PinX1的原核表达、纯化及其与hTERT的相互作用

Prokaryotic Expression and Purification of PinX1 and its Interaction with hTERT

目的:原核表达人Pin2/TRF1结合蛋白X1(PinX1),确定该蛋白与人端粒酶逆转录酶(hTERT)催化亚基的相互作用。方法:用PCR方法从乳腺文库中扩增PinX1基因的编码序列,克隆至pET-32a载体构建重组质粒pHisPinX1,经双酶切鉴定后转化大肠杆菌BL21并进行诱导表达,SDS-PAGE和Western印迹检测His-PinX1的表达;用His磁珠纯化His-PinX1,在人肾胚细胞293T内检测His-PinX1与hTERT的相互作用。结果:扩增得到980 bp的PinX1基因;Western印迹检测表明,相对分子质量约57×103的His-PinX1获得表达,且纯化的His-PinX1与hTERT具有相互作用。结论:表达并纯化得到了与hTERT相互作用的His-PinX1,为深入研究PinX1的功能奠定了基础。

Objective： To express Pin2/TRFl-interacting protein X1 （PinXl） in E.coli, and verify interaction be tween purified His-PinX1 and human telomerase reverse transcriptase（hTERT）. Methods： The coding sequence of PinX1 was amplified by PCR and fused in frame with the coding region of the pET-32a vector to generate pHis PinX1. His-PinX1 was expressed in E.coli BL21 and identified by SDS-PAGE and Western blotting. The interac tion between purified PinX1 and hTERT was detected in 293T cell. Results： The 980 bp PinX1 gene was insert ed into pET-32a vector, and its expression product with relative molecular weight 57x10~ was identified by SDS PAGE and Western blotting. Purified His-PinX1 interacted with hTERT in 293T cell. Conclusion： The recombi- nant PinX1 which can interact with hTERT has been obtained, laying a foundation for further study on the func tion of PinX1.