Cloning And Expression Analysis Of Wap65-2 From Miiuy Croaker

Miiuy croaker, Miichthys miiuy, is the lower water carnivorous fish in the warm coastal areas. It is one of the important farmed fish in Zhejiang deep sea-cage. However, as stocking density increases and environment deterioration, a variety of bacterial, parasitic and viral diseases have occurred. The warm temperature acclimation protein (Wap65) in teleost fish shares high structural similarities with mammalian hemopexins and has been found in teleosts as a multifunctional protein, involving in multiple physiological and biochemical processes, such as temperature adaptation, pathogen infection, heavy metala pollution and other stress related to the external environment.The aim of the work is to study the molecular characteristics of miiuy croaker Wap65-2 (mWap65-2) and its potential involvement in immune responses after bacterial infection. We obtained Wap65-2 gene from the cDNA library of liver. Using 5'Rapid Amplification of cDNA ends, we got its full-length cDNA, 1613 nucleotides in length. It contains a large open reading frame (ORF) and predicts to produce a 447-amino-acid protein with a molecular weight of 50.7 kDa. A signal peptide of 19 residues is in the N-terminus. Sequence comparison analysis showed that mWap65-2 was most similar to Wap65-2 from Harpagifer antarcticus, up to 70.4% amino acid identity. Phylogenetic analysis indicated that mWap65-2 grouped tightly with those fish Wap65-2-type sequences. Following Vibrio harveyi infection, liver Wap65-2 mRNA expression change was determined by quantitative real-time PCR (qRT-PCR) method. Wap65-2 transcripts were significantly up-regulated after bacteremia infected, suggesting that Wap65 might be involved in the immune response of miiuy croaker as a kind of acute phase response protein. Besides, the prokaryotic expression plasmid pET22b-Wap65-2 was constructed and the aim protein was induced by IPTG. The SDS-PAGE analysis showed that the aim protein can be highly expressed with an expected size. A polyclonal antiserum raised to the aim protein reacted strongly with itself by Western blot.