Geneanalysisand Function Characterization Ofthe Variable Merozoite Surfaceantigen (VMSA) Gene Familyofbabesiaorientalis

Babesia orientalis,a protozoan hemoparasite of phylum Apicomplexa,is transmitted by Rhipicephalus haemaphysaloides.This parasite causes water buffalo babesiosis which is endemic in central and southern China,and water buffalo is the only known host.Buffalo infected can develop typical clinical signs such as fever,anemia,jaundice,hemoglobinuria and even to death.Similar to the characteristics observed in other hemoparasites,extracellular merozoites exhibit reversible attachment to host red blood cells（RBCs）,furthermore,reorientation of the merozoite enables organization of the apical organelles close to the attachment interface for the formation of tight junctions.During invasion,the RBC membrane undergoes invagination and forms a parasitophorous vacuole（PV）.After completion of a parasitic invasion into erythrocytes,the PV disintegrates and resides freely within the host RBC cytoplasm.Parasites produce two merozoites by binary fission.After the occurrence of erythrocyte lysis,merozoites are released and the invasion of new RBCs occurs.Therefore,focus has been directed towards the prevention and control of Babesia by blocking this process.At the initial step of erythrocyte invasion,Babesia parasites reportedly utilize glycosyl phosphatidyl inositol（GPI）anchor antigens to enable attachment to target cells,but no specific functions have been identified in the researches of Babesia.The purpose of this study was to identify,clone,and characterize the BoMSA gene family and explore its functions.And the main research work include:1.Characterization of the VMSA gene family of Babesia orientalisAll the reported VMSA genes of Babesia spp.were obtained from Gen Bank,and multiple alignments were performed by using conserved regions to blast the Babesia orientalis genome database.Five MSA genes（named MSA-2a1,MSA-2a2,MSA-2c1,MSA-1,and MSA-2c2,respectively）were identified,sequenced,and cloned from B.orientalis,which were shown to encode proteins with open reading frames ranging in size from 266（MSA-2c1）to 317（MSA-1）amino acids.All the five proteins contain an MSA-2c superfamily conserved domain,with an identical signal peptide and glycosyl phosphatidyl inositol（GPI）-anchor for each of them.The five proteins were also predicted to contain B cell epitopes,with only three for BoMSA-2c1,the smallest protein in the Bo VMSA family,while at least six for each of the others.Notably,the predicted three-dimensional model of BoMSA-2c1 is also different fromother members of this family.These results indicated the BoMSA-2c1 participate in the growth of B.orientalis.2.Molecular function of merozoite surface antigen 2c1BoMSA-2c1 without signal peptide was amplified and cloned into p E-sumo,and expressed in the BL21.Then,the recombinant BoMSA-2c1 protein was purified to prepare polyclonal antiserum.BoMSA-2c1 was identified in the lysate of B.orientalis-infected water buffalo erythrocytes with a molecular weight of 36 k Da,corresponding to the expected molecular mass of BoMSA-2c1.The result of r BoMSA-2c1 with positive serum revealed that BoMSA-2c1 can elicit an immune response to B.orientalis-infected water buffalo.Immunofluorescence staining results showed that BoMSA-2c1 was expressed only on extracellular merozoites,as signals were undetectable in intracellular parasites.RBC binding assay suggested that BoMSA-2c1 helps specific adhesion to water buffalo erythrocytes.An in vitro cytoadherence assay using a eukaryotic expression system was performed for further verification of the adhesion and inhibition ability of BoMSA-2c1;HEK 293T cells with BoMSA2c1 expressed on cell surfaces exhibit adhesion to red blood cells by rosettes assay,and this ability can be neutralized by anti-r BoMSA2c1 serum.These results suggested BoMSA-2c1 protein has an important role in the initial binding to host erythrocytes during parasite invasion.3.Establishment of gene editing system of Babeisa.In order to study the functions of MSA family genes more intuitively,a gene editing method of Babesia on the basis of in vitro culture was established.First,the optimal concentration of blasticidin（BSD）on Babesia was determined.Then the p BS-EGBE plasmid was designed for integration into the ef-1αlocus of B.gibsonigenome by double cross-over homologous recombination,which can stably express the recombinant protein of GFP-BSD.Linearized plasmid was transfected by4D Nucleofector ^TMinto in vitro cultured B.gibsoni and 0.4μg/m L was added for drug selection two days after transfection.GFP-expressing parasites were observed by fluorescence microscopy as early as two weeks after drug selection.Genome integration was confirmed by PCR and sequencing analysis.Finally,the single-copy gene of Sera I was knockout and identified by both PCR,q PCR and Western blot,indicated the gene editing method was successfully established in the Babesia.In summary,this study identified the genes of the VMSA family of Babesia orientalis,and also verified that BoMSA-2c1,as a member of this family,plays an important role in the invasion of parasites.This research fills the gap of adhesion factors during the invasion of Babesia and provides new ideas and vaccine candidate factors for Babesia research.The establishment of a Babesia gene editing method in this process provided great convenience for the study of Babesia.