Study of Heat Shock Cognate 70 in Penaeus monodon- Gene Cloning, Molecular Characterization and Immune Function Assay

• Main Authors: Wan-Yu Lo, 羅婉瑜
• Other Authors: Yen-Ling Song
• Format: Others
• Language: en_US
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/57054852855372743664

博士 === 國立臺灣大學 === 動物學研究所 === 93 === We cloned the cDNA of the heat shock cognate 70 (hsc70) gene of tiger shrimp (Penaeus monodon). It was 2207 bp long and included a 1959 bp coding region, a 40 bp flanking region at the 5’ end and a 208 bp flanking region at the 3’end. The deduced, 652 amino acid sequence had a molecular mass of 71,481 Da and an estimated isoelectric point (pI) of 5.2. Based on phylogenetic analysis, the gene is clustered with the Hsc70 proteins of invertebrates and vertebrates. In native gel electrophoresis, recombinant P. monodon Hsc70 expressed in an E. coli system is tightly associated with S- carboxymethylated α-lactalbumin ( CMLA ), which indicating that Hsc70 probably functions as a chaperone. In an in vitro ATPase assay, recombinant Hsc70 hydrolyzed ATP to ADP and increased hydrolysis activity by binding to unfolded peptide, CMLA. In situ hybridization using an antisense riboprobe revealed that the hsc70 gene was active in most tissues of unstressed shrimp. Expression of hsc70 mRNA in hemocytes increased 2 to 3-fold at the first hour after shrimp experienced heat shock and recovery 0.5 hour. Hsc70 mRNA decreased gradually to the background level. Cloning and characterizing the P. monodon hsc70 gene is the first, crucial step in studying the relationship of heat shock proteins to the stress or immune responses of shrimp. In the immune function study, Hsc70 protein was appeared mainly in granulocytes and colocalized with envelope protein of white spot syndrome virus (WSSV) in the cytoplasmic granules by immune-gold staining. In contrast, no envelope protein was observed in hyalinocytes and only few Hsc70 were found there. A 28kDa protein of WSSV envelope protein was coimmunoprecipitated with Hsc70 in hemocyte lysates of infected shrimp indicates a specific relationship between the Hsc70 and WSSV envelope protein. Expression of Hsc70 was enhanced in hemocytes drawn from infected shrimp. Highest expression of Hsc70 was found at 15 hrs post infection then dramatically decreased to the level even lower than the constitutively expressed at 36 and 48 hrs post infection. The caspase-3 like proteases activity in hemocytes drawn from infected shrimp increased significantly and the highest at 48 hrs post infection. The data suggested the apoptosis in hemocytes of infected shrimp was caspase dependent. The lowest expression of Hsc70 accompanied by the highest caspase-3 like proteases activity, during the infectious course, implicates the potential antiapoptotic activity of Hsc70. However, the effect was not noted in subcuticular epithelium and gill of the WSSV infected shrimp. Conceivably, the Hsc70 of shrimp may play an important role in the regulation of antiapoptosis function in the WSSV infected shrim