Cloning,Expression And Immunomodulatoryeffects Of A Serine Proteinase From Haliotis Discus Hanai

As a traditional Chinese precious seafood,abalone has been loved and sought after by people.Recently,with the rapid economic growth and the improvement of people's living standard,the demand of abalone is increasing.However,sea water pollution,high density breeding and other factors lead to a large outbreak of abalone disease,resulting in huge economic losses.The prevention of abalone diseases is an urgent problem in the abalone aquaculture industry.Vibrio parahaemolyticus has been the main pathogen of abalone culture.The main way to solve the problem is to investigate the innate immune system of abaloneand to explore the function of key genes and proteins involved in innate immune responses.Serine proteinase plays an important role in regulation of immuneological functions.Therefore,the full-length cDNA of serine protease(Hdh-SP)from abalone(Haliotis discus hannai)was cloned using nested-PCR and RACE technique.The full-length cDNAof Hdh-SP gene was 1107 bp including a 5'UTR of 111 bp,a 3'UTR of 39 bp and an open reading frame(ORF)of 957 bp that encoding for 319 amino acid residues with an estimated molecular weight of 34.0 kDa and a theoretical pI of 6.89.The amino acid sequences of SP showed a high degree of identity(54%)to that from Crassostrea gigas and lower degree of identities(50%,47% and 40%)to those from Azumapecten farreri,Sinonovacula constricta and Pinctada fucata.The secondary structure of Hdh-SP was mainly composed of ?-helices(15.72%)and random coil(50.63%).Phylogenetic analysis revealed that Hdh-SP was closely related to a serine proteinase from Crassostrea gigas.Furthermore,prokaryotic expression plasmid pET28a-SP was constructedand transformed into BL21(DE3)cells.The recombinant Hdh-SP(32 kDa)protein,expressed in inclusion bodies,was purified with Ni-NTA Spin column.The recombinant protein,without enzyme activity was used for preparing a specific polyclonal antibody.For purpose of acquiring active Hdh-SP,we used the strategy to increase the yield of soluble recombinant protein by the baculovirus-insect cell expression system.SDS-PAGE and Westernblot analysis indicated that the molecular mass of active recombinant SP in cell supernatantwas about 32 kDa..The expression of Hdh-SP mRNA and protein in abalone hemocytes,hepatopancreas,muscle,gonad,gill and mantle was detected by quantitative real-time PCR and Western blot.The highest expression level of Hdh-SP was found in hepatopancreas,followed by muscle and gonad.After Vibrio parahaemolyticus challenge,the relatively transcript and protein levelsof Hdh-SPin hemocytes,hepatopancreas and muscle tissues increased significantly.Compared to other tissues,the highest expression levels were found in hepatopancreas at 12 h post-infection,while muscle and hemocytes expressed highest level of Hdh-SP at 24 h post-infection.These results indicated that Hdh-SP might be engaged in the immune response of abalone against Vibrio parahaemolyticus infection.Meanwhile,the expression levelsofcysteinyl aspartate specific proteinase 8(Caspase 8),Allograft inflammatory factor(ALIn Fa),macrophage expressed protein(MEP)and Nuclear factor ?B(Rel/NF-?B)were determined.Thefour immune-related genes were significantly upregulated after Vibrio parahaemolyticus infection,indicating that the four immune factors were involved in the immune response of abalone.To further explore the role of Hdh-SP in immune regulation,RNAi was used to silence the Hdh-SP gene.The results showed that the relative expression levels of Rel/NF-?B,MEP,ALInFa and caspase 8 genes decreased in different degrees.The results suggest that Hdh-SP was involved in abalone immune response by regulating the expression of immune factors.Furthermore,the apoptosis of hemocytes was decreased when the expression of Hdh-SP was inhibited,indicating that Hdh-SP participated in cell apoptosis by regulating the expression of caspase 8 gene.In this study,the role of Hdh-SP in abalone immune response was investigated.The results showed that Hdh-SP was significantly upregulated when challenged with Vibrio parahaemolyticus.Hdh-SP was supposed to be involved in immune response of by regulating the expression of Rel/NF-?B,MEP,ALIn Fa and Caspase 8 genes.In this context,this study was helpful to elucidate the mechanism of anti-pathogen immune response of abalone and served as an important pathogen-control reference for its aquaculture.