Preliminary Study On The Biological Characteristics Of Toxoplasma Gondii Surface Antigen Glycoprotein 1-Related Sequence SRS47D

As an opportunistic pathogenic and zoonotic parasite,Toxoplasma gondii can infect all nucleated cells of human and more than 200 kinds others vertebrate animals.Feline is its terminal host.Human and others livestock and poultry are its intermediate hosts.Toxoplasmosis distributes worldwide and brings significant challenge to public health security.The life cycle of Toxoplasma includes five stages,tachyzoite,bradyzoite,gametophyte,cyst and schizonts.In intermediate host,tachyzoite multiplicate rapidly in the nucleated cells by the form of binary fission or gemmation when the live environment is suitable.In the terminal host,microgametocyte and microgametocyte combine and then develop to oocysts in epithelial cells of intestinal villi.The infection forms of the parasite are variety.It can be infected by the digestive tract mucosa,placenta,blood,damaged skin,etc.,and then reaches to the nucleated cells of whole body through the blood circulation system.As a food-borne parasite,infection by digestive system is the most common ways and almost one-third of the world's people infected.As an opportunistic pathogenic parasite,the infection and course of the disease are obviously influenced by host's immunity state.The sera positive rate in developed regions is lower than it in less developed regions.Accompanying the number increase of companion animals such as dogs and cats,the infection rate of Toxoplasma gondii through food contaminated by cat and dog feces is also increased.Raw and cooked foods have not been separated at the food production process is also increases the probability of Toxoplasma infection by cross-contamination.The immunogenicity and potential function of surface antigen glycoprotein 1-related sequence SRS47 D of Toxoplasma gondii were firstly predict with online bioinformatics softwares.The first,second and third structures,protein constitution,epitope and potential function of the protein were predicted using online software ProtParam,SOSUI,SOPMA,TMHMM,MotifScan and SYFPEITHI.The results showed that SRS47 D protein was composed with 376 amino acid residues with molecular mass of approximately 40 ku.In its secondary structure,the percent rate of consisted ?-helixes,?-folds,?-corners and random coils were 23.14%,19.68%,2.39% and 54.79%,respectively.There were a signal peptide sequence,18 regions of amino acid residues with high level of hydrophilicity(critical value:1.9),8 regions with high level of flexible(critical value: 2)and 17 regions with high level of surface accessibility(critical value: 1.9),but no transmembrane domain in the protein.The SRS47 D protein post-translational modification sites contained two glycogen synthase kinase sites and two O-GlcNAc glycosylation sites,but no N-GlcNAc and C-GlcNAc glycosylation sites.Comparing with the tertiary structure of SAG1 protein,there were overlaps at the ?-sheet regions.The protein also contains 24 potential CTL epitopes,13 potential Th cell epitopes and 10 B cell epitopes.These results indicated that the SRS47 D protein maybe have good immunogenicity and a potential vaccines candidate molecular for toxoplasmosis.In order to express the coding gene of SRS47 D,determine the protein location in T.gondii tachyzoite,SRS47 D gene was amplified by RT-PCR and the prokaryotic expression plasmidpColdI-SRS47 D was constructed by inserting the SRS47 D gene into the prokaryotic expression vector pCold?.The recombinant plasmid pColdI-SRS47 D was then transformed into Escherichia coli BL21(DE3)and induced to express with IPTG.The recombinant SRS47D(rSRS47D)protein was detected by SDS-PAGE,western blot,and purified by affinity chromatography.The antisera of rSRS47 D were prepared by immunizing the purifying recombinant protein in New Zealand white rabbits,and their titers were detected by indirect ELISA.The localization of SRS47 D in T.gondii tachyzoite was detected by immunofluorescence with the antisera.The results showed that the recombinant plasmid pColdI-SRS47 D was constructed and induced to expressed in E.coli BL21(DE3)successfully.The molecular weight of recombinant protein was about 55 ku.Western blot analysis showed that the recombinant protein could react with sera from mice infected with T.gondii.The titers of rabbit antisera could reach 1:51 200.Immunofluorescence analysis showed that the SRS47 D protein was mainly located at the surface of T.gondii tachyzoite,especially at the outer membrane of the anterior and posterior end.These results laid a foundation for further study on the characteristics and functions of the Toxoplasma SRS47 D.To explore the interaction between T.gondii SRS47 D and SRS37 A,SRS23,and their interaction model,the bimolecular fluorescent complementary plasmid of SRS47 D,SRS37A,SRS23 genes,and the2 code gene fragments of SRS47 D and SRS37 A structural domains were constructed,respectively,and co-transfected to Vero cells.The interaction relationships were detected by fluorescent test.The techniques of immunofluorescence co-localization and laser confocal microscopy were also used to observe the interaction relationship.The results showed that 7 bimolecular fluorescent complementary plasmids were constructed successfully.Bimolecular fluorescence complementation experiments showed that there was interaction between SRS47 D and SRS37 A,but no interaction between SRS47 D and SRS23.Both SRS47D-D1 and SRS47D-D2 interacted with SRS37A-D1 and SRS37A-D2,respectively.Immunofluorescence co-localization analysis showed that the SRS47 D and SRS37 A protein were mainly co-located at the surface of T.gondii tachyzoite,especially at the outer membrane of the anterior and posterior end.Based on these results,the author speculated that there were close relationships between the two domains of SRS47 D and SRS37 A,but there was no interaction between SRS47 D and SRS23.The immunoprotective effects of rSRS37 A,rSRS47D and union use of rSRS37 A and rSRS47 D to artificial infection of mice of T.gondii virulent strain were firstly tested.The antibody levels and its resistant to invasion of tachyzoite in vitro,spleen lymphocyte proliferation ability,the levels of cytokines and T lymphocyte subtypes,and survival time of immunized mice after challenging with T.gondii RH strain were tested,respectively.The results showed that extremely significant IgG levels were produced in mice immunized with rSRS37 A,rSRS47D,rSRS37A+rSRS47D protein(p<0.0001).Anti-rSRS47 D IgG could significantly block the invasion of tachyzoite of T.gondii RH strain in vitro at50?100?200?300?400 ?g/mL concentrations(p<0.05),and the blocking effects were enhanced companying with the increase of its concentration.All three test groups could stimulate proliferation of splenic lymphocytes of immunized mice extremely significant(p<0.0001),but no differences wereobserved in control groups(p>0.05).The secretion of IL-17 A in rSRS47 D and rSRS37A+rSRS47D proteins immunized groups' mice were all significantly increased at 2 weeks after the first immunization(p<0.05).At the same time,the IL-6 secretion levels were just begun to increasing.At 1 week after the second immunization,the level of IL-17 A secretion levels were found to decrease from 2 weeks after the first immunization.At that time,IL-6 secretion levels reached the highest level.After the third immunization,IL-6 secretion levels changed companying with the changes of IL-17 A secretion levels,indicating that the IL-6 expression were laged behind IL-17 A.The secretion levels of TNF of all 3 test groups were increased at first,then decreased partially and increased another times at 6 w post first immunization.The secretion of IFN-? in rSRS37 A immunized group was extremely significant increased at 2 w post first immunization(p<0.0001).All 3 test groups and adjuvant group were extremely significant increased at 3 w post first immunization(p<0.0001),and then decreased.After challenging with T.gondii RH strain,the BABL/c mice in 3 control groups were all died between days 7post infection(D7PI)and D9 PI,and there was no significant difference between them(P>0.05).While mice immunized with rSRS37 A,rSRS47D and rSRS37A+rSRS47D died between D8 PI and D10 PI,between D9 PI and D12 PI and between D11 PI and D15 PI,respectively,and significantly difference were found on survive times between 3 tested recombinant protein immunization groups and control groups(p<0.05).These results indicated that there were obvious immune protection effects of rSRS37 A,rSRS47D,rSRS37A+rSRS47D for mice T.gondii infection.In summary,the structure and potential function of T.gondii surface antigen glycoprotein 1-related sequence SRS47 D protein were predicted with online bioinformatics softwares.The recombinant SRS47 D protein was expressed in vitro in prokaryotic expression system and the SRS47 D protein was mainly located at the surface of T.gondii tachyzoite,especially at the outer membrane of the anterior and posterior end.There were interaction relationships between T.gondii SRS47 D and SRS37 A,between 2 domains of SRS47 D and 2 domains of SRS23 proteins.Immunization of rSRS37 A,rSRS47D and rSRS37A+rSRS47D had partly immunoprotective effects for T.gondii infection mice.The results of these studies laid the foundation for further study of the function and characteristics of SRS47 D protein and the use of protein to develop new prevention and control strategy for toxoplasmosis.