Molecular Cloning and Expression Analysis of Heat Shock Protein 90 (Hsp90) of the Mud Crab, Scylla Paramamosain

摘要

Heat shock protein 90 (HSP90), functions as a molecular chaperone in protein biosynthesis play an important role in signal transduction, immune responses and embryogenesis. However, the function research of HSP90 in invertebrates is limited. To further understand the role and mechanism of HSP90 in immune and stress response, we have isolated the the fragment of HSP90 homologue from mud crab Scylla paramamosain through transcriptome sequencing of mixture of hepatopancreas and hemocytes, The full-length cDNA of HSP90 is harvested through RACE technology and it contains 73 bp 5’terminal UTR, 577 bp 3’terminal UTR and 2373 bp open reading frame which encoding a 790-amino-acid protein. BLASTP results demonstrate that SpHSP90 share high identity with Tribolium castaneum and Locusta migratoria and other reported crustacean (61%-73%). Phylogenetic tree based on HSP90 proteins show that SpHSP90 and Daphnia pulex form a cluster within the invertebrate group while other reported HSP90s of vertebrate are grouped into another branch. The tissue distribution was tested by quantitative real-time PCR and SpHSP90 was detected express in all test tissues, the results showed that HSP90 was constitutively expressed in Scylla paramamosain . The highest expression of HSP90 was found in hepatopancreas, the following was in heart, and the lowest expression of it was found in hemocyte. The expression patterns of SpHSP90 after challenged by the Staphylococcus aureus , Vibrio harveyi and WSSV were also analyzed by qRT-PCR and the result showed that the expression level of SpHSP90 was enhanced after challenged with the Gram-positive bacteria ( Staphylococcus aureus ), Gram-negative bacteria ( Vibro harveyi ) and virus (white spot syndrome virus, WSSV). These results suggested that SpHSP90 involved in the immune response against invading pathogens. Furthermore, SpHSP90 was expressed by prokaryotic expression system and purified by His Bind resin chromatography.