人PNGase片段的克隆表达和多克隆抗体制备.pdfVIP

第33卷 第1期 贵 阳 医 学 院 学 报 VO1．33 No．1 2008年2月 JOURNAL OF GUIYANG MEDICAL COLLEGE 2oo8．2 人PNGase片段的克隆表达和多克隆抗体制备水 赵国杰h ，郝凤进2，王 彪2，张丽君h” (1．北京大学生命科学学院，北京 100871；2．中国医科大学生物化学与分子生物学教研室，辽宁沈阳 110001) [摘 要]目的：利用分子生物学技术克隆、表达、纯化PNGase的N端片段，并制备其多克隆抗体。方法：利 用内切酶从人PNGase全长质粒上切下PNGase N端97个氨基酸的编码序列克隆入pGEX4tl载体，进行原核系 统诱导表达并纯化出目的蛋白，用该蛋白免疫大白兔制备其多克隆抗体，经免疫印记检测。结果：重组的PN— Gase N端片段经测序显示构建成功，制备的抗体可以特异性识别小鼠组织内源性PNG~e。结论：本研究结果 为进一步的研究奠定了基础。 [关键词]分子，克隆；电泳；兔；多肽：PNG~e；寡糖酶；抗体制备 [中图分类号]Q783．2 [文献标识码]A [文章编号]1000-2707(2008)01-0005-05 Cloning，Expression and Polyclone Antibody Preparation of Human PNGase Gene Fragment ZHAO Guojie ，HAO Fengjin 。WANG Biao ，ZHANG Lijun (J．College ofLife Sciencses，Peking Univers ，Bering 100871，China；2．Department ofBiochemistry and Molecular Biology，School ofBasic Medial Sciences，China Medical University，Shenyang 110001，Liaoning，China) [Abstract]Objective：To clone，express，and purify the N terminal fragment of human PNGase with molecular biological techniques，and to prepare polyclone antibodies against it．Methods：The N ter— minal fragm ent of human PNGase was cut from PNGase reconstructive plasmid，inserted into vector pGEX4tl，and expressed in prokaryotic system．The produced protein was purified and its antibody was produced by immunizing rabbit with it．The obtained antibodies were tested by using Western blot． Results：The N terminal fragm ent of human PNGase was cloned，expressed，and purified successfully， and Western blot revealed the antibody prepared could recognize PNGase in mouse tissue specifically． Conclusion：The results of this research provide good basis for further studies． [Key words]cloning，molecular；electrophoresis；rabbits；peptide：N—glycanase；oligase；antibody preparation PNGase被称为多肽：Ⅳ一寡糖酶