Studies On Japanese Encephalitis Virus NS1 Subunit Vaccine

Japanese encephalitis(JE)is an insect-borne zoonotic disease caused by Japanese encephalitis virus(JEV)infection.The disease can cause fatal encephalitis in humans,with a fatality rate of 30%.Nearly 50% of survivors are having neurological sequelae;at the same time,the disease can cause reproductive disorders in breeding swine,mainly causing miscarriage and stillbirth in pregnant sows.Boar orchitis and semen quality decline in breeding boar,seriously endanger the pig industry.At present,the swine vaccine sold on the market is a live attenuated vaccine,which has the potential threat of anti-virulence,so it is of great significance to develop a new safer vaccine.NS1 is an important non-structural protein of JEV,can induce a humoral and cell-mediated immune response,and through soluble complement binding activity induce cross-protection,avoiding the antibody-dependent enhancement(ADE)effect.The purpose of the study was to prepare a subunit vaccine by expressing the NS1 protein of JEV and evaluate its immune effect in the animal model.The details are as follows:1.Expression and purification of the NS1 protein of JEVJEV(GX1209 strain)was proliferated,RNA was extracted,and the NS1 gene was amplified by RT-PCR.The NS1 gene was inserted into the lentiviral expression vector,and then the 293 T cell line stably expressing NS1 protein was constructed by co-transfection and drug screening.The recombinant NS1 protein was purified by nickel column affinity purification.The results showed that the expression of NS1 protein in the 293 T cell line was low,which could not meet the requirements of subunit vaccine preparation.To obtain a large amount of NS1 protein,the study continues to try the baculovirus expression system.The NS1 gene was cloned into the baculovirus expression vector to construct the recombinant baculovirus r Bacmid-JEV-NS1.Then the recombinant baculovirus was transfected into sf9 cells.The recombinant baculovirus was successfully constructed by Western Blot and IFA assay.The High5 cells were infected by recombinant baculovirus.The NS1 protein was purified by nickel column in the culture medium,and the purification effect of NS1 protein was verified by SDSPAGE and Western Blot assay.The results showed that NS1 protein could express in the baculovirus expression system and could be secreted into culture medium.2.Manufacture of inactivated vaccine and subunit Vaccine of JEVTo compare the immune effect of JEV NS1 subunit vaccine and inactivated vaccine,firstly we need to prepare inactivated vaccine of JEV.The study explored the conditions of suspension culture of ST cells and established the technological parameters for the proliferation of JEV GX strain by suspended ST cells,And then,the JEV GX strain was inactivated by β-propiolactone.The inactivated vaccine of JEV was manufactured by adding VG201 adjuvant.Secondly,the purified NS1 protein was combined with Freund adjuvant to manufacture the NS1 subunit vaccine.3.Evaluation of immune protective efficacy of NS1 protein subunit vaccineEvaluation of immune protective effect of JEV NS1 protein subunit vaccine using mice models.Firstly,four-week-old C57BL/6 female mice were randomly divided into six groups:PBS control group,commercial attenuated vaccine group,inactivated vaccine group,and NS1 protein subunit vaccine group including 50 μg vaccine group,100 μg vaccine group,and 200μg vaccine group.Mice were immunized by subcutaneous injection of the neck.Among them,the attenuated vaccine group was immunized only once,and the other groups were immunized once every two weeks.Secondly,the antibody titer of NS1 protein in serum was detected by ELISA assay after the first and second immunization.The results showed that NS1 antibody was produced in all groups except the PBS group,and the level of NS1 antibody was positively correlated with the dose of the NS1 protein.On day 14 of post-secondary immunization,all mice were challenged with JEV P3 strain.On day 7 post-challenge,the PBS group showed clinical symptoms such as mental depression and general tremors.At this time,the viral load and the level of inflammatory factors in the brain tissue of mice were detected.The results showed that a large number of JEV appeared in the brain tissue of mice in the PBS group,and the expression of inflammatory factors increased significantly,while JEV could hardly be detected in the brain tissue of mice immunized with the vaccine,and the expression level of inflammatory factors was significantly lower than that in PBS group.After that,the brain tissue was stained with HE and IHC,and the results showed that the pathological changes in brain tissue in the vaccine group were significantly less than those in the PBS group.The results of the challenge protection test showed that the protective efficiency of JEV NS1 protein subunit vaccine in mice reached 90%,while that of inactivated Japanese encephalitis vaccine and commercial attenuated vaccine reached 100%.In summary,the JEV NS1 protein was obtained by the baculovirus expression system,and the subunit vaccine of NS1 protein was manufactured.In the mouse model,the study confirms that the JEV NS1 protein subunit vaccine has a good immune protection effect and has the potential to be developed as a new type of Japanese encephalitis virus vaccine.