The Study For The Life Cycle And Polyclonal Antibody Aimed To EhSWP7 Of Enterocytozoon Hepatopenaei

Enterocytozoon hepatopenaei(EHP)is an obligate intracellular parasite that can infect a variety of economically valuable shrimp species,belonging to Microsporida,Enterocytozoonidae,Enterocytozoon,shown to infect only the tubule epithelial cells of the crustacean hepatopancreas.Since EHP was discovered in 2001,EHP infection has been reported in major Litopenaeus vannamei aquaculture areas of the world.However,there is still a lack of profound understanding of EHP life cycle,cell adhesion and infection mechanisms.For the composition of spore wall proteins,current studies have found that spore wall proteins contain at least 7 proteins,and the first spore wall protein of EHP has been identified and reported.We use molecular biological detection and transmission electron microscopy to study the life history of this pathogen and predicted the secreted proteins of the EHP genome with the Eu Sec Pred2.0 pipeline and analyze the length of these secreted proteins,the length of the signal peptide,and the amino acid distribution at the cleavage site,and annotated the function of secreted proteins.Then,the sequence and structure of EhSWP7,one of the predicted secreted proteins,is analyzed.The prokaryotic expression vector of EhSWP7 is constructed to express the recombinant protein.EhSWP7 polyclonal antibody is prepared by immunizing animals.After verifying the specificity of polyclonal antibodies by western blotting,indirect immunofluorescence assay(IFA)is performed to determine the localization of EhSWP7.The main results of this research as follows: 1.Preliminary study of the life cycle of EHPBy transmission electron microscopy,the life cycle of EHP can be divided into three stages: the infective stage,the proliferative stage,and the sporogonic stage.This study also shows that fish and shellfish act as carriers of EHP,and that healthy shrimp can be infected with EHP by feeding on diseased hepatopancreatic tissue.Early proliferating cells are characterized by unique electron-lucent lamellar inclusions(ELI).Most proliferating cells divides first by binary fission,followed by multiple nuclear divisions.An electron-dense disk(EDD)structure is observed at the beginning of the sporogonic stage.At early sporogonial plasmodia,EDD is randomly distributed in the cytoplasm;however,as the density of EDD increases,the number of nuclei increases substantially after multiple divisions.Subsequently,development enters into the late sporogonial plasmodium stage.At this stage,EDD is stacked together,fused,and elongated,forming an arc to wrap the nucleus,and finally forming a polar filament.Then the anchoring disk structure is connected to the fixed polar tube by the manubroid,to form a polar sac-anchoring disk complex.When the plasma membrane sinks inward,dense secretions thickens the plasma membrane,this signals the beginning of sporogonial division to form sporoblasts.Sporoblasts are composed of a nucleus,a polar filament with 4-5 spirals,a polar sac-anchoring disk complex,and ELI.Sporoblasts continue to develop into spores and the mature spores gradually developed a thick spore wall.Different developmental stages are observed within a single hepatopancreatic cell.Some mature spores are expelled by exocytosis.Based on the results of this and previous studies,we are able to depict the full life cycle of EHP.This result could provide the basis for future research on the prevention and control of EHP.2.Genome-wide Prediction and Analysis of Secreted Proteins of EHPWe predict the secreted proteins of the EHP genome with sequence analysis software and online websites and analyze the length of these secreted proteins,the length of the signal peptide,and the amino acid distribution at the cleavage site,and annotate the function of secreted proteins.The results show that the length of the secreted proteins ranged from 30 to 400 amino acid residues,the signal peptides covered approximately 9～32 amino acids,and the cleavage sites of the signal peptides were mainly composed of hydrophobic amino acids.Motif analysis reveales a NV[VT][IK]CA[ED][SA] motif in signal peptides.Functional annotation of proteins reveales that a variety of key proteins are involved in adhesion and infection of microsporidia,regulation of cell cycle,and immune response.3.Antibody preparation and localization of EhSWP7.By the bioinformatics method,EhSWP7 is predicted to have a signal peptide and the signal peptide has high hydrophobicity.The protein has no potential glycosylation modification sites,but has several phosphorylation modification sites.There are no known functional domains in the protein sequence.The polyclonal antibodies is prepared by the expression and purification of the prokaryotic protein EhSWP7.The results of western blotting showes that the antibody is highly specific and sensitive.IFA showed that rabbit polyclonal antibodies could localize EhSWP7 to the entire spore wall.EhSWP7 was expressed throughout the spore wall,suggesting that it may play an important role in adhesion to host cells and mediating invasion.In summary,the life cycle of EHP was constructed,the secreted proteins of EHP were predicted,and the location of EhSWP7 was analyzed.The results of this paper are helpful to understand the infection mechanism of EHP to host and provide basis for the prevention and treatment of EHP in the future.