贵州医科大学学报

2008, No.130(01) 5-9

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人PNGase片段的克隆表达和多克隆抗体制备
Cloning, Expression and Polyclone Antibody Preparation of Human PNGase Gene Fragment

赵国杰;郝凤进;王彪;张丽君;
ZHAO Guojie1, HAO Fengjin2, WANG Biao2, ZHANG Lijun1(1.College of Life Sciencses, Peking University, Beijing 100871, China; 2.Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, China Medical University, Shenyang 110001, Liaoning, China)

摘要(Abstract):

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PNGaseN端97个氨基酸的编码序列克隆入pGEX4t1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-GaseN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PNGase。结论:本研究结果为进一步的研究奠定了基础。
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 terminal fragment of human PNGase was cut from PNGase reconstructive plasmid, inserted into vector pGEX4t1, 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 fragment 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.

关键词(KeyWords): 分子,克隆;电泳;兔;多肽:PNGase;寡糖酶;抗体制备
cloning, molecular; electrophoresis; rabbits; peptide:N-glycanase; oligase; antibody preparation

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基金项目(Foundation): 国家自然科学基金资助(No30570900)

作者(Author): 赵国杰;郝凤进;王彪;张丽君;
ZHAO Guojie1, HAO Fengjin2, WANG Biao2, ZHANG Lijun1(1.College of Life Sciencses, Peking University, Beijing 100871, China; 2.Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, China Medical University, Shenyang 110001, Liaoning, China)

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DOI: 10.19367/j.cnki.1000-2707.2008.01.004

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