| Bovine viral diarrhea/mucous membrane virus(BVDV)causes serious respiratory tract,gastrointestinal tract and reproductive multi-system diseases in cattle,ranging from subclinical and transient infections to persistent infections,which has brought huge economic losses to the world cattle industry.Vaccination is one of the main prevention strategies for the disease.Currently,the commonly used vaccine is the whole BVDV inactivated vaccine.However,with the continuous variation of the virus,the immune effect of inactivated vaccine can not meet the clinical prevention and control of the disease,and there is an urgent need to develop a new BVDV vaccine.BVDV E0,E2 and NS5A are important antigenic proteins,which are expected to be developed into new subunit vaccines.Objective:To explore the use of escherichia coli recombinant expression of BVDV NS5A,E0,E2,mE2T(tandemE2)protein to develop subunit vaccine,and to improve the immune protection effect of inactivated vaccine by the combination of antigen protein and inactivated vaccine,and to provide theoretical basis for the development of new BVDV subunit vaccine.Methods:(1)BVDV NS5A,E0,E2 and mE2T plasmids were transformed into escherichia coli.After induced expression by IPTG,ni-nta Resin nickel column was purified and prepared.(2)Purified antigen protein was mixed with Freund complete adjuvant to prepare subunit vaccine.Mice were immunized with E0 subunit vaccine,E2 subunit vaccine,commercial inactivated vaccine,E2 combined with commercial inactivated vaccine and PBS control group(3)for 3 times,with an interval of 14 days for each immunization.ELISA antibody titer and white blood cell count were performed for each immunization.(4)Before challenge,immune indexes in serum and body weight of mice were detected using mouse ELISA kit.After challenge,the changes of immune indexes and body weight of mice were detected again,and the white blood cell count after challenge was carried out.(5)The spleen of mice was removed aseptically to separate splenic lymphocytes,and the proliferation of lymphocytes was detected by MTT.The neutralization test of antibody was carried out by immobilizing the virus and diluting the antiserum.On the7th and 14th day after challenge,mixed tissue samples were collected for QRT-PCR analysis of BVDV copy number expression.(6)BVDV-negative sheep in each group were immunized with vaccine for 3times,with an interval of 14 days.After each immunization,blood samples were collected from jugular groove,ELISA antibody titer detection and white blood cell count.(7)Before challenge,sheep ELISA kit was used to detect the immune indexes in sheep serum and the weight of sheep.On the 14th day after the third immunization,sheep were challenged.After the challenge,the changes of immune indexes and body weight of sheep were detected again,and the white blood cell count after the challenge was conducted.(8)The whole blood lymphocytes of sheep were isolated and the proliferation of lymphocytes was detected by MTT.The neutralization test of antibody in sheep serum was carried out by diluting antiserum with fixed virus.On the 7th and 14th days after challenge,RNA was extracted from whole blood of sheep and reversed into c DNA for QRT-PCR,and the expression changes of BVDV copy number were analyzed.Results:(1)high concentration of bvdv-1a virus was obtained,and the concentration of bvdv-1a virus was1×10~8/ml after three times of TCID50 determination.(2)BVDVNS5A,E0,E2,mE2T proteins were purified by Ni-Nta Resin nickel column,and the reactivity was good by WB test.(3)After three times of immunization,the antibody level of BALB/C mice was significantly increased,and the antibody level of combined vaccine was significantly higher than that of single component subunit vaccine and inactivated vaccine.But the white blood cell count of the blank control group was significantly lower than that of each vaccine group after challenge.(4)The immune indexes and white blood cell count of mice were significantly increased compared with the blank control group.The immune indexes after challenge were significantly different from those before challenge,and the body weight of each group was not significantly changed before and after challenge.(5)Cell test:MTT detection of lymphocyte proliferation and antibody neutralization test by fixed virus-dilution antiserum method verified that the corresponding protein of mouse spleen lymphocyte and the stimulation of inactivated virus can stimulate the proliferation of spleen lymphocytes.Mice serum BVDV virus had good neutralization ability.(6)Experiments on sheep susceptible to BVDV showed that sheep immunized with subunit vaccine could still elicit higher antibody levels.White blood cell counts in the blank control group were also significantly lower than those in each vaccine group.(7)The immune indexes of sheep after challenge were significantly improved compared with those before challenge.The weight of sheep in all vaccine groups had no significant difference compared with that before challenge,but the weight of sheep in blank control group decreased significantly after challenge.(8)Cell test:MTT detection of lymphocyte proliferation and antibody neutralization test by fixed virus-dilution antiserum method verified that lymphocytes isolated from sheep whole blood could stimulate splenic lymphocyte proliferation under the stimulation of corresponding protein and inactivated virus.Sheep serum had good neutralization ability to BVDV virus.Qrt-pcr verified that BVDV subunit vaccine had certain protective effect on mice and sheep,and the BVDV copy number in mice decreased significantly compared with the control group.Among them,the copy quantity of BVDV virus in qRT-PCR verification of combined vaccine was very low.Conclusion:BVDV functional proteins NS5A,E0,E2 and ME2T were successfully expressed in EScherichia coli system.It was confirmed that the vaccine could significantly increase the antibody level in mice.The vaccine also significantly increased antibody levels in sheep tested in susceptible animals.BVDV virus replication was reduced by qRT-PCR and cell and animal tests.The effect of the combined vaccine group was better than the other groups,which laid a foundation for the preparation of BVDV vaccine. |
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