Cloning and expression of tumor necrosis factor (TNF alpha) cDNA from red seabream Pagrus major

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	Cloning and expression of tumor necrosis factor (TNF alpha) cDNA from red seabream Pagrus major
	Cai, ZH ; Song, LS ; Gao, CP ; Wu, LT ; Qiu, LH ; Xiang, JH
	2003-12-01
发表期刊	ACTA BIOCHIMICA ET BIOPHYSICA SINICA
ISSN	0582-9879
卷号	35 期号:12 页码:1111-1116
文章类型	Article
摘要	A fragment of TNFalpha cDNA sequence from red seabream was cloned by homology cloning approach with two degenerated primers which were designed based on the conserved regions of other animals' TNF sequences. The sequence was elongated by 3' and 5' RACE to get the full length CDS sequence. This sequence contained 1264 nucleotides that included a 5' UTR of 85 bp, a 3' UTR of 514 bp and an open reading frame (ORF) of 666 bp which could encode 222 amino acids propeptide. In 3' UTR, there were several mRNA instability motifs and three endotoxin-responsive sequences, but the sequence lacked the polyadenylation signal. The deduced peptide had a clear transmembrane domain, a TNFalpha family signature and a TNF2 family profile. The cell attachment sequence and the glycosaminoglycan attachment sites were also found in the sequence. The red seabream TNF sequence shared relatively high similarity with both mammalian TNFalpha and TNFbeta by multiple sequence alignments. Phylogenetic analysis showed that the piscine TNFalpha were located independently in a different branch compared with mammalian TNFalpha and TNFbeta. Based on the primary and secondary structure analysis and gene expression study, we could concluded that the red seabream TNF should be a TNFalpha, not TNFbeta. RT-PCR was used to study TNFa transcript expression. 24 h after the red seabream was challenged by Vibrio anguillarum, the RS TNFalpha transcript expression were detected in blood, brain, gill, heart, head kidney, kidney, Ever, muscle and spleen. Results showed that TNFalpha mRNA was constitutively expressed in parts of the tissues both in stimulated and unstimulated fish and the expression could be enhanced after the pathogen infection.; A fragment of TNFalpha cDNA sequence from red seabream was cloned by homology cloning approach with two degenerated primers which were designed based on the conserved regions of other animals' TNF sequences. The sequence was elongated by 3' and 5' RACE to get the full length CDS sequence. This sequence contained 1264 nucleotides that included a 5' UTR of 85 bp, a 3' UTR of 514 bp and an open reading frame (ORF) of 666 bp which could encode 222 amino acids propeptide. In 3' UTR, there were several mRNA instability motifs and three endotoxin-responsive sequences, but the sequence lacked the polyadenylation signal. The deduced peptide had a clear transmembrane domain, a TNFalpha family signature and a TNF2 family profile. The cell attachment sequence and the glycosaminoglycan attachment sites were also found in the sequence. The red seabream TNF sequence shared relatively high similarity with both mammalian TNFalpha and TNFbeta by multiple sequence alignments. Phylogenetic analysis showed that the piscine TNFalpha were located independently in a different branch compared with mammalian TNFalpha and TNFbeta. Based on the primary and secondary structure analysis and gene expression study, we could concluded that the red seabream TNF should be a TNFalpha, not TNFbeta. RT-PCR was used to study TNFa transcript expression. 24 h after the red seabream was challenged by Vibrio anguillarum, the RS TNFalpha transcript expression were detected in blood, brain, gill, heart, head kidney, kidney, Ever, muscle and spleen. Results showed that TNFalpha mRNA was constitutively expressed in parts of the tissues both in stimulated and unstimulated fish and the expression could be enhanced after the pathogen infection.
关键词	Red Seabream (Pagrus Major) Tumor Necrosis Factor (Tnf Alpha) Cloning Mrna Expression
学科领域	Biochemistry & Molecular Biology ; Biophysics
收录类别	SCI
语种	英语
WOS记录号	WOS:000187534300010
引用统计	被引频次：16[WOS] [WOS记录] [WOS相关记录]
文献类型	期刊论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/3242
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.Ocean Univ Qingdao, Qingdao 266003, Peoples R China 3.Tianjin Agr Coll, Tianjin 300381, Peoples R China
推荐引用方式 GB/T 7714	Cai, ZH,Song, LS,Gao, CP,et al. Cloning and expression of tumor necrosis factor (TNF alpha) cDNA from red seabream Pagrus major[J]. ACTA BIOCHIMICA ET BIOPHYSICA SINICA,2003,35(12):1111-1116.
APA	Cai, ZH,Song, LS,Gao, CP,Wu, LT,Qiu, LH,&Xiang, JH.(2003).Cloning and expression of tumor necrosis factor (TNF alpha) cDNA from red seabream Pagrus major.ACTA BIOCHIMICA ET BIOPHYSICA SINICA,35(12),1111-1116.
MLA	Cai, ZH,et al."Cloning and expression of tumor necrosis factor (TNF alpha) cDNA from red seabream Pagrus major".ACTA BIOCHIMICA ET BIOPHYSICA SINICA 35.12(2003):1111-1116.

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