Cloning and characterization of orange-spotted grouper (Epinephelus coioides) CD9, and its expression analysis in response to GIV infection
Date Issued
2012
Date
2012
Author(s)
Yang, Zheng-Lin
Abstract
Orange-spotted grouper (Epinephelus coioides) is one of the high valued target species in aquaculture. The fiercest menace to the grouper industry is the breakout of virus disease. GIV (grouper iridovirus) is an icosahedron DNA virus and could cause highly lethal rate from fry to adult stage. In 2011, our laboratory found that the orange-spotted grouper kidney and spleen CD9 gene expression level could increase obviously after GIV intraperitoneal injection. Also, the tendency of CD9 gene expression was in accordance with GIV as we injected poly I:C. But there was no significance increasing phenomenon while we used LPS as artificial stimulator. This research engages in revealing the characteristic of orange-spotted grouper CD9 gene and discusses the interplay between GIV and CD9. CD9 , a surface glycoprotein, belongs to the tetraspanin family. The members of this family appear to form large integrated signaling complexes or tetraspanin-enriched microdomains (TEM) by their associated molecules. Tetraspanins are also known to play roles in pathology of infectious disease such as malara and numerous viral infections. We designed three CD9 gene specific primers to sequence the 5‘ end of orange-spotted grouper CD9 gene. Base on the partial sequence form the orange-spotted kidney cDNA library databank, we obtained the complete sequence and deduced the open reading frame length of orange-spotted grouper CD9 gene to 720 bp. We analyzed the 239 amino acid sequence which spans the plasma membrane four times, producing two extracellular loops, and processes a conserved CCG motif. We compared multiple sequence alignmernt of orange-spotted grouper CD9 to that of other vertebrate, and plotted the phylogenic tree to realize the conservation of orange-spotted grouper CD9 gene. Moreover, we produced the CD9 recombinant protein to generate polyclonal mouse anti-CD9 antibody. Meanwhile, we also tansfected the CD9 expression vectors into Hela and GK cells to explore CD9 subcellular localization. The immunochemistry observation showed that the CD9 fusion protein is mainly expressed on the nucleus and plasma membrane. Finally ,we challenged GK cell with GIV (moi=1) in 12 hrs time course and semi-quantated the RNA expression level of CD9 by RT-PCR. The result gives the sign of the fact that GK could alter the CD9 transcription magnitude according to the GIV infection elapsed time.
Subjects
Tetraspanin
orange-spotted grouper
GIV
SDGs
Type
thesis
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