Molecular cloning, genomic organization and functional analysis of an anti-lipopolysaccharide factor from Chinese mitten crab Eriocheir sinensis

doi:10.1016/j.dci.2007.11.008

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	Molecular cloning, genomic organization and functional analysis of an anti-lipopolysaccharide factor from Chinese mitten crab Eriocheir sinensis
	Li, Chenghua 1,2; Zhao, Jianmin 1; Song, Linsheng 1; Mu, Changkao 1; Zhang, Huan 1; Gai, Yunchao 1; Qiu, Limei 1; Yu, Yundong 1; Ni, Duojiao 1; Xing, Kezhi 3
刊名	DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY
	2008
卷号	32 期号:7 页码:784-794
关键词	Eriocheir Sinensis Anti-lipopoly-saccharide Factor Northern Blot Quantitative Real-time Pcr Recombinant Protein Bactericidal Activity
ISSN号	0145-305X
DOI	10.1016/j.dci.2007.11.008
文献子类	Article
英文摘要	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.
学科主题	Immunology ; Zoology
URL标识	查看原文
语种	英语
WOS记录号	WOS:000255152100006
公开日期	2010-12-24
内容类型	期刊论文
源URL	[http://ir.qdio.ac.cn/handle/337002/5593]
专题	海洋研究所_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
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.