Research On The Mechanism Of Scavenger Receptor Class B Resisting White Spot Syndrome Virus In Procambarus Clarkii

Procambarus clarkii as an invertebrate,mainly relies on innate immunity to resist the invasion of pathogens,and pattern recognition receptors are the key to invertebrate identification of pathogenic microorganisms to initiate innate immunity.White spot syndrome caused by White Spot Syndrome Virus(WSSV)is one of the most important viral pathogens that endanger the development of the crustacean aquaculture industry.Scavenger receptor B(Scavenger Receptor B,SRB)as a pattern recognition receptor has been reported as a function of resisting pathogens in other invertebrates,but the research on SRB resisting viruses and its molecular mechanism is less.In this study,we used Procambarus clarkii as the research object,cloned the SRB gene,and named it PcSRB.When crayfish were stimulated by WSSV,we found that the expression level of PcSRB in the important tissues of crawfish increased significantly.The RNA interference of PcSRB and the WSSV challenge experiment showed that the survival rate of crayfish in the experimental group was significantly reduced and the content of WSSV in the body was significantly increased,while the phagocytosis of WSSV by hemocytes was reduced.It shows that PcSRB may have the function of resisting viruses in the crayfish.In order to further study the mechanism of PcSRB against viruses,we prokaryotic expression and purification of recombinant protein PcSRB.Through immunoelectron microscopy,it was found that rPcSRB can bind to WSSV.PcSRB is expressed in non-receptive cells,and it is found that PcSRB can bind to WSSV.Pull down experiments showed that PcSRB can recognize WSSV structural proteins VP19,VP26 and VP28,but not VP24.Through intermolecular interaction experiments,it is proved that PcSRB can interact with the lipid molecule Cholesta-3,5-diene(CD)on the surface of WSSV.The hydrophobic domain of PcSRB(amino acids 132-195)was deleted by mutation,and it was found that the mutant protein could not bind WSSV structural protein and lipid molecules,indicating that the hydrophobic domain of PcSRB recognizes the structural protein and lipid molecules of the virus.We also used Chloroquine(CLQ)to inhibit the function of lysosomes and found that the content of WSSV in shrimps increased significantly,indicating that lysosomes are involved in the elimination of WSSV.