带myc标签的人造血相关PBX相互作用蛋白真核表达载体的构建及其功能研究

Construction of myc- Tagged Human HPIP Eukaryotic Expression Vector and its Activity Detection

目的:构建带myc标签的造血相关PBX相互作用蛋白(HPIP)的真核表达载体,获得myc-HPIP融合蛋白,并对其生物学功能进行初步检测。方法:以本实验室保存的pcDNA3.0-HPIP质粒为模板,采用PCR技术扩增HPIP编码序列,将其插入pXJ-40-myc载体,通过Western印迹检测表达情况;将重组质粒与空载体分别转染人肝癌HepG2细胞,通过Western印迹检测其对AKT、ERK信号通路中AKT、ERK磷酸化水平的影响。结果:双酶切和测序结果表明myc-HPIP真核表达质粒构建成功,转染HepG2细胞后表达了myc-HPIP融合蛋白;Western印迹证明myc-HPIP可升高AKT、ERK的磷酸化水平。结论:构建了带myc标签的人HPIP真核表达载体,为进一步研究HPIP在肿瘤发生发展中的功能奠定了基础。

Objective： To construct the eukaryotic expression vector of human hematopoietic PBX-interacting protein（HPIP） labeled with myc tag and detect its activity. Methods： The human HPIP gene was obtained from pcDNA3.0-HPIP plasmid by PCR and cloned into the pXJ-40-myc vector. Human HepG2 cells were transfected with the recombinant plasmid myc-HPIP and the expression was detected by Western blot. The phosphorylation levels of AKT and ERK, which were supposed to be upregulated by myc-HPIP, were identified by Western blot in HepG2 cells. Results： HPIP eukaryotic expression vector labeled with myc tag was successfully constructed by dou- ble digestion identification. The inserted fragment was confirmed correct by sequencing. The expression of myc- HPIP in human HepG2 cells was identified by Western blot. In addition, Western blot results showed that myc- HPIP protein could up-regulate the phosphorylation levels of AKT and ERK. Conclusion： The eukaryotic expression vector myc-HPIP was successfully constructed and myc-HPIP was expressed in human HepG2 cells. Moreover, myc-HPIP could up-regulate the phosphorylation level of AKT and ERK, which laid foundation for the further study of the role of HPIP in cancer development and progression.