体,转染乳腺癌MDA-MB-231细胞,表达并鉴定其编码蛋白PinX1。方法采用RT-PCR法扩增PinX1基因的编码序列,将其克隆至pM D18-T载体中构建T-PinX1质粒,NOT1和Xhol1双酶切T-PinX1质粒与PUB6/V5-HIS A载体后,连接并构建真核表达载体PUB6/V5-HIS A-PinX1,采用序列分析鉴定阳性质粒转染乳腺癌MDA-MB-231细胞,采用Western blot鉴定PinX1蛋白的表达。结果经酶切、序列分析鉴定证实成功构建PinX1基因的真核表达载体;Western blot检测结果证实,成功完成了该表达载体在乳腺癌MDA-MB-231细胞内的特性外源性表达。结论本研究成功构建了PinX1基因的真核表达载体,并完成了该表达载体在乳腺癌MDAMB-231细胞内的特性外源性表达。为后期进一步研究PinX1基因在乳腺癌发生发展中的作用及PinX1蛋白表达与乳腺癌预后的关系奠定了基础。
Objective To observe the effect of PinX1 gene on the regulation of telomerase activity of breast carcinoma cell line by constructing the full-length PinX1 gene into an eukaryotic expression vector,and to identify the coding protein expression in human breast cancer MDA-MB-231 cell line. Methods The coding sequence of PinX1 was amplified by one step RT-PCR and cloned into p MD18-T vector to construct T-PinX1 plasmid. NOT1 and Xhol1 digested TPinX1 plasmid and the vector of PUB6 / V5-HIS A,then the recombinant eukaryotic expression vector PUB6 / V5-HIS APinX1 was constructed,and transfected into breast cancer MDA-MB-231 cells. The expression of the PinX1 gene in MDAMB-231 cells was identified by Western blot. Results The recombinant eukaryotic expression vector PUB6 / V5-HIS APinX1 was successfully constructed. The expression of PinX1 gene was detected by Western blot. Conclusions The PinX1 gene recombinant eukaryotic expression vector has been successfully constructed and expressed in human breast cancer cells. This work would lay a foundation for further study on the function of PinX1 protein and its interaction with other proteins in breast cancer.
出处
《实用老年医学》
CAS
2015年第12期1000-1003,共4页
Practical Geriatrics
