Molecular Cloning and Expression Analysis of Tumor Necrosis Factor Alpha (TNF-α) Gene in Larval Stage of Orange-Spotted Grouper (Epinephelus coioides)

• Main Authors: Wai-San Lam, 林惠珊
• Other Authors: Han-You Lin
• Format: Others
• Language: en_US
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/11031177850078696663

碩士 === 國立成功大學 === 生物科技研究所碩博士班 === 96 === Epinephelus coioides (E. coioides, orange-spotted grouper) is an important farmed fish in Taiwan; there is a paramount problem of raring fish is pathogen infection, especially it occurs at larval stage. Fish larvae were not able to against the pathogen invasion because its immune system was incompletely developed. Innate immune response is the first line to defense pathogens invasion by inducing inflammatory reaction, numerous of mediator and immunocytes are intervening in it, TNF-α is the most important cytokines and is secreted in the earliest inflammation reaction, TNF-α gene was found in some of bony fish; however, the gene of most fish is still unknown. In this study, a TNF-α gene of E. coioides was cloned, the gene was 1516 base pairs (bp) long with 4 exons and 3 introns. The length of open reading frame is 762 bp encoding 253 amino acids with the molecular weight of putative protein was about 27 kDa, the TNF family signature, transmembrane domain, TACE cutting site and two cysteins residues which involved in tertiary structure formation were predicted. Subsequently, the TNF-α mRNA expression level in ontogeny of fish and different organs was evaluated by quantitative reverse transcriptase real time PCR (qRT-PCR). The result shown the expression level of TNF-α mRNA raised after 19 dpf of fries. It indicated that the innate immune system was activated at that stage. The development and functions of immunity on larval fish will be comprehended in this study expectantly, furthermore, designing a proper and efficient strategy to prevent infection of larval stage of fish form the information of this study.