Construction Of Toxoplasma Gondii SRS29C Nucleic Acid Vaccine And Comparative Immunoprotective Study Of SRS29C And SAG1 Combination

Toxoplasma gondii is an intracellular parasitic protozoan parasite that infects all nucleated cells except for red blood cells.Since the developmental history of Toxoplasma gondii is complex,the morphology of each stage is different,the host range is wide and the toxoplasmosis caused by different stages of Toxoplasma gondii infection is complex,therefore,pre-vaccination with the safe,stable,and protective vaccine is the most effective measure to control Toxoplasma gondii infection and reduce the economic loss in animal husbandry.Currently,nucleic acid vaccines are being widely investigated in Toxoplasma gondii control,and several nucleic acid vaccine candidate antigens have shown good protection in various studies.The Toxoplasma gondii family of surface antigenic proteins(SAG1-related sequences,SRS)are well-studied DNA vaccine candidates that possess functions in host cell recognition,adhesion,invasion,and activation of host immune responses.The aim of this study was to construct a nucleic acid vaccine with Toxoplasma gondii SRS29C as the target gene,and also to explore the nucleic acid vaccine with Toxoplasma surface protein SRS29C and the combined gene of SRS29C and SAG1,and to evaluate its immunoprotective effect against Toxoplasma gondii.The SRS29C gene fragment was amplified by PCR and cloned into the eukaryotic expression vector p EGFP-N1,and the recombinant plasmid p EGFP-SRS29C was constructed.The plasmid was identified by enzymatic digestion,and the eukaryotic cells were also transfected with the plasmid,and the expression of the target protein was detected by Western blot method.The objective of this study was to develop a nucleic acid vaccine targeting the Toxoplasma gondii SRS29C gene,as well as to investigate the potential of a nucleic acid vaccine containing both the Toxoplasma surface protein SRS29C and the combined gene of SRS29C and SAG1,and to evaluate its effectiveness in providing immunity against Toxoplasma gondii in mice.The SRS29C gene fragment was amplified using PCR and inserted into the eukaryotic expression vector p EGFP-N1,resulting in the creation of the recombinant plasmid p EGFP-SRS29C.The plasmid was verified through enzymatic digestion,and eukaryotic cells were transfected with the plasmid.The expression of the target protein was then analyzed via the Western blot method.Mice were inoculated with the p EGFP-SRS29C and p EGFP-SAG1 single gene nucleic acid vaccines,as well as the combined vaccine,in a total of three rounds of immunization.The empty vector p EGFP-N1 and PBS served as control groups.Blood serum was collected from the mice prior to immunization and at 2,4,and 6 weeks post-vaccination.The Ig G levels in the serum were measured using ELISA.The splenic lymphocytes of mice were stimulated with Toxoplasma gondii tachyzoite antigen,and the expression of splenic lymphocyte factor was analyzed using an ELISA kit.The survival time of the mice was observed and recorded through an in vivo attack experiment to assess the efficacy of the vaccine.The results indicated that a1119 bp fragment of the SRS29C gene was successfully amplified via PCR.Double digestion identified both the 1119 bp target fragment and the 4773 bp vector fragment.Furthermore,the recombinant plasmid p EGFP-SRS29C was transfected,and the extracted protein exhibited a target protein strip at 66 ku as detected by Western blot analysis.The results of the SRS29C and SAG1 gene vaccine test showed that the Ig G content in serum was significantly higher in the p EGFP-SRS29C group and the co-immunization group compared with the PBS group and the empty vector group,the Ig G potency induced in the co-immunization group was higher than that in the p EGFP-SRS29C group and the p EGFP-SAG1group,the splenic The proliferation number of lymphocytes was higher in the p EGFP-SRS29C and co-immunization groups than in the PBS and empty vector groups,and the CD4~+/CD8~+T ratio was higher than in the PBS and empty vector groups.The expression of IFN-γand TNF-αwas significantly higher in splenocytes of the p EGFP-SRS29C and co-immunization groups after antigen stimulation.The attack experiment showed that the mice in the PBS and empty vector groups died within 9 d after attack,the mice in the p EGFP-SRS29C group survived longer until 18 d,the mice in the p EGFP-SAG1 group survived longer until 21 d,and the mice in the co-immunization group survived longer until 24 d.The mice in the p EGFP-SRS29C group survived longer than the mice in the p EGFP-SAG1 group.It was demonstrated that the constructed Toxoplasma gondii nucleic acid vaccine p EGFP-SRS29C and the combined gene vaccine could induce certain humoral and cellular immune responses in mice and enhance the ability of mice to resist Toxoplasma gondii infection,and it was also confirmed that SRS29C was a protective antigen and the combined immunity of SRS29C and SAG1 was more effective than the immunity alone,which provided a new target for further development of Toxoplasma gondii nucleic acid vaccine.