Molecular cloning and expression analysis of heat-shock protein 70 in orange-spotted grouper Epinephelus coioides following heat shock and Vibrio alginolyticus challenge

Abstract

In this study, the complementary (c)DNA encoding heat-shock protein 70 (Hsp70) of orange-spotted grouper Epinephelus coioides (OsgHsp70) was cloned. OsgHsp70 was 2206 bp and encoded 652 amino acids with predicted molecular mass of 70·89 kDa and theoretical isoelectric point of 5·48. Three Hsp70 family signatures, bipartite nuclear localization signal sequence (NLS) and cytoplasmic characteristic motif (EEVD) were observed in the OsgHsp70, which shared high similarity in amino-acid sequences with the Hsp70 gene of other vertebrates. The results indicated that the OsgHsp70 is a member of the heat-shock protein 70 family. The Hsp70 messenger (m)RNA expressions were quantified by real-time PCR following heat shock, bacterial infection and immunization with formalin-killed Vibrio alginolyticus, a kind of bacterial pathogen that causes septicaemia. Hsp70 mRNA expression in gill, kidney, spleen, thymus gland, muscle and total-blood samples increased at first and then decreased gradually following heat shock. A similar time-dependent pattern was observed following V. alginolyticus pathogen challenge, in which Hsp70 mRNA expression peaked at 24 h after live bacterial infection and 3 days after dead bacterial vaccination. The results indicated that the Hsp70 gene was inducible and involved in the fish immune response.