Study On The Immune Function Of Extracellular Domain Of Down Syndrome Cell Adhesion Molecule In Chinese Mitten Crab(Eriocheir Sinensis)

Chinese mitten crab(Eriocheir sinensis),also known as river crab or crab,has become the main freshwater economic breeding crab for fishermen to increase production and income in China because of its large scale and high output value.However,with the increasing scale of aquaculture,in addition to germplasm degradation,it also faces a huge risk of frequent diseases.Therefore,increasing the research on the immune defense mechanism of Chinese mitten crab can not only provide theoretical guidance for the disease prevention and control of the crab,but also enrich and expand the theory of crustacean immunology and promote the development of green and healthy breeding.E.sinensis,as an invertebrate,is different from vertebrates in immune mode.It lacks specific immunity and relies only on innate immunity.As a star gene in Chinese mitten crab and even arthropods,Dscam is famous for its high molecular diversity.It plays an important role in the nervous system regulation and immune defense of Chinese mitten crab.It is involved in various immune defense pathways such as cell signal transduction,cell phagocytosis,and antimicrobial peptide production.Based on the results of previous studies,this study further explored the extracellular region of Dscam,focusing on the functions of the non-variable part(Dscam-Ig8-FNIII6)and the variable part(Dscam-Ig1-Ig7)in the immune defense of E.sinensis.The related functions of Dscam extracellular non-variable region and variable region were verified by recombinant protein technology,bacterial binding,bacteriostasis and phagocytosis experiments.The results showed that Dscam extracellular non-variable region could bind to bacteria and had direct bactericidal function and could eliminate bacteria in hemolymph by promoting phagocytosis of blood cells,thus helping to prolong the survival time of crabs.The extracellular variable region of Dscam(three variable exons are exon 4.3,6.46 and 14.18)can also improve the survival rate of crabs by promoting blood cell phagocytosis and bacterial clearance,but it has no inhibitory effect on bacterial growth,which is significantly different from the non-variable region.In view of the uncertainty of the number of exon alternative splicing,Illumina sequencing was used in this study for deep mining again.The results showed that exon 6(Ig3 domain)could produce at least 57 alternative splicing,while exon 14(Ig7domain)could produce 20 alternative splicing.Among them,exon 6 had 23 more alternative splicings than before,and exon 14 had 2 more alternative splicings than before.In addition,sequencing results also showed that Dscam may have variable exon 4,exon 6 or exon 14 skipping.Based on the results of this study,it was confirmed that there may be more than 57,000 potential protein isomers in the extracellular region of Dscam,which greatly enriched the Dscam protein subtype library of E.sinensis.In the resequencing of three variable exons,it was found that there was a very high proportion of individual alternative splicing in exon 14,so we focused on it.The results showed that the function of Ig7 domain was similar to that of the whole extracellular domain of Dscam,and it played an important role in the phagocytosis and clearance of pathogens in E.sinensis.Phagocytosis usually has corresponding receptors.Therefore,we used the T7 phage library to screen the interacting proteins of Ig7,and found 16 potential interacting proteins,including VIP36 and p Ig R,which were further verified by immunofluorescence,Co-IP and pull-down experiments.The results showed that neither VIP36 nor p Ig R interacted with Dscam,indicating that Ig7 may have other potential receptors or only indirectly regulate phagocytosis.In summary,this study explored the immune function of the non-variable and variable parts of the extracellular region of the Chinese mitten crab Dscam.This study not only helps to better understand the complex immune function of the extracellular domain of Dscam in E.sinensis,but also complements the mechanism of immune defense in E.sinensis,and also provides important evidence for the involvement of Dscam in invertebrate immune defense.