Cloning of HSP60 gene from Epinephelus akaara and its express characterization before and after vibrionic stressed

As a major member of heat shock protein families, HSP60 can not only be served as a molecular chaperone to promote protein disaggregation and proper refolding, but also be regarded as a threatened signal under stress to elicit a potent proinflammatory response in cells of the innate immune system. Based on the EST sequences from cDNA library, the full length cDNA of HSP60 from E.akaara was successfully obtained by RACE technique. The full-length of HSP60 is 2351bp, including 75bp of 5'UTR, 539bp of 3'UTR, and a 1737bp open reading frame encoding a protein of 578 amino acids. The alignment result shows that the nuclear acid sequence and amino acids sequence of HSP60 from E. akaara share high identity with other vertebrate homologs. The phylogenetic tree based on the HSP60 amino acid sequences is congruent with their evolutionary relationships, which indicates that HSP60 may be an appropriate marker for the phylogenetic research among interspecies. The E. akaara HSP60 gene contains 9 introns and 10 exons, the same as other teleosteans. The conserved sequence, gene structure and 3D protein structure show that HSP60 may play an important role in life activities. Real-time PCR was also conducted to detect the express characterization of the E. akaara HSP60 gene. The result shows that HSP60 can be detected in various tissues, and after Vibrio harveyi-challenging, the expression of HSP60 in the liver increases dramatically, most of other tissues are also detected increasing to a great extent, which supports that HSP60 maybe play a role in fish innate immune response.