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| Molecular cloning, genomic organization and functional analysis of an anti-lipopolysaccharide factor from Chinese mitten crab Eriocheir sinensis | |
| Li, Chenghua1,2; Zhao, Jianmin1; Song, Linsheng1; Mu, Changkao1; Zhang, Huan1; Gai, Yunchao1; Qiu, Limei1; Yu, Yundong1; Ni, Duojiao1; Xing, Kezhi3 | |
| 2008 | |
| 发表期刊 | DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY
 |
| ISSN | 0145-305X |
| 卷号 | 32期号:7页码:784-794 |
| 文章类型 | Article |
| 摘要 | Anti-lipopolysaccharide factor (ALF) represents one kind of basic proteins, which binds and neutralizes LPS and exhibits strong antibacterial activity against Gram-negative R-type bacteria. The ALF gene of Chinese mitten crab Eriocheir sinensis (Milne Edwards, 1853) (denoted as EsALF) was identified from haemocytes by expressed sequence tag (EST) and PCR approaches. The full-length cDNA of EsALF consisted of 700 nucleotides with a canonical polyadenylation signal-sequence AATAAA, a polyA tail, and an open-reading frame of 363 bp encoding 120 amino acids. The high similarity of EsALF-deduced amino acid sequence shared with the ALFs from other species indicated that EsALF should be a member of ALF family. The mRNA expression of EsALF in the tissues of heart, gonad, gill, haemocytes, eyestalk and muscle was examined by Northern blot analysis and mRNA transcripts of EsALF were mainly detected in haemocytes, heart and gonad. The temporal expression of EsALF in haemocytes after Vibrio anguillarum challenge was recorded by quantitative real-time RT-PCR. The relative expression level of EsALF was up-regulated rapidly at 2 h post-injection and reached 3-fold to that in blank group. After a drastic decrease to the original level from 4 to 8h, the expression level increased again and reached 4-fold to that in the blank group at 12 h post-injection. The genomic DNA sequence of EsALF gene consists of 1174bp containing three exons and two introns. The coding sequence of the EsALF mature peptide was cloned and expressed in Escherichia coli BL21(DE3)-pLysS to further elucidate its biological functions. The purified recombinant product showed bactericidal activity against both Gram-positive (G(+)) and Gram-negative (G(-)) bacteria, which demonstrated that the rEsALF was a broad-spectrum antibacterial peptide. All these results indicated that EsALF was an acute-phase protein involved in the immune responses of Chinese mitten crab, and provided a potential therapeutic agent for disease control in aquaculture. (c) 2007 Elsevier Ltd. All rights reserved.; Anti-lipopolysaccharide factor (ALF) represents one kind of basic proteins, which binds and neutralizes LPS and exhibits strong antibacterial activity against Gram-negative R-type bacteria. The ALF gene of Chinese mitten crab Eriocheir sinensis (Milne Edwards, 1853) (denoted as EsALF) was identified from haemocytes by expressed sequence tag (EST) and PCR approaches. The full-length cDNA of EsALF consisted of 700 nucleotides with a canonical polyadenylation signal-sequence AATAAA, a polyA tail, and an open-reading frame of 363 bp encoding 120 amino acids. The high similarity of EsALF-deduced amino acid sequence shared with the ALFs from other species indicated that EsALF should be a member of ALF family. The mRNA expression of EsALF in the tissues of heart, gonad, gill, haemocytes, eyestalk and muscle was examined by Northern blot analysis and mRNA transcripts of EsALF were mainly detected in haemocytes, heart and gonad. The temporal expression of EsALF in haemocytes after Vibrio anguillarum challenge was recorded by quantitative real-time RT-PCR. The relative expression level of EsALF was up-regulated rapidly at 2 h post-injection and reached 3-fold to that in blank group. After a drastic decrease to the original level from 4 to 8h, the expression level increased again and reached 4-fold to that in the blank group at 12 h post-injection. The genomic DNA sequence of EsALF gene consists of 1174bp containing three exons and two introns. The coding sequence of the EsALF mature peptide was cloned and expressed in Escherichia coli BL21(DE3)-pLysS to further elucidate its biological functions. The purified recombinant product showed bactericidal activity against both Gram-positive (G(+)) and Gram-negative (G(-)) bacteria, which demonstrated that the rEsALF was a broad-spectrum antibacterial peptide. All these results indicated that EsALF was an acute-phase protein involved in the immune responses of Chinese mitten crab, and provided a potential therapeutic agent for disease control in aquaculture. (c) 2007 Elsevier Ltd. All rights reserved. |
| 关键词 | Eriocheir Sinensis Anti-lipopoly-saccharide Factor Northern Blot Quantitative Real-time Pcr Recombinant Protein Bactericidal Activity |
| 学科领域 | Immunology ; Zoology |
| DOI | 10.1016/j.dci.2007.11.008 |
| URL | 查看原文 |
| 收录类别 | SCI |
| 语种 | 英语 |
| WOS记录号 | WOS:000255152100006 |
| 引用统计 | |
| 文献类型 | 期刊论文 |
| 条目标识符 | http://ir.qdio.ac.cn/handle/337002/5592 |
| 专题 | 实验海洋生物学重点实验室 |
| 作者单位 | 1.Chinese Acad Sci, Inst Oceanol, Qingdao 266071, Peoples R China 2.Chinese Acad Sci, Yantai Inst Coastal Zone Res Sustainable Dev, Yantai 264003, Peoples R China 3.Tianjin Agr Coll, Tianjin 300381, Peoples R China |
| 第一作者单位 | 中国科学院海洋研究所 |
| 推荐引用方式 GB/T 7714 | Li, Chenghua,Zhao, Jianmin,Song, Linsheng,et al. Molecular cloning, genomic organization and functional analysis of an anti-lipopolysaccharide factor from Chinese mitten crab Eriocheir sinensis[J]. DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY,2008,32(7):784-794. |
| APA | Li, Chenghua.,Zhao, Jianmin.,Song, Linsheng.,Mu, Changkao.,Zhang, Huan.,...&Xing, Kezhi.(2008).Molecular cloning, genomic organization and functional analysis of an anti-lipopolysaccharide factor from Chinese mitten crab Eriocheir sinensis.DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY,32(7),784-794. |
| MLA | Li, Chenghua,et al."Molecular cloning, genomic organization and functional analysis of an anti-lipopolysaccharide factor from Chinese mitten crab Eriocheir sinensis".DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY 32.7(2008):784-794. |
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