【摘要】 目的表达并纯化出包含所有功能基序的端粒酶催化亚基(hTERT)融合蛋白,为深入研究端粒酶作用机制、制备抗hTERT抗体和肽库筛选以端粒酶为靶的小分子抑制剂奠定基础。方法自行设计引物,以pLPC-hTERT为模板扩增出包括端粒酶发挥反转录功能所需的全部8个基序长为1 310 bp的基因片段,将PCR扩增的此片段克隆至带有6个连续组氨酸标签的原核表达载体pET-32a中,IPTG诱导表达后,用SDS-PAGE和Western-Blot(WB)检测确定表达出端粒酶反转录酶功能区融合蛋白,以8 M尿素溶解以包涵体形式存在的目的蛋白,用金属鳌合层析柱纯化融合蛋白并对之进行复性。结果经PCR、酶切、测序、SDS-PAGE和WB鉴定证实成功构建了表达质粒pET32a-hTERT,鉴定和测序结果亦证实包含全部8个基序的基因片段,凝胶电泳和WB确认表达出特异性的目的蛋白条带。结论成功构建pET32a-hTERT表达质粒,实现在大肠杆菌中高效表达,亲和层析纯化获得特异性的hTERT重组蛋白。更多还原

【Abstract】 Objective To construct,express,and purify human telomerase reverse transcriptase(hTERT) functional motifs protein.Methods A 1.3kb foreign DNA insert of telomerase catalytic subunit gene containing all 8 motifs was cloned and amplified by PCR into expression vector of pET-32a.The recombinant plasmid was induced by IPTG for 4 h and produced 59 kD recombinant protein which appeared in the form of inclusion body.This inclusion body was dissolved in 8 mol/L urea and purified by affinity chromatography using Ni-NTA resin under denaturing conditions.Purified hTERT protein was identified by SDS-PAGE and Western-Blot analysis.Results The obtained target gene fragment was 1 310bp.The recombinant pET32a-hTERT was expressed in E.coli at high level as inclusion body,accounting for 18% of the total bacterial proteins.The relative molecular mass of the protein appeared to be 59 kD by SDS-PAGE.Purification of recombinant protein was carried out by Ni-NTA affinity chromatography.The identification of recombinant hTERT was confirmed by SDS-PAGE and Western-Blot analysis.Conclusion Identification results revealed that expression plasmid pET32a-hTERT was constructed successfully and recombinant protein of hTERT functional region was expressed and purified correctly.更多还原