| Toxoplasma gondii is an important intracellular parasite that is distributed worldwide and can infect warm-blooded animals almost all.At present,the population structure and hybridization between different strains of T.gondii are abundantly in worldwide.There is a high degree of geographical distinction among the genotypes.Chinese I(TOXODB#9)genotype is dominant and widely spreads in China.The Chinese I strain,which may cause immune responses of host specifically,had unique pathogenicity compared with other strains.The mutual transformation of T.gondii tachyzoites and bradyzoites is the central link in the pathogenesis of toxoplasmosis.The morphological similarities between the two,but there is significant difference in molecular levels.There are different period-specific antigens in different stages.At present,some period-specific molecules have been identified,the specific period of T.gondii can be determined by identifying the period-specific genes at a molecular level.The BIN3 protein(Bicoid-Interacting Protein 3)of Drosophila plays an important role in embryonic development.BIN3 protein in T.gondii was predicted to relate to the development or polarity of T.gondii because the motif sequence of BIN3 in T.gondii is similar to Drosophila.However,the study of BIN3 protein in T.gondii is currently limited to online prediction,and the study of the BIN3 protein in the Chinese I strain is still blank.In this study,the following experiments were used to locate the BIN3 protein,study the protective immune reaction in Chinese I strain and construction of BIN3 gene knockout strain of Chinese I genotype Wh3 strain.The experimental data provide a foundation for the further study of the biological characteristics of the Chinese I genotype.Bioinformatics analysisBIN3 protein was analyzed initially by online databases such as NCBI,TOXODB and online software provided by websites such as Exacy and CBS,including basic physical and chemical properties,secondary structure,and post-transcription modifications.The results showed that the theoretical molecular weight of BIN3 protein was 84.38 kD;The theoretical isoelectric point(PI)is 7.65;The protein has no transmembrane structure and does not contain signal peptides.The probability of the BIN3 protein located in the nucleus is 78.3%.The BIN3protein has 3 N-terminal glycosylation sites;there is 6 tyrosine residue,and multiple serine and threonine residues;Acylation exists.The BIN3 protein secondary structure includes helix,sheet,turn,and coil.In the front and back ends of this protein,the helix structure and the sheet structure are abundant and interspersed with binding;the coil structures are mainly concentrated in the middle of protein;the turn structures are evenly distributed in the protein.Preparation of BIN3 protein monoclonal antibodyThe primers were designed by Primer software.The BIN3 gene from the Chinese I genotype Wh3 strain of T.gondii was cloned.The expression vector pET28a-BIN3 and recombinant BIN3 protein were obtained.The monoclonal antibody was prepared by method of cell fusion and preparation of the hybridoma.The results showed that three monoclonal antibodies,AbM-BIN3:#43,AbM-BIN3:#42 and AbM-BIN3:#7 were obtained.The titer of ascites was analyzed by ELISA,AbM-BIN3:#43 and AbM-BIN3:#7 all reached 1:100,000,0 units or more,AbM-BIN3:#42 was close to 1:100,000,0,AbM-BIN3:#43 has higher titer.Localization of the BIN3 protein in Chinese I genotype Wh3 strainThe cryopreserved T.gondii Chinese I strain was thawed and cultured in Vero cells.After 2-3 generations,the culture medium was replaced by the RPIM-1640medium with pH=8.2 and final serum concentration was 2%.SAG1,a tachyzoite stage specific gene,BAG1,a bradyzoite stage-specific gene,and SAG2C,the early-bradyzoite stage specific gene were used to identify the induced parasites.Localization of the BIN3 protein by indirect immunofluorescence.The results showed that the Chinese I genotype Wh3 strain exhibited pseudocysts and cysts in alkaline conditions after parasites were induced,and the bradyzoite stage specific proteins were expressed.The BIN3 protein is expressed in free-state and pseudocysts tachyzoites,and cysts of the Chinese I genotype Wh3 strain.The BIN3 protein is located in the cytoplasm of the tachyzoites in the free-state,secreted between the parasite and the pseudocyst membrane in pseudocysts.The cysts induced under alkaline culture conditions were viewed under LSCM,which showed that the Bin3protein,represented by the green fluorescence,was uniformly distributed in the cyst wall,presenting a complete circular boundary.Protection of recombinant BIN3 protein against Chinese I genotype Wh3 strainIn this study,the protective efficacy of recombinant T.gondii Bicoid-Interacting Protein 3(rTgBIN3)against T.gondii Chinese I strain infection in Balb/c mice(aged6 weeks)was evaluated,the vaccinated mice were injected with 50?g of rTgBIN3.Balb/c mice were subcutaneously immunized with rTgBIN3 or PBS three times with2 week intervals.Two weeks after the last immunization,the humoral and cellular immune responses were evaluated.The immunized mice were challenged with tachyzoites of Chinese I strain 14 days after the last immunization.In challenge study,all mouse challenged with 1×10~5 tachyzoites except control group,observed their physical appearance daily.The results showed that the rTgBIN3-immunized mice showed higher IgG titers and increased levels of specific antibodies against the recombinant protein(P<0.05)than control group mice,and high levels of interferon-?and interleukin-2(P<0.05).After challenge,the rTgBIN3-immunized mice survived longer than the control groups for 72h.Our result showed that TgBIN3 immunization induced protective immune responses.Construction of BIN3 gene knockout strain of Chinese I genotype Wh3 strainConstruction of BIN3 motif sequence knockout strain by CARSPR/Cas 9.BIN3-gRNA primers were designed and pSAG1-Cas9-suprt plasmid were constructed.The DHRR homologous sequence was prepared.Ethylamine was used for drug screening.The BIN3 gene knockout strain was determined in molecular and protein level.The PCR result showed that the DHFR fragment was inserted into the target gene.The Western blot result showed that the BIN3 protein was not expressed in knockout strain.The BIN3 gene knockout strain of Chinese I genotype Wh3 strain was constructed successful.BIN3 gene knockout strain grow slowly than wild strain in Vero cells.The gap for the number of parasites was reached peak after 72h by challenged with the parasites in a same amount(P<0.05).The number of BIN3 gene knockout strain was about 1.25×10~6 less than wild strain.After challenge,the BIN3gene knockout strain challenged mice survived longer than the wild strain groups for24h.In this study,the bradyzoite-specific protein was induced to express in T.gondii Chinese I genotype Wh3 strain,which cannot be cystogenous in animal models.It exhibited cysts in alkaline conditions after it was induced.The BIN3 protein is expressed in free-state and pseudocysts tachyzoites,and cysts of the Chinese I genotype Wh3 strain.The result of rTgBIN3 against Chinese I genotype Wh3 strain showed that rTgBIN3 immunization induced protective immune responses,indicating that rTgBIN3 might be a promising vaccine candidate for combating toxoplasmosis Chinese I strain.It is has a great significance to the prevention and treatment of T.gondii in China.The construction of BIN3 knockout strain also provides a basis for further study on the biological characteristics of the Chinese I genotype. |
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