| Bovine viral diarrhea virus(BVDV)is a single-stranded positive-sense RNA virus encapsulated by an envelope,belonging to the genus Pestivirus of the family Flaviridae.BVDV can cause persistent infection after invading the body,causing incalculable losses to the cattle industry worldwide.At present,the main measure to prevent BVDV is to vaccinate with good immune effect.In China,inactivated vaccines are mainly used for immunization and prevention.However,no E2 subunit vaccine of BVDV is put into production and use.In this study,the E2 gene of BVDV epidemic strains in Sichuan was analyzed,some E2 genes of BVDV-1 were expressed in prokaryotic cells and subunit vaccines were prepared,and their immune effects were evaluated.The results were as follows:1.Sequence analysis of E2 gene of BVDV epidemic strains in SichuanBVDV was detected by RT-PCR in 275 cattle fecal samples collected from three regions of Sichuan in 2021.The results showed that the total detection rate of BVDV was 27.64%(76/275),and evolutionary result analysis showed that all BVDV-1a subtypes.The complete E2 gene of 9 strains was successfully amplified from 15 positive samples selected from 3regions and cloned and sequenced.The results showed that the nucleotide length between E2 genes of 9 strains was about 1073 ～ 1100 bp,the homology was about 72.4% ～ 99%,and the amino acid homology was about 61.5% ～ 100%.Amino acid mutation site analysis and antigenic determinant prediction were performed on the E2 gene of the amplified 9 E2 proteins and the vaccine strain SMU-Z6/1a/SC/2016 in our laboratory.The results showed that the antigenic determinants of the 9 positive strains(RL6,LZ5,R3-5,LZ3,R1-1)were identical to those of SMU-Z6/1a/SC/2016 in residues 146-312.The results showed that the nine E2 genes had the closest genetic relationship with the vaccine strain SMUZ6/1a/SC/2016 isolated in our laboratory,and all of them clustered in a clade with BVDV-1a and had consistent genotypes.In order to understand the recombination phenomenon of E2 gene in 9 strains,gene recombination analysis was performed.The results showed that only the E2 gene of strain GK2 was recombined,but the strain R3-14 was amplified by the main parent based experiment after recombination,and the secondary parent was SMUZ6/1a/SC/2016,all of which were BVDV-1a type.The results of this experiment showed that BVDV-1a was widespread and prevalent in cattle herds in Sichuan,providing a theoretical basis for the prevention and control and clinical diagnosis of BVDV in Sichuan.2.Prokaryotic expression,purification and indirect ELISA of BVDV-1 E2 gene proteinAccording to the E2 sequence results,part of the E2 gene of vaccine strain SMUZ6/1a/SC/2016 isolated in our laboratory was selected for amplification,induced expression,solubility analysis,purification and refolding,and identified by Western blot.The results showed that the recombinant prokaryotic expression vector of E2 was successfully constructed.The recombinant strain successfully expressed E2 recombinant protein in the form of inclusion body after induced with 0.3 m M IPTG at 37℃ for 6 h.The size was about40 KDa.It could react with rabbit anti-SMU-Z6/1a/SC/2016 hyperimmune serum and had good reactogenicity.In order to establish an indirect ELISA method for the detection of BVDV-1 antibody,the BVDV E2 recombinant protein prepared in this study was used as antigen,and the conditions such as antigen coating concentration and primary antibody were optimized to establish an indirect ELISA method.The optimization results were that the coating concentration of E2 protein was 1 μg/m L and the dilution of primary antibody was1:100,and OD450 nm ≥ 0.357 was judged as positive.This method provides a technical means for the evaluation of antibody levels induced by subsequent E2 subunit vaccines in animals.3.Evaluation of immune effect of BVDV-1 E2 recombinant protein subunit vaccineTo prepare the BVDV-1 E2 subunit vaccine,MONTANIDE ISA 201 VG BVDV-1 E2 recombinant protein subunit vaccine was obtained by fully emulsifying VG adjuvant with an equal volume of purified E2 recombinant protein,and tested.The results showed that the BVDV-1 E2 recombinant protein subunit vaccine was successfully prepared without contamination,with good stability and safety.In order to evaluate the immune effect of BVDV-1 E2 subunit vaccine,New Zealand white rabbits were immunized with 200 μg protein/rabbit,and the growth and decline of rabbit serum antibody after immunization were detected by antibody neutralization test and indirect ELISA.The results of ELISA showed that the highest antibody level in serum was3.39 after 14 days of the second immunization,and the neutralizing antibody titer reached the highest at 1:186 at 21 days of the second immunization.Beef cattle were immunized intramuscularly in the neck at a dose of 1 mg protein/head,and the growth and decline of bovine serum antibodies were detected by the same method.The results showed that the ELISA antibody titer of the collected sera after immunization of cattle was found to reach the highest 3.3671 at 14 days after the second immunization.The neutralizing antibody titer of bovine serum was determined.The neutralizing antibody titer of E2 subunit vaccine could be detected after the first immunization.The neutralizing antibody titer reached more than1:285 at 7 days after the second immunization.The antibody titer reached the highest 1:2185and the lowest 1:89 at 28 days after the second immunization,indicating that the prepared E2 subunit vaccine could effectively stimulate rabbits and cattle to produce higher levels of specific antibodies.In order to further study whether E2 subunit vaccine could induce protection in mice,in this study,SPF BALB/c mice were immunized,and 21 days after the second immunization,each mouse was intragastrically challenged with 1 m L of the vaccine strain SMUZ6/1a/SC/2016 isolated in our laboratory,and the blood routine and fecal viral load of the mice were measured after challenge.The results showed that on the 5th and 9th day after challenge,the contents of white blood cells,lymphocytes and platelets in the E2 subunit vaccine group were higher than those in the inactivated vaccine blank challenge group and lower than those in the negative control group.The fecal virus load of mice after challenge was determined by quantitative fluorescence RT-PCR.The results showed that the fecal copy number of mice in the E2 subunit vaccine group was significantly reduced by 9-fold compared with the negative control group(p < 0.01),indicating that the E2 subunit vaccine prepared in this study could stimulate mice to produce specific antibodies and effectively inhibit the massive replication of BVDV virus in mice.It provides some reference data for the preparation of BVDV E2 subunit vaccine. |
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