Preparation Of Toxoplasma RTgPRF Protein Vaccine And Research On Its Protective Effect

Objective:In this study,the anti-rTgPRF polyclonal antibody was prepared according to the immune program established by the research group,and then we monitored its effects on the growth,apoptosis and necrosis of normal cells and Toxoplasma gondii infected cells in vitro.To evaluate the changes of specific antibodies and CD4+/CD8+after rTgPRF liposome or rTgPRF combined with Freund’s adjuvant immunization of C57BL/6J mice,and further evaluate the effects of the two immunization methods on the survival time,histopathology,levels of reproductive hormones,cytokines,and apoptosis-related proteins in mice infected with Toxoplasma gondii,and then to explore the protective efficacy and mechanism of rTg PRF vaccine,which provided theoretical support and experimental basis for its further application and development.Methods:The rTgPRF protein was expressed in large quantities by transferring pET30a（+）-TgPRF plasmid into E.coli BL21（DE3）,purified by Ni column and anion column through the AKTA system,and then freeze-dried.First,anti-rTgPRF polyclonal antibody was prepared for in vitro activity detection.BALB/c-3T3 fibroblasts were plated and then added with anti rtgprf serum（1:40）or rabbit pre immune serum（1:40）respectively,while the normal control was only supplemented with culture medium,and the effect of anti-rTgPRF antibody on the growth,necrosis and apoptosis of normal cells was dynamically monitored by Incucyte Zoom^TMsystem at 1h intervals.The effect of anti-TgPRF antibody on Toxoplasma gondii-infected cells was also tested.The cells were plated and pre-cultured for 12 h,and then the uninfected wells were set into normal control and antiserum control（1:40 anti-rTgPRF serum）;6×10³ tachyzoites（RH strain）were added into the infection wells,which were divided into negative control（1:40 rabbit pre-immune serum）,simultaneous intervention group（anti-r TgPRF serum was added during infection）,delayed intervention group（anti-r TgPRF serum was added 12 h after infection）,and cell growth,apoptosis and necrosis were monitored at 1 hour intervals.The rTgPRF liposomes with particle sizes of 200 nm and 400 nm were prepared by film dispersion method,and their encapsulation efficiency was detected by low-temperature ultracentrifugation.Further,the protective effect of rTgPRF liposome and rTgPRF combined with Freund’s adjuvant vaccine in vivo was studied.C57 mice were divided into PBS control,blank adjuvant control,blank liposome control and pure protein group（20μg per mouse）;rTgPRF combined with Freund’s adjuvant group was divided into 10μg and 20μg dose groups;rTg PRF liposome group was divided into 4 dose groups according to particle size and dose:10μg,20μg group with 400 nm particle size and 10μg,20μg group with 200 nm particle size.Mice in each group were immunized subcutaneously three times at two-week intervals,during which blood was collected from the tail vein of the mice to detect IgG and subtypes.Two weeks after the last immunization,the spleen cells of three mice in each group were taken for CD4+/CD8+T lymphocyte detection.The remaining mice were intraperitoneally infected with 5×10² tachyzoites（RH strains）,blood was collected from tail vein after 8⁹ days of infection,serum was collected by centrifugation for ELISA detection of reproductive hormone and chip detection of cytokines.After fixing the liver and testis of mice,pathological sections were made.The tachyzoites load of testis,liver and brain of mice was detected by fluorescence quantitative PCR,and 17 kinds of organ cytokines and 21 kinds of apoptosis-related proteins were detected by microarray.Results:1.The r TgPRF protein was soluble expression,and the reconstituted lyophilized rTgPRF protein was detected by SDS-PAGE and showed that the protein was intact.In vitro experiments showed that the anti-rTgPRF antibody was non-cytotoxic and significantly inhibited the necrosis during normal BALB/C-3T3 fibroblast proliferation or caused by Toxoplasma gondii infection,and promotes its apoptosis.2.The entrapment efficiency of rTgPRF liposome suspension prepared by the PVC filter membrane was about 85%.r TgPRF combined with Freund’s adjuvant could induce the increase of IgG1,IgG2b and IgG2c（P<0.05）,while rTgPRF liposome immunization was mainly based on IgG2,in which the antibody level of 400 nm r TgPRF liposome was better than that of 200nm liposome（P<0.05）,and the ratio of CD4+/CD8+can be increased by each immune mode.3.The protective experiment of mice showed that rTgPRF combined with Freund’s adjuvant and rTgPRF liposome could prolong the survival time of mice.Among them,the protective effect of liposome vaccine with 200 nm particle size was poor,while that of liposome vaccine with 400 nm particle size was more effective than Freund’s adjuvant vaccine in reducing organ（testis,liver,brain）tachyzoites burden（P<0.05）,and the pathological damage was also lighter.Toxoplasma gondii infection can reduce the levels of testosterone（T）and dihydrotestosterone（DHT）in the testis of mice,while increase the levels of follicle-stimulating hormone（FSH）,but these hormone levels have no significant improvement after different forms of immune protection（P>0.05）.Immunization with rTgPRF combined with Freund’s adjuvant can further increase the pro-inflammatory factors（IFN-γ,IL-6,IL-23 P19）in the serum of mice infected with Toxoplasma gondii（P<0.05）,while liposome with 400 nm particle size can increase the proinflammatory and anti-inflammatory factors（IL-10,TGF-β1 and IL-22）（P<0.05）.Liposome with 400 nm particle size vaccine also showed differences in local immune regulation of the organs,compared with the combined adjuvant group,its proinflammatory factors and apoptosis in testis were significantly increased（P<0.05）,proinflammatory factors and apoptosis in brain tissue also increased slightly but no difference（P>0.05）,while proinflammatory factors and apoptosis in liver tissue were significantly weakened（P<0.05）.Conclusion:1.Anti-rTgPRF antibody has the effect of directly inhibiting the proliferation of Toxoplasma gondii tachyzoites in vitro,which is closely related to its activity of inhibiting cell death and promoting apoptosis.2.Liposomes with different particle sizes have obvious differences in the activation of immune response.The immune protection effect of rTgPRF liposomes with 400 nm particle size is better than that with200 nm particle size.3.The rTgPRF protein vaccine has a good protective effect against Toxoplasma gondii in mice,and the rTg PRF liposome vaccine with 400 nm particle size is more effective in reducing organs（testis,liver,brain）tachyzoite burden.The rTgPRF combined with adjuvant immunization was mainly Th1 type,while the rTgPRF liposome vaccine promotes the balance of serum pro-inflammatory/anti-inflammatory cytokines and also reflects the organ specificity of immune regulation.However,rTgPRF liposome vaccine not only balances the balance of pro-inflammatory/anti-inflammatory cytokines in serum,but also reflects the organ specificity of immune regulation,and can promote apoptosis through Fas-Cytochrome c-Caspase pathway,which may be related to its effective reduction of organ tachyzoite burden the effective reduction of organ load.