| Toxoplasma gondii(T.gondii)is an obligate intracellular parasitic protozoan that poses a threat to one-third of the global population.It is one of the world’s most successful zoonotic parasites because of its host range,high infection rate,and co-existence with its host.T.gondii has serious effects on people with deficient immune systems(such as AIDS patients,malignant tumor patients and organ transplant patients),causing encephalitis,hydrocephalus,myocarditis,retinal choroiditis and other illnesses.Pregnant women infected with T.gondii may have malformations and miscarriages.Additionally,T.gondii may alter the risk tolerance of the host and be associated with mental illness,suicidal or accidental tendencies.Nevertheless,there is no ideal drug or vaccine for the prevention and control of toxoplasmosis.T.gondii secretes a variety of effector proteins during invasion of host cells or growth and development to achieve nutrient acquisition and immune evasion.Secretory proteins of T.gondii play a key role in mediating host-pathogen interactions.In this study,we screened three novel secretory proteins of T.gondii(Toxo DB accession number:TGGT1＿242240,TGME49＿210095,and TGRH88＿028240)utilizing the bioinformatic methods with preliminary identification and analysis,prepared corresponding mouse monoclonal antibodies and rabbit polyclonal antibodies,analyzed their expression characteristics and localization distribution in different invasion stages of T.gondii by realtime fluorescence quantitative PCR,western blot,and indirect immunofluorescence techniques.Purposes:1.To screen for novel secretory proteins of T.gondii by bioinformatic methods,and perform preliminary characterization.2.To construct the prokaryotic expression vectors p ET-28a(+)-TGGT1＿242240,p ET-28a(+)-TGME49＿210095 and p ET-28a(+)-TGRH88＿028240,and express the corresponding proteins,and then analyzed purified proteins immunoreactivity.3.To prepare corresponding rabbit polyclonal antibodies and mouse monoclonal antibodies,and analyze their reactivity with antigen after purification.4.To analyze the expression characteristics of the target proteins in different invasion stages of T.gondii and their localization in tachyzoites.Methods:1.Bioinformatics tools were used to analyze the sequence information,physicochemical properties,structure of TGGT1＿242240,TGME49＿210095 and TGRH88＿028240.2.The c DNA of T.gondii RH strain was extracted,and the target genes were obtained by PCR amplification.The genes were then inserted into the prokaryotic expression vector p ET-28a(+),and transferred into the expression bacterium E.coli BL21(DE3)after sequencing.The purified proteins were obtained by affinity chromatography using Ni column,and their immunoreactivity were analyzed by western blot using rabbit anti-T.gondii positive serum as the primary antibody.3.Rabbits and mice were immunized with purified r TGGT1＿242240,r TGME49＿210095 and r TGRH88＿028240 to prepare rabbit polyclonal antibodies and murine monoclonal antibodies.Balb/c mice with high titer were selected for fusion,and subclonal screening was performed using the limited dilution method.Murine monoclonal antibodies were prepared in large quantities by in vivo induction,and the antibodies were purified by ammonium sulfate precipitation.Their reactivity with the antigen was then identified by western blot.4.T.gondii tachyzoites were collected at different invasion times and different stages(adhesion penetrating cell membrane,invasion,intracellular increment,egress,and free)using the temperature conversion method.Their expression characteristics were analyzed by q PCR and WB,and their localization in tachyzoites was identified by IFA.Results:1.The bioinformatics analysis showed that TGGT1＿242240,TGME49＿210095 and TGRH88＿028240 have signal peptides but no transmembrane structural domains and are classical secretory proteins.TGGT1＿242240 and TGME49＿210095 have better B-cell epitopes.The sequence similarity between TGGT1＿242240 and T.gondii ROP protein kinase(Tg ROPK)is 100%,and TGME49＿210095 has 50% sequence similarity with T.gondii c AMP-dependent protein kinase(AMPK),presumably may perform functions similar to them.2.We constructed plasmids,p ET-28a(+)-TGGT1＿242240,p ET-28a(+)-TGME49＿210095 and p ET-28a(+)-TGRH88＿028240,and obtained recombinant protein TGME49＿210095 with good reactogenicity.3.The corresponding rabbit polyclonal antibodies,G-PAb,M-PAb and R1-PAb,were prepared.Nine monoclonal antibodies with good reactivity with the antigen,1G-MAb,1MMAb,2M-MAb,3M-MAb,5M-MAb,6M-MAb,M2-MAb,R1-MAb,2R1-MAb,were obtained.4.The q PCR results at different stages showed that the m RNA expression of TGME49＿210095 was significantly higher at 20 min post-invasion compared to 40 min(p<0.01),and the expression of TGRH88＿028240 at egress was significantly higher than that of the control(p<0.05).The western blot results showed that TGGT1＿242240 was expressed at the egress stage.Based on the analysis,it was speculated that TGME49＿210095 might be involved in invasion,while TGGT1＿242240 and TGRH88＿028240 might be involved in egress.The IFA results showed that at 4 hours post-invasion of tachyzoite invasion into HFF cells,TGGT1＿242240 was relatively diffuse and anteriorly biased,while TGME49＿210095and TGRH88＿028240 were mainly concentrated in the head and neck region.Conclusions:1.Three novel secretory proteins,TGGT1＿242240,TGME49＿210095 and TGRH88＿028240,were obtained,and bioinformatics analysis revealed that TGGT1＿242240and TGME49＿2100953 have kinase structural domains.2.The prokaryotic expression vectors,p ET-28a(+)-TGGT1＿242240,p ET-28a(+)-TGME49＿210095 and p ET-28a(+)-TGRH88＿028240,were successfully constructed,and the corresponding proteins were expressed and purified.Additionally,rabbit polyclonal antibody and nine strains of monoclonal antibody with good reactivity to antigen were prepared,which laid a foundation for further investigation of their biological characteristics.3.We analyzed the expression characteristics and localization of three novel secretory proteins(TGGT1＿242240,TGME49＿210095 and TGRH88＿028240)at different stages of T.gondii invasion into host cells. |
