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学科主题: Biochemical Research Methods; Chemistry ; Analytical
题名: Application of methyl parathion hydrolase (MPH) as a labeling enzyme
作者: Yang, Wei1, 2; Zhou, Ya-Feng1; Dai, He-Ping3; Bi, Li-Jun4; Zhang, Zhi-Ping1; Zhang, Xiao-Hua3; Leng, Yan1; Zhang, Xian-En1
通讯作者: Zhang, XE, Chinese Acad Sci, Wuhan Inst Virol, State Key Lab Virol, Wuhan 430071, Peoples R China
关键词: methyl parathion hydrolase ; labeling enzyme ; single-chain variable fragment ; fusion protein ; white spot syndrome virus
刊名: ANALYTICAL AND BIOANALYTICAL CHEMISTRY
发表日期: 2008-04-01
DOI: 10.1007/s00216-008-1987-y
卷: 390, 期:8, 页:2133-2140
收录类别: SCI
文章类型: Article
部门归属: [Yang, Wei; Zhou, Ya-Feng; Zhang, Zhi-Ping; Leng, Yan; Zhang, Xian-En] Chinese Acad Sci, Wuhan Inst Virol, State Key Lab Virol, Wuhan 430071, Peoples R China; [Yang, Wei] Chinese Acad Sci, Grad Sch, Beijing 100049, Peoples R China; [Dai, He-Ping; Zhang, Xiao-Hua] Chinese Acad Sci, Inst Hydrobiol, Wuhan 430072, Peoples R China; [Bi, Li-Jun] Chinese Acad Sci, Natl Lab Biomacromol, Inst Biophys, Beijing 100101, Peoples R China
WOS标题词: Science & Technology ; Life Sciences & Biomedicine ; Physical Sciences
类目[WOS]: Biochemical Research Methods ; Chemistry, Analytical
研究领域[WOS]: Biochemistry & Molecular Biology ; Chemistry
摘要: Methyl parathion hydrolase (MPH) is an enzyme that catalyzes the degradation of methyl parathion, generating a yellow product with specific absorption at 405 nm. The application of MPH as a new labeling enzyme was illustrated in this study. The key advantages of using MPH as a labeling enzyme are as follows: (1) unlike alkaline phosphatase (AP), horseradish peroxidase (HRP), and glucose oxidase (GOD), MPH is rarely found in animal cells, and it therefore produces less background noise; (2) its active form in solution is the monomer, with a molecular weight of 37 kDa; (3) its turnover number is 114.70 +/- 13.19 s(-1), which is sufficiently high to yield a significant signal for sensitive detection; and (4) its 3D structure is known and its C-terminal that is exposed to the surface can be easily subjected to the construction of genetic engineering monocloning antibody-enzyme fusion for enzyme-linked immunosorbent assay (ELISA). To demonstrate its utility, MPH was ligated to an single-chain variable fragment (scFv), known as A1E, against a white spot syndrome virus (WSSV) with the insertion of a [-(Gly-Ser)(5)-] linker peptide. The resulting fusion protein MPH-A1E possessed both the binding specificity of the scFv segment and the catalytic activity of the MPH segment. When MPH-A1E was used as an ELISA reagent, 25 ng purified WSSV was detected; this was similar to the detection sensitivity obtained using A1E scFv and the HRP/Anti-E Tag Conjugate protocol. The fusion protein also recognized the WSSV in 1 mu L hemolymph from an infected shrimp and differentiated it from a healthy shrimp.
英文摘要: Methyl parathion hydrolase (MPH) is an enzyme that catalyzes the degradation of methyl parathion, generating a yellow product with specific absorption at 405 nm. The application of MPH as a new labeling enzyme was illustrated in this study. The key advantages of using MPH as a labeling enzyme are as follows: (1) unlike alkaline phosphatase (AP), horseradish peroxidase (HRP), and glucose oxidase (GOD), MPH is rarely found in animal cells, and it therefore produces less background noise; (2) its active form in solution is the monomer, with a molecular weight of 37 kDa; (3) its turnover number is 114.70 +/- 13.19 s(-1), which is sufficiently high to yield a significant signal for sensitive detection; and (4) its 3D structure is known and its C-terminal that is exposed to the surface can be easily subjected to the construction of genetic engineering monocloning antibody-enzyme fusion for enzyme-linked immunosorbent assay (ELISA). To demonstrate its utility, MPH was ligated to an single-chain variable fragment (scFv), known as A1E, against a white spot syndrome virus (WSSV) with the insertion of a [-(Gly-Ser)(5)-] linker peptide. The resulting fusion protein MPH-A1E possessed both the binding specificity of the scFv segment and the catalytic activity of the MPH segment. When MPH-A1E was used as an ELISA reagent, 25 ng purified WSSV was detected; this was similar to the detection sensitivity obtained using A1E scFv and the HRP/Anti-E Tag Conjugate protocol. The fusion protein also recognized the WSSV in 1 mu L hemolymph from an infected shrimp and differentiated it from a healthy shrimp.
关键词[WOS]: SPOT SYNDROME VIRUS ; PSEUDOMONAS SP WBC-3 ; IMMUNOASSAY ; ANTIBODY ; SHRIMP ; ELIMINATION ; CONJUGATION ; PROTEINS ; ANTIGENS ; CLONING
语种: 英语
WOS记录号: WOS:000254755700019
ISSN号: 1618-2642
Citation statistics:
内容类型: 期刊论文
URI标识: http://ir.ihb.ac.cn/handle/152342/8188
Appears in Collections:中科院水生所知识产出(2009年前)_期刊论文

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作者单位: 1.Chinese Acad Sci, Wuhan Inst Virol, State Key Lab Virol, Wuhan 430071, Peoples R China
2.Chinese Acad Sci, Grad Sch, Beijing 100049, Peoples R China
3.Chinese Acad Sci, Inst Hydrobiol, Wuhan 430072, Peoples R China
4.Chinese Acad Sci, Natl Lab Biomacromol, Inst Biophys, Beijing 100101, Peoples R China

Recommended Citation:
Yang, Wei; Zhou, Ya-Feng; Dai, He-Ping; Bi, Li-Jun; Zhang, Zhi-Ping; Zhang, Xiao-Hua; Leng, Yan; Zhang, Xian-En.Application of methyl parathion hydrolase (MPH) as a labeling enzyme,ANALYTICAL AND BIOANALYTICAL CHEMISTRY,2008,390(8):2133-2140
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