Study On Clone,expression And Impacts Of Immune Related Gene Of Matrix Protein Of Infectious Hematopoietic Necrosis Virus Isolated From Rainbow Trout(Oncorhynchus Mykiss)

In 2016,juvenile rainbow trout from Sichuan province with suspected Infectious Hematopoietic Necrosis Virus(IHNV)were found.The experiment could be judged preliminarily by RT-PCR?cell culture?sequence analysis and artificial infection trial.Base on these results,the gene of Matrix ptrotein was cloned and purified by prokaryotic expression for effects of related immune gene in vitro and vivo.The results are as follows: 1 Isolation and identification of IHNV from rainbow troutIn 2016,an outbreak of acute infectious disease in rainbow trout farm caused 80% mortality with dark skin and proptosis in Sichuan province,Chengdu.RT-PCR was used to detect common viral infectious disease in diseased fish.After blasting search in Genbank,the spleen and kidney tissue suspension was inoculated to the fathead monnow cell line for observing the typical cytopathic effect(CPE).The collected virus suspension was used to inject intraperitoneally into 20 trouts and the mortality was calculated.Then,G protein gene was cloned into T vector for sequencing analysis.The results showed that the PCR was positive.With blast in Genbank,the sequences had a 99% identity to nucleoprotein gene of IHNV.CPE could be found after three blind passages in FHM cells.The animal experiment with injecting intraperitoneally into 20 trouts showed 86.67% mortality in 14 days with similar symptoms of naturally infected fish.A clear amplification band from RT-PCR could be found.The chapter isolate pathogenic agentIHNV successfully from diseased rainbow trout,further showing the agent is IHNV by artificial infection experiment RT-PCR.2 Cloning and expression of matrix protein of IHNVThe gene sequence of matrix protein was cloned with genomic RNA of IHNV used as templat.The sequences of matrix were analyzed by bioinformatics software.The results found that M protein was 585 bp with 195 amino acids and one complete ORF.The predicted protein molecular mass was 21.9 kDa.Besides,domain analysis revealed that M protein contained a conserved domain of Rhabdo_matrix and it did not contain signal peptide cleavage sites and transmembrane regions with higher hydrophobicity.The results of the phylogenetic tree showed that M and IHNV HLJ-09 were isolated from a single branch of JRt genotype.Then,the purified matrix protein was obtained by prokaryotic expression and their specificity was also analyzed.The results revealed the induction temperature is 37?,IPTG concentration is 0.1 mM and the induction time is 4h.Under these conditions,recombinant protein M can be found in sediment with 38 kDa.The results of the western-blotting showed that M had good specificity.3 Study on the immunoregulation of recombinant matrix protein of IHNVIn order to studying the immunoregulation effect of M protein,recombinant protein from last chapter was used to stimulate cultured rainbow trout head kidney monocytes/ macrophages for 4 h,8 h,12 h and 24 h,respectively.The RNA was extracted from each cell at each time point.The expression of MHC II,MHC I,IL-1?,TNF ?,IFN-I,IRF1,CD4 and CD8 was detected by qPCR.The results showed that the recombinant protein could suppress the expression of the eight genes.Besides,the recombinant protein was immunized with healthy and cultured trout for 8 h,1 d,3 d,1 w and 2 w,respectively.The RNA was extracted from the head kidney tissue at each time point,and The expression of MHC II,MHC I,IL-1?,TNF ?,IFN-I,IRF1,CD4 and CD8 was detected by qPCR.The results showed that M protein could also suppress the expression of these genes.