Evaluation Of Immune Efficacy Of CSFV E2 Subunit Vaccine And Study Of Itsefficacy Evaluation Of IL-2 As Adjuvant

Classical swine fever(CSF)is one of the most important infectious diseases restricting the development of pig industry in China all the time.Although we have bred an attenuated vaccine,which is very safe and has a good immune effect,but CSF breaks year by year instead of disappearing.Which contributed to this is that there is no way to distinguish immunized pigs from unimmunized ones in general farms.All in all,there is an urgent need for a candidate way to distinguish immunized pigs from unimmunized ones.So in my study,we confirmed the protective efficacy of the E2 subunit vaccine expressed by baculovirus builded in our lab before.Further on,we builded another recombinant baculovirus which is integrated into the IL-2 gene.The expressed IL-2 could be used as a cytokine adjuvance.1.Evaluation of immune efficacy of CSFV E2 subunit vaccine in pigsBased on the recombinant baculovirus expressing E2 protein of CSFV,which was builded in our lab before,we expanded an experiment with pigs to test the immune efficacy of E2 subunit vaccine.The recombinant baculovirus Ac-CSFV-E2 was used to infect sf9 cells,and cells were collected by centrifugation 96 hours later.Then E2 protein was quantified by Western blot analysis and emulsified the turbid liquid with ISA 201 adjuvant.The final product contains 40μg E2 proteins per immunizing dose(2ml).Pigs were immunized with CSFV E2 subunit vaccine and boosterd 20 days later.Then pigs were challenged with CSFV Shimen strain.ELISA antibody levels and CSFV copies number in blood and other tissues were detected though all the process.Pathological changes in pigs after challenge were observed by HE staining and immunohistochemistry.The experimental results showed that pigs immunized with E2 subunit inactivated vaccine did not show an elevated body temperature and skin lesions,but kept good mental state and appetite after challenged with velogen strain.The antibody inhibition rate in serum produced by CSFV was higher than 70%three weeks after immunization.Fluorescent quantitative RT-PCR results showed that the copy number of classical swine fever virus was under detection in whole blood;histopathological observation of immunity showed no pathological changes in all organs.The experiment confirmed that CSFV E2 subunit vaccine expressed by recombinant baculovirus can protect pigs against virulent strain infection.2.IL-2 as E2 subunit vaccine cytokine adjuvantAfter testing and verifying the protective effect of the E2 subunit vaccine expressed by baculovirus,we builded another recombinant baculovirus expressing swine IL-2 which can be used as a cytokine adjuvant.Then we explain another experiment to test whether IL-2 expressed by recombinant baculovirus can improve the immune efficacy of E2 subunit vaccine.The expression of IL-2 was detected by western-blot.Expression time was optimal.Then protein was mixed with equal volumes of adjuvant ISA 201.Animal experiments were performed in rabbits.Rabbits were Immuned with E2 protein expressed by baculovirus and IL-2 protein,and rabbits were boostered 21 days later.Control groups were immunized with baculovirus expression of E2 protein only and commercial vaccine.After 42 days,rabbits were challenged with commercial cell source 100 RID50 C strain.All rabbits were killed 14days after challenge.During the whole process,rabbits’body temperature was detected by measuring the rectal temperature every 6 hours,serum was collected to detect the specific antibody against E2 protein.Other detection index including copies number of CSFV in whole blood and the expression amount of IFN-y and IL-4 were detected every two days.Data was analysed to test the effectiveness of IL-2 on immunity of E2 subunit vaccine.After the attack,,body temperatures were stable in the whole progress except the PBS group.The copy number of CSFV in blood of different time was detected by fluorescence quantitative RT-PCR.And CSFV copies were under detection in all groups expect for PBS group.The results show that in the rabbit model,porcine IL-2 E2 subunit vaccine did not show obvious immune enhancement effect.