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目的:构建带His标签的人造血相关PBX相互作用蛋白(HPIP)的原核表达载体,获得His-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,将其插入载体p ET-28a(+)中,经Bam HⅠ和HindⅢ双酶切鉴定后转化大肠杆菌Rossate株进行小量诱导,挑选能诱导出His-HPIP的菌液进行融合蛋白的纯化,采用SDS-PAGE和Western印迹检测融合蛋白的纯化效果,采用GST pull-down技术对蛋白的生物学功能进行初步鉴定。结果:双酶切和测序结果表明His-HPIP原核表达质粒构建成功;His pull-down实验证实His-HPIP蛋白和雌激素受体α存在相互作用,说明生物学活性良好。结论:原核表达并纯化出His-HPIP融合蛋白,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 a prokaryotic expression vector containing His-tagged hematopoietic-related PBX interacting protein (HPIP), obtain the His-HPIP fusion protein, and conduct a preliminary detection of its biological function. Methods: The pcDNA3.0-HPIP plasmid in this laboratory was used as a template to amplify the HPIP coding sequence by PCR and inserted into vector p ET-28a (+). The recombinant plasmid was identified by restriction endonucleases Bam HI and Hind III, Escherichia coli Rossate strain was induced in a small amount. His-HPIP-competent bacteria were selected to purify the fusion protein. SDS-PAGE and Western blotting were used to detect the purification of the fusion protein. The GST pull-down technique was used to analyze the protein biology Function for initial identification. Results: The double digestion and sequencing results showed that the His-HPIP prokaryotic expression plasmid was constructed successfully. The His pull-down assay confirmed the interaction between His-HPIP protein and estrogen receptor α, indicating that the biological activity is good. Conclusion: The prokaryotic expression and purification of His-HPIP fusion protein provide the basis for further study on the function of HPIP in tumorigenesis.