Evaluation Of Homologous And Heterologous Immune Effects Of Recombinant Adenovirus And Recombinant Protein Of GRA4 Gene Of Toxoplasma Gondii

Toxoplasma gondii is an obligate intracellular parasite and can infect various vertebrates,such as humans,birds,marine mammals,etc.After T.gondii infection,the host is usually asymptomatic,but in some patients,swollen lymph nodes in the neck or eye infection.Toxoplasma infection in pregnant women can lead to miscarriage or congenital malformations,affecting the fetus’s brain,eyes,or other organs.Toxoplasmosis can cause pneumonia,encephalitis,and even death in immunocompromised patients.In animal husbandry,T.gondii can cause miscarriage and neonatal deformity in domestic animals,causing severe harm to animal husbandry.The existing drugs for the treatment of toxoplasmosis have considerable side effects and poor curative effects and have no impact on the parasites in the cysts.A safe and effective vaccine can effectively prevent toxoplasmosis.This study aimed to construct a recombinant adenovirus expression vector（294-Tg GRA4）and a prokaryotic expression vector（p ET30a-Tg GRA4）carrying the Tg GRA4（T.gondii dense granule antigen4）gene of T.gondii and to package the recombinant adenovirus Ad5-Tg GRA4 in vitro and express the Tg GRA4 protein in vitro.Homologous booster immunization（Prime-Boost）and heterologous Prime-Boost immunization strategies were used to immunize mice to evaluate their immune effects.First,in this study,a recombinant adenovirus expressing the Tg GRA4 gene was successfully constructed,and then the protein of Tg GRA4 could be expressed in HEK293 cells.The Tg GRA4 gene was amplified by the PCR method.The recombinant shuttle vector of p CR259-Tg GRA4 was constructed.The PCR identification and gene sequencing showed that the obtained Tg GRA4 gene was 100%homologous with the reference sequence（M76432.1）.The recombinant adenovirus vector 294-Tg GRA4 was obtained by homologous recombination.The 294-Tg GRA4 vector was successfully constructed by Nde I restriction enzyme digestion.The linearized 294-Tg GRA4 by Pac I vector was transfected into HEK293 cells to obtain recombinant adenovirus Ad5-Tg GRA4.Through PCR,IFA,and Western Blot detection,it was proved that the recombinant adenovirus Ad5-Tg GRA4 was successfully packaged at the gene and protein level and could express Tg GRA4 protein.The determination of virus content showed that the titer of the primary recombinant adenovirus was 2.85×10⁸TCID₅₀/m L.Secondly,in this study,we constructed the prokaryotic expression vector p ET30a-Tg GRA4,expressed and purified the Tg GRA4 His-Tag fusion protein.The Tg GRA4gene was directionally cloned into the p ET30a（+）expression vector.PCR identification and gene sequencing showed that the obtained Tg GRA4 gene had 100%homology with the reference sequence（M76432.1）.The identification of the p ET30a-Tg GRA4 by the Eco R I and Xho I double digestion,SDS-PAGE electrophoresis,and Western Blot methods showed that the prokaryotic expression vector p ET30a-Tg GRA4 was successfully constructed and could express Tg GRA4 protein,which was named p Tg GRA4.Prepared recombinant adenovirus and recombinant protein,BALB/c mice were immunized with homologous Prime-Boost group（p Tg GRA4 alone group and Ad5-Tg GRA4 alone group）and heterologous Prime-Boost group（Ad5-Tg GRA4/p Tg GRA4）,respectively.ELISA detected specific antibody levels and cytokines concentrations in the serum of the immunized mice.The results showed that the levels of Ig G,Ig G1,and Ig2a antibodies in the heterologous Prime-Boost group（Ad5-Tg GRA4/p Tg GRA4）were significantly higher than those in the homologous Prime-Boost group（Ad5-Tg GRA4 and p Tg GRA4）;The level of IFN-γin the Ad5-Tg GRA4/p Tg GRA4 group was significantly higher than that in the p Tg GRA4 group,but not significantly different from the Ad5-Tg GRA4 group;The level of IL-4 in Ad5-Tg GRA4/p Tg GRA4group was significantly higher than that in Ad5-Tg GRA4 group and p Tg GRA4 group.After immunization in the Ad5-Tg GRA4/p Tg GRA4 group,strong cellular and humoral immunity was induced in mice.The PLK-GFP strain challenge experiment of T.gondii was carried out,and the Ad5-Tg GRA4/p Tg GRA4 group significantly improved the survival rate of mice.The formation of brain cysts in mice could not be completely inhibited,but there was a tendency to reduce the burden of brain cysts significantly.Conclusion:The heterologous Prime-Boost immunization strategy used in this experiment,with recombinant adenovirus as the primary immunization and recombinant protein for enhanced immunization,has shown a good effect in the prevention of toxoplasmosis.