上海交通大学学报（医学版）

›› 2009, Vol. 29 ›› Issue (10): 1152-.

• 论著（基础研究） • 上一篇    下一篇

人细胞内Ⅰ型血小板激活因子乙酰水解酶重组蛋白的构建、纯化及酶活性研究

陈晓莹1, 徐 静2, 杨军伟3, 张怡轩1   

  1. 1. 沈阳药科大学 生命科学与生物制药学院, 沈阳 110016；2. 上海医药高等专科学校基础部, 上海 201318；3. 中国科学院 上海生命科学研究院植物生理生态研究所, 上海 200032
  • 出版日期:2009-10-25 发布日期:2009-10-26
  • 通讯作者: 张怡轩, 电子信箱: zhangyixuan@syphu.edu.cn。
  • 作者简介:陈晓莹（1984—）, 女, 硕士生；电子信箱: chxychxy@hotmail.com；徐静（1981—）, 女, 助教, 硕士；电子信箱：xujing_x@126.com。
  • 基金资助:

    国家自然科技资源平台项目（2005DKA21203）

Construction, purification and substrate specificity identification of recombinant human platelet-activating factor acetylhydrolase isoformⅠ

CHEN Xiao-ying1, XU Jing2, YANG Jun-wei3, ZHANG Yi-xuan1   

  1. 1. School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang 110016, China；2. Shanghai Institute of Health Sciences, Shanghai 201318, China；3. Institute of Plant Physiology and Ecology, Shanghai Institute for Biological Sciences, Shanghai 200032, China
  • Online:2009-10-25 Published:2009-10-26
  • Supported by:

    National Natural Science and Technology Resource Platform Program, 2005DKA21203

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摘要：

目的 构建人细胞内Ⅰ型血小板激活因子乙酰水解酶(PAF-AH) 重组蛋白并加以纯化，利用不同底物研究酶活性。方法 细胞内Ⅰ型PAF-AH β亚基基因进行PCR扩增，目的基因经纯化、连接载体转化大肠杆菌DH5α感受态细胞构建重组蛋白，提取质粒行琼脂糖凝胶电泳、NdeⅠ和HindⅢ双酶切及测序鉴定。鉴定正确的重组质粒转化大肠杆菌BL21 (DE3) pLysS感受态细胞诱导蛋白表达和纯化，15%SDS-PAGE和Western blotting鉴定。分别以乙酸苯酯、l-O-hexadecyl-2-deoxy-2-thioacetyl-sn-glycero-3-phosphocholine（2-Thio PAF）和1-myristoyl-2-(4-nitrophenylsuccinyl) phosphatidylcholine为底物（后两者为血浆型PAF-AH的商业化底物），应用分光光度法检测纯化后重组蛋白的酶活性，以血浆型PAF-AH作为阳性对照。结果 人细胞内PAF-AHⅠ型β亚基重组蛋白构建正确，纯化后在大肠杆菌感受态细胞中诱导表达。与阳性对照血浆型PAF-AH比较，纯化后的重组蛋白同样能够水解乙酸苯酯，具有芳香酯酶活性；能快速水解2-Thio PAF，但不能水解1-myristoyl-2-(4-nitrophenylsuccinyl) phosphatidylcholine。结论 成功构建人细胞内Ⅰ型PAF-AH重组蛋白，其对血浆型PAF-AH的两个商业化底物具有完全不同的水解能力，以此可快速区分细胞内和血浆型PAF-AH。

关键词: Ⅰ型血小板激活因子乙酰水解酶, 血浆型血小板激活因子乙酰水解酶, 重组蛋白, 纯化, 酶活性, 底物

Abstract:

Objective To construct and purify the recombinant protein of platelet-activating factor acetylhydrolase (PAF-AH) isoformⅠ, and study the enzyme activity by different substrates. Methods The β subunit of PAF-AH isoformⅠwas cloned and expressed in E. coli. Exogenously expressed recombinant protein was purified to SDS-PAGE homogeneity, and its activity was identified by arylesterase detection. Phenylacetate, l-O-hexadecyl-2-deoxy-2-thioacetyl-sn-glycero-3-phosphocholine (2-Thio PAF) and 1-myristoyl-2-(4-nitrophenylsuccinyl) phosphatidylcholine (the latter two were commercial plasma PAF-AH substrates) were used for the substrate identification. The plasma type PAF-AH was served as positive control. Results Recombinant protein of β subunit of PAF-AH isoformⅠwas successfully constructed and expressed in E. coli after purification. Compared with positive control, the recombinant protein could hydrolyze phenylacetate and 2-Thio PAF, but could not hydrolyze 1-myristoyl-2-(4-nitrophenylsuccinyl) phosphatidylcholine. Conclusion Recombinant protein of β subunit of PAF-AH isoformⅠcan be successfully constructed. There are differences in the substrate specification to the two commercial PAF substrates for PAF-AH isoformⅠand plasma type PAF-AH, which provides a quick method to differentiate PAF-AH isoformⅠfrom plasma type PAF-AH.

Key words: platelet-activating factor acetylhydrolase isoformⅠ, plasma platelet-activating factor acetylhydrolase,
recombinant protein, purification, enzyme activity, substrate

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