Development Of Genetic Engineering Subunit Vaccine Against Bovine Viral Diarrhea And Its Immune Effect Evaluation

Bovine viral diarrhea（BVD）is an acute,febrile,highly contagious infection caused by the Bovine viral diarrhea virus（BVDV）.The main symptoms of this disease are fever,diarrhea,reproductive disorders,immune dysfunction and so on.In the world,BVDV is divided into three genotypes:BVDV-1,BVDV-2 and BVDV-3,while in China,the main incidence is BVDV-1.BVDV is a member of the Distemper genus of flaviviridae.Its gene length is about 12.5kb.It is a single-stranded plus stranded RNA virus with a capsule membrane.This disease is one of the most important bovine infectious diseases in the world,with high morbidity and low mortality,causing huge economic losses every year.BVDV has 4 structural proteins and 8 non-structural proteins.The structural protein Erns（E0）is highly conserved and has neutral epitopes.Structural protein E2 is the main determinant of BVDV antigen,which is highly conserved and has neutralizing activity.Therefore,E0 and E2 structural proteins can be said to be the main immune antigens of BVDV.Most studies at home and abroad are carried out from these two proteins,and this study mainly focuses on the research and development of novel subunit vaccine of BVD.At present,the traditional attenuated vaccines used for prevention and control of BVD cannot be identified and diagnosed after immunizing animals,and there is a possibility of virulence returning,which has certain defects.Therefore,it is of great significance for the prevention and control of BVD in China to study a safe and effective vaccine against the epidemic strains.In order to develop an effective genetic engineering subunit vaccine of BVDV and lay a foundation for the effective prevention,control and identification of infection by BVD,we designed E0 sequence,E2 sequence,E2 and E0 protein fusion sequences by using CHO suspension cells to express structural proteins.The target proteins expressed were all above clear and soluble secreted,and the purity after purification could reach more than 95%.The expression products were identified by SDS-PAGE,Western blotting and other methods,and the recombinant protein was emulsified with Freustrin’s complete adjuvant and Freustrin’s incomplete adjuvant,and immunized New Zealand white rabbits showed good immunogenicity,and the animal challenge test results showed that the immunized groups could protect the body lesions.The main research contents are as follows:1.The E0 and E2 gene sequences of BVDV-1 genotype were queried in Gen Bank,and the E0 and E2 protein signal peptides and transmembrane regions were analyzed and predicted by Signal P 4.1 Server and TMHMM Server v.2.0 software.Finally,the expression strategies of BVDV-E0,BVDV-E2 and BVDV-E2-E0 proteins were determined based on the gene sequence of NADL strain of BVDV-1 standard strain.The recombinant eukaryotic expression plasmids pc DNA3.1-BVDV-E0,pc DNA3.1-BVDV-E2 and pc Dna3.1-BVdv-E0 of BVDV E0,E2 and E2-E0 were constructed successfully.2.The constructed eukaryotic recombinant plasmids pc DNA3.1-BVDV-E0,pc DNA3.1-BVDV-E2 and Pc Dna3.1-BVdv-E0 were transfected into CHO suspension cells by homologous recombination method,and the supernatant was secreted and expressed.In the transient transfection process of CHO suspended cells,three key parameters,such as the preparation of transfection complex,amount of DNA and cell density at transfection time,were optimized in sequence.The supernatant of cells on day 12 was obtained by centrifugation.SDS-PAGE identification showed that the recombinant BVDV proteins were all successfully expressed in the supernatant.2.The constructed eukaryotic recombinant plasmids pc DNA3.1-BVDV-E0,pc DNA3.1-BVDV-E2 and Pc Dna3.1-BVdv-E0 were transfected into CHO suspension cells by homologous recombination method,and the supernatant was secreted and expressed.In the transient transfection process of CHO suspended cells,three key parameters,such as the preparation of transfection complex,amount of DNA and cell density at transfection time,were optimized in sequence.The supernatant of cells on day 12 was obtained by centrifugation.SDS-PAGE identification showed that the recombinant BVDV proteins were all successfully expressed in the supernatant.3.The supernatant of CHO suspension cell culture was collected and purified by nickel column affinity chromatography.The protein purity of the samples was up to 95%by SDS-PAGE and Western blotting,and the results showed that the recombinant protein could react specifically with the positive serum of BVDV and His Tag antibody.The concentration of purified recombinant protein was detected by BCA protein quantitative kit.The concentration of E0 protein was 0.886 mg/m L,that of E2 protein was 1.228 mg/m L and that of E2-E0 protein was 1.303 mg/m L.4.This study took New Zealand white rabbits as the research object to study the pathogenicity of BVDV-NADL standard toxin.After infection,the New Zealand white rabbits showed clinical symptoms of increased body temperature and decreased body weight,and nasal and anal detoxification could be detected in the first 5 days after infection.In addition,the virus was detected in liver,spleen,lung,kidney,trachea and other organs in the first 5 days after infection,which proved that the pathogenicity test of BVDV-NADL standard virus in New Zealand White rabbits was valid.5.Animal immunization tests in this study demonstrated that the three recombinant proteins combined with Woolf’s complete adjuvant and Woolf’s incomplete adjuvant were used to immunize New Zealand white rabbits,respectively.Serum antibody was positive on the 7th day after immunization and continued to the 28th day after immunization,and the serum antibody titer level of E0 protein immunized group could reach 1:128 000.The titer level of serum antibody in the E2 group and the E2-E0 group could reach 1:1 024 000 and 1:51 000 respectively,and the serum antibodies in the three groups could react specifically with the BVDV virus infected in MDBK cells.It was further confirmed that the three purified proteins had good immunogenicity and specificity.The results of virus neutralization experiment showed that immunizing rabbits with recombinant E0 protein induced the production of virus-neutralizing antibody,and the titer of neutralization was Log₁₀（405）=2.54;immunizing rabbits with recombinant E2 protein induced the production of virus-neutralizing antibody,and the titer of neutralization was Log₁₀（413）=2.59;immunizing rabbits with recombinant E2-E0 protein induced the production of virus-neutralizing antibody.The neutralizing titer is Log₁₀（447）=2.80.6.In this animal immune challenge protection test,it was found that after infecting each immunized group of New Zealand White rabbits with BVDV-NADL standard strain by nasal spray and intravenous injection,body weight of the immunized group increased,body temperature showed no significant changes,and the number of peripheral blood white blood cells and lymphocytes showed no significant changes.The typical heat response temperature and the viral load carried in the organs of New Zealand white rabbits showed that E2-E0 immunized group<E2 immunized group<commercial immunized group<E0 immunized group<control challenge group.The viriemia period,nasal and anal detoxication period of the three protein immunized group and the commercialized vaccine immunized group of New Zealand white rabbits were significantly shorter than the control challenge group.The same blood viral load and nasal detoxification volume were significantly lower than that of the control challenge group.HE staining of liver,kidney,lung and spleen of the immune group and the commercial vaccine immune group showed no significant pathological changes,while the control group showed significant pathological changes.In conclusion,the BVDV-E0 proteins,BVDV-E2 proteins and Bv DV-E2-E0proteins prepared by CHO suspension cell culture of eukaryotic expression system have good reactivity and immunogentility in vitro and in vivo in New Zealand White rabbit model animals,and can provide good protection for the model animals.It provides the basis for the research and development of novel subunit vaccine of BVD.