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Cloning and infection response of tumour-necrosis factor alpha in large yellow croaker Pseudosciaena crocea (Richardson)

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Abstract:

In this article, reverse transcriptase polymerase chain reaction (RT-PCR) and real-time PCR were used to clone the tumour-necrosis factor alpha (TNF-α) gene of the large yellow croaker Pseudosciaena crocea (Richardson) and to analyse its response to bacterial infection. The full-length cDNA of TNF-α contained a 5′-untranslated region (5′-UTR) of 68 bp, followed by an open reading frame (ORF) of 759 bp and a 3′-UTR of 512 bp. The ORF was capable of encoding 252 amino acids with an estimated molecular mass of 28 kDa. In the 3′-UTR, there were two putative endotoxin-responsive motifs and five mRNA instability motifs. The deduced peptide had a transmembrane domain and a TNF-α family signature. Multiple sequence alignments indicated that P. crocea TNF-α shares relatively high similarity with both teleost and mammalian TNF-α. Phylogenetic analysis showed that teleost TNF-α was located independently in a different branch compared with mammalian TNF-α. Real-time PCR analysis demonstrated that the expression of TNF-α in the head kidney of the P. crocea injected with Vibrio parahaemolyticus was most significantly higher than that of the control group at 2 and 4 days. In blood, at 2 days post-injection, TNF-α expression was significantly higher than that of the control group, while at 4 days post-injection, it was most significantly higher than that of the control group.

Keywords: RT-PCR; TNF-α; large yellow croaker; real-time PCR

Document Type: Regular Paper

DOI: http://dx.doi.org/10.1111/j.1095-8649.2008.01945.x

Affiliations: 1: The Key Laboratory of Science and Technology for Aquaculture and Food Safety, Fisheries College, Jimei University, Xiamen 361021 China 2: Molecular Biosciences Research Group, Department of Chemistry & Biochemistry, Texas State University, TX 78666, U.S.A.

Publication date: October 1, 2008

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