| Subtractive phage display technology identifies zebrafish marcksb that is required for gastrulation | |
| Wang, Yan-Wu1,2; Wei, Chang-Yong2,3; Dai, He-Ping2; Zhu, Zuo-Yan1,2; Sun, Yong-Hua2; Zhu, ZY (reprint author), Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan 430072, Peoples R China. | |
| 2013-05-25 | |
| Source Publication | GENE
 |
| ISSN | 0378-1119 |
| Volume | 521Issue:1Pages:69-77 |
| Abstract | 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.; 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. |
| Subtype | Article |
| Keyword | Zebraflsh Phage Display Single-chain Fv Fragment Marcks Gastrulation Movements |
| Department | [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 |
| DOI | 10.1016/j.gene.2013.03.028 |
| WOS Headings | Science & Technology ; Life Sciences & Biomedicine |
| Funding Organization | 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] ; 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] ; 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] ; 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] |
| Indexed By | SCI |
| Language | 英语 |
| WOS Research Area | Genetics & Heredity |
| WOS Subject | Genetics & Heredity |
| WOS ID | WOS:000319032500010 |
| WOS Keyword | C-KINASE SUBSTRATE ; DIFFERENTIALLY EXPRESSED GENES ; PEPTIDE LIBRARIES ; ENUCLEATED EGGS ; CELL POLARITY ; PROTEIN ; MOVEMENTS ; ANTIBODY ; CONSTRUCTION ; DOWNSTREAM |
| Funding Organization | 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] ; 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] ; 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] ; 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] |
| Citation statistics | |
| Document Type | 期刊论文 |
| Identifier | http://ir.ihb.ac.cn/handle/342005/19307 |
| Collection | 鱼类生物学及渔业生物技术研究中心_期刊论文 |
| Corresponding Author | Zhu, ZY (reprint author), Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan 430072, Peoples R China. |
| Affiliation | 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 GB/T 7714 | Wang, Yan-Wu,Wei, Chang-Yong,Dai, He-Ping,et al. Subtractive phage display technology identifies zebrafish marcksb that is required for gastrulation[J]. GENE,2013,521(1):69-77. |
| APA | Wang, Yan-Wu,Wei, Chang-Yong,Dai, He-Ping,Zhu, Zuo-Yan,Sun, Yong-Hua,&Zhu, ZY .(2013).Subtractive phage display technology identifies zebrafish marcksb that is required for gastrulation.GENE,521(1),69-77. |
| MLA | Wang, Yan-Wu,et al."Subtractive phage display technology identifies zebrafish marcksb that is required for gastrulation".GENE 521.1(2013):69-77. |
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