Construction And Immunization Experiment Of Pseudorabies Virus Expressing SAG1 From Toxoplasma Gondii

Toxoplasma gondii is an obligate intracellular parasite,capable of infecting a variety of mammals and birds.Primary infection during pregnancy can cause severe neonatal malformations and ocular complications in the fetus,and the infection often results in a fatal encephalitis in immunocompromised human.T.gondii is an important food-borne parasite,whose main routes of transmission from animals to humans are through meat,mainly from pigs and lambs,and oocysts shed by cats into the environment.Toxoplasmosis is also of veterinary importance,since infection during pregnancy,especially in sheep and pigs,often results in abortion, causing considerable economic losses.Vaccine against infection in human is no available,but an effective vaccine preventing infection in animals used for food would block the main transmission route to humans. Although several types of vaccine against toxoplasmosis,which include attenuated and inactivated vaccine, subunit vaccine,genetically engineering vaccine and DNA vaccine,have been developed and tested for their immunological effects in animal models,few have been licensed for use,principally due to biosafety concerns or poor efficacy.Inactivated vaccines and subunit vaccines are found only moderate protective efficacy.A live attenuated vaccine has been available for veterinary use for several years in some countries,but it is expensive, causes side effects,and has the possibility to revert to pathogenic strain.It is necessary to develop cheap and effective vaccine agaist T.gondii infection in animals.In the present study,we describe development of recombinant pseudorabies virus expressing SAG1 based on PRV Bartha K-61 and the ability of the recombinant virus inducing protective immunity against virulent T.gondii RH strain challenge in a BALB/c mouse model.Construction of eukaryotic expression vector The main protective antigen SAG1 of T.gondii was amplified by PCR,and cloned into eukaryotic expression vector pcDNA3.1,which was identified by restriction enzymes digestion and sequence analysis,to construct eukaryotic expression vector pcDNA/SAG1.Construction of rPRV/SAG1 SAG1 expression cassette containing CMV promoter and BGH ployA signal was obtained by MluⅠand PvuⅡdigestion,and subcloned into the SphⅠ/NdeⅠsite of the transfer vector p8KD deleting PK/gG gene of pseudorabies virus to generate the intermedial transfer vector p8KD/SAG1. The recombinant plasmid p8KD/SAG1 was co-transfection with the genome of recombinant virus rPRV/LacZ digested with EcoRI into Vero cell using Lipofectamine^TM 2000 reagent.A recombinant PRV recombinant containing SAG1 of T.gondii,designated as rPRV/SAG1,was constructed after several cycles of blue plaque purification.Identification of rPRV/SAG1 The genome DNA and total RNA from rPRV/SAG1 was extracted, respectively,and PCR and RT-PCR were preformed using the specific primers for T.gondii SAG1.The results shown that the expected fragment was to be found.Western blotting and indirect immunofluorescence assay (IFA) indicated that the SAG1 expressed by rPRV/SAG1could be recognized by the positive serum against T. gondii.And the rPRV/SAG1 was demonstrated to have good genetic stability.Immunization experiment To test the safety and efficacy of vaccine,animal immunity test was performed in BALB/c mice.The total IgG and cytokines of IL-2,IL-4,1L-10,IFN-γwere determined by ELISA.The results indicated the pcDNA/SAG1 and rPRV/SAG1 could induce higher IgG antibody;and produce higher levels of IL-2 and IFN-γin mice,compared with the control group.The challenge experiments indicated that there was an obvious mortality delay in all the immunized groups,and rPRV/SAG1 could produce partial protection against virulent strain challenge.These results demonstated that expression of protective antigens of T.gondii in PRV Bartha K-61 is a novel approach towards the development of a vaccine against animal toxoplasmosis.