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题名: Subtractive phage display technology identifies zebrafish marcksb that is required for gastrulation
作者: Wang, Yan-Wu1, 2; Wei, Chang-Yong2, 3; Dai, He-Ping2; Zhu, Zuo-Yan1, 2; Sun, Yong-Hua2
通讯作者: Zhu, ZY (reprint author), Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan 430072, Peoples R China.
关键词: Zebraflsh ; Phage display ; Single-chain Fv fragment ; Marcks ; Gastrulation movements
刊名: GENE
发表日期: 2013-05-25
DOI: 10.1016/j.gene.2013.03.028
卷: 521, 期:1, 页:69-77
收录类别: SCI
文章类型: Article
部门归属: [Wang, Yan-Wu ; Zhu, Zuo-Yan] Wuhan Univ, Coll Life Sci, Wuhan 430072, Peoples R China ; [Wang, Yan-Wu ; Wei, Chang-Yong ; Dai, He-Ping ; Zhu, Zuo-Yan ; Sun, Yong-Hua] Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan 430072, Peoples R China ; [Wei, Chang-Yong] Univ Chinese Acad Sci, Beijing 100049, Peoples R China
WOS标题词: Science & Technology ; Life Sciences & Biomedicine
资助者: State Key Fundamental Research of China [2010CB126306]; National Natural Science Foundation of China [31222052, 30771100]; Innovation research projection of Institute of Hydrobiology, Chinese Academy of Sciences [220314]
类目[WOS]: Genetics & Heredity
研究领域[WOS]: Genetics & Heredity
摘要: In the present study, we used a phage display technique to screen differentially expressed proteins from zebrafish post-gastrula embryos. With a subtractive screening approach, 6 types of single-chain Fv fragments (scFvs) were screened out from an scFv antibody phage display library by biopanning against zebrafish embryonic homogenate. Four scFv fragments (scFv1, scFv3, scFv4 and scFv6) showed significantly stronger binding to the tailbud embryos than to the 30%-epiboly embryos. A T7 phage display cDNA library was constructed from zebrafish tailbud embryos and used to identify the antigens potentially recognized by scFv1, which showed the highest frequency and strongest binding against the tailbud embryos. We acquired 4 candidate epitopes using scFv1 and the corresponding genes showed significantly higher expression levels at tailbud stage than at 30%-epiboly. The most potent epitope of scFv1 was the clone scFv1-2, which showed strong homology to zebrafish myristoylated alanine-rich C-kinase substrate b (Marcksb). Western blot analysis confirmed the high expression of marcksb in the post-gastrula embryos, and the endogenous expression of Marcksb was interfered by injection of scFv1. Zebrafish marcksb showed dynamic expression patterns during embryonic development. Knockdown of marcksb strongly affected gastrulation movements. Moreover, we revealed that zebrafish marcksh is required for cell membrane protrusion and F-actin alignment. Thus, our study uncovered 4 types of scFvs binding to zebrafish post-gastrula embryos, and the epitope of scFv1 was found to be required for normal gastrulation of zebrafish. To our knowledge, this was the first attempt to combine phage display technique with the embryonic and developmental study of vertebrates, and we were able to identify zebrafish marcksb that was required for gastrulation. (c) 2013 Elsevier B.V. All rights reserved.
英文摘要: In the present study, we used a phage display technique to screen differentially expressed proteins from zebrafish post-gastrula embryos. With a subtractive screening approach, 6 types of single-chain Fv fragments (scFvs) were screened out from an scFv antibody phage display library by biopanning against zebrafish embryonic homogenate. Four scFv fragments (scFv1, scFv3, scFv4 and scFv6) showed significantly stronger binding to the tailbud embryos than to the 30%-epiboly embryos. A T7 phage display cDNA library was constructed from zebrafish tailbud embryos and used to identify the antigens potentially recognized by scFv1, which showed the highest frequency and strongest binding against the tailbud embryos. We acquired 4 candidate epitopes using scFv1 and the corresponding genes showed significantly higher expression levels at tailbud stage than at 30%-epiboly. The most potent epitope of scFv1 was the clone scFv1-2, which showed strong homology to zebrafish myristoylated alanine-rich C-kinase substrate b (Marcksb). Western blot analysis confirmed the high expression of marcksb in the post-gastrula embryos, and the endogenous expression of Marcksb was interfered by injection of scFv1. Zebrafish marcksb showed dynamic expression patterns during embryonic development. Knockdown of marcksb strongly affected gastrulation movements. Moreover, we revealed that zebrafish marcksh is required for cell membrane protrusion and F-actin alignment. Thus, our study uncovered 4 types of scFvs binding to zebrafish post-gastrula embryos, and the epitope of scFv1 was found to be required for normal gastrulation of zebrafish. To our knowledge, this was the first attempt to combine phage display technique with the embryonic and developmental study of vertebrates, and we were able to identify zebrafish marcksb that was required for gastrulation. (c) 2013 Elsevier B.V. All rights reserved.
关键词[WOS]: C-KINASE SUBSTRATE ; DIFFERENTIALLY EXPRESSED GENES ; PEPTIDE LIBRARIES ; ENUCLEATED EGGS ; CELL POLARITY ; PROTEIN ; MOVEMENTS ; ANTIBODY ; CONSTRUCTION ; DOWNSTREAM
语种: 英语
WOS记录号: WOS:000319032500010
ISSN号: 0378-1119
Citation statistics:
内容类型: 期刊论文
URI标识: http://ir.ihb.ac.cn/handle/342005/19307
Appears in Collections:鱼类生物学及渔业生物技术研究中心_期刊论文

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作者单位: 1.Wuhan Univ, Coll Life Sci, Wuhan 430072, Peoples R China
2.Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan 430072, Peoples R China
3.Univ Chinese Acad Sci, Beijing 100049, Peoples R China

Recommended Citation:
Wang, YW; Wei, CY; Dai, HP; Zhu, ZY; Sun, YH.Subtractive phage display technology identifies zebrafish marcksb that is required for gastrulation,GENE,2013,521(1):69-77
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