| Pseudorabies virus(PRV)and Porcine reproductive and respiratory syndrome virus(PRRSV)are two important pathogens affecting the world pig industry development.They cause pseudorabies(PR)in pigs and Porcine reproductive and respiratory syndrome(PRRS).PR and PRRS are the major viral infectious diseases that cause reproductive failure in sows,respiratory system disorders in fattening pigs,and fatal neurological disorders in newborn piglets.These two kinds of infectious diseases are widely found in various types of pig farms in China,leading to huge economic losses to the pig husbandry and restricting the development of China's pig industry seriously.Outbreaks of PRRS in pigs have occurred in many countries and regions of the world,resulting in economic losses and serious biosecurity issue.In order to control the epidemic and outbreak of PRRS,every country has invested large amounts of funds and resources and taking passtive measures in the world.It is the key to control infection diease to insist that prevention is more important than treatment.Despit various PRRS vaccines since its outbreak in china,inactivated and attenuated live vaccines are still the mainstay.The protection rate of PRRS vaccine is not high,it can only play a partial protection for PRRSV infection.To make up for the shortcoming of PRRS vaccine,it is imperative that developing new PRRS vaccine.In recent years,with the development of genetic engineering technology,the research and development of PRRS genetic engineering vaccines has become a research focus.Compared with inactivated vaccines and attenuated live vaccines,genetically engineered vaccines have more advantages as new vaccines.Recombinant live vector vaccines and nucleic acid vaccines are the key to the development of PRRS genetic engineering vaccines.Since the end of 2011,pseudorabies has re-emerged in pig farms that have been vaccinated with live vaccines.It was shown that PRV had varivanted at different genetic locus by virus islation and sequencing analysis and resulting in virulence to change.Combined with the results of clinical investigation,it was found that PR attenuated live vaccine in the market cannot provide 100% protection against the current PRV variant strains.Therefore,it is particularly important to develop a novel PR attenuated live vaccine based on current epidemic strains.Based on the successful application of PR gene deleted vaccines,the use of PRV strains as vectors to construct recombinant PRV with expressing exogenous immunogenic genes has become a research focus of pseudorabies gene engineering vaccines.In this study,membrane proteins GP5 and M related to PRRSV neutralizing antibody production were selected as the inserted foreign immunogenic genes.GP5,M,modified GP5(GP5m)and porcine interleukin-18 genes(IL-18)was cloned into the eukaryotic expression vector PTK and five recombinant plasmids were constructed:(1)p TK-GP5-M(2)p TK-GP5-M-IL-18(3)p TK-GP5m(4)p TK-GP5m-IL-18(5)p TK-IL-18.Initial immunity efficacy tests were performed in rabbit.On this basis,five recombined pseudorabies virus r PRV(1)r PRV-g E-/TK-/GP5+/M+(2)r PRV-g E-/TK-/GP5+/M+/IL-18+(3)r PRV-g E-/TK-/GP5m+(4)r PRV-g E-/TK-/GP5m+/IL-18+(5)r PRV-g E-/TK-/IL-18+ were constructed by homologous recombination in 293 T cells and immunogenicity study were performed in this laboratory.To study the immunogenicity of the five recombinant viruses,45 female test mice were randomly divided into 9 groups of 5 in each group.group 1: r PRV-g E-/TK-/GP5+/M+;group 2: r PRV-g E-/TK-/GP5+/M+/IL-18+;group 3: r PRV-g E-/TK-/GP5m+ group 4: r PRV-g E-/TK-/GP5m+/IL-18+;group5 r PRV-g E-/TK-/IL-18+;group6: 105.5TCID50 r PRV-TK-/g E-;group7: 105.41 TCID50 PRRSV VR2332;group8: 105.5 TCID50 PRV HB-98;group9: DMEM.The immunization route was subcutaneous injection plus intranasal immunization and boosted once every 4 weeks.The blood was collected on the day of the first immunization.Indirect ELISA was performed once a week to detect the specific anti-PRRSV ELISA antibody levels.Neutralization test was performed biweekly to detect the levels of anti-PRRSV and PRV-specific neutralizing antibodies;Spleen T lymphocytes were isolated from mice on 4 weeks after the second immunization.T lymphocyte proliferation in vitro and the expression assay of T lymphocyte cytokines IFN-??IL-4?IL-10 were performed to evaluate the cellular immunity level.The anti-PRRSV specific neutralizing antibody assay results showed that the anti-PRRSV neutralizing antibody level of the PRRSV VR2332 vaccine strain reached 3log2 at 28 days after the first immunization;The neutralizing antibodies levels of r PRV-g E-/TK-/GP5+/M+,r PRV-g E-/TK-/GP5+/M+/IL-18+ and r PRV-g E-/TK-/GP5m+/IL-18+ reaching 3log2;r PRV-g E-/TK-/GP5m+ and r PRV-g E-/TK-/IL-18+ anti-PRRSV neutralizing antibodies were not detected.The anti-PRRSV specific ELISA assay results showed that the experimental group r PRV-g E-/TK-/GP5+/M+,r PRV-g E-/TK-/GP5+/M+/IL-18+,r PRV-g E-/TK-/GP5m+/IL-18+,r PRV-g E-/TK-/GP5m+ anti-PRRSV ELISA Ig G antibody levels without significant difference peaked one week after the second immunization and persisted for 2-3 weeks.In contrast,ELISA Ig A antibodies are lower than Ig G in serum.The anti-PRV specific neutralizing antibody assay results showed that neutralization antibody of PRV HB-98 vaccine strain reached 4log2,;and the neutralizing antibody in the r PRV-TK-/g E-group was 5log2;PRV-g E-/TK-/IL-18+ test group is higher,and the level of neutralizing antibody up to 6log2.The results of the assay showed that clinically isolated strains of the PRV epidemic strain with gene-deleted can produce higher anti-PRV neutralizing antibodies and IL-18 can effectively enhance the body's specific humoral immunity.The assay of T-lymphocyte cytokines expression showed that the proportion of CD4+ and CD8+ T-lymphocytes in the IL-18 co-expressing group increased and the cytokine expression level was higher,resulting in more intense humoral,cellular,and mucosal immune responses.This study lays a solid foundation for the development of recombinant live vector vaccines that are effective against PRRS and PR for vaccination through the mucosal immune route. |
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