| Bovine viral diarrhea virus(BVDV)is one of the most important viruses affecting the cattle industry worldwide.The International Committee on Taxonomy of Viruses divides BVDV into three genotypes,BVDV-1,BVDV-2 and BVDV-3(Ho Bi-like),of which BVDV-1 is the earliest discovered in my country and has the widest spread.The main way to prevent bovine viral diarrhea-mucosal disease in my country is to inoculate inactivated vaccine,but due to the rapid mutation rate of BVDV,the protection of existing vaccines is reduced.The E2 protein(gp55)of bovine viral diarrhea virus is the main antigenic protein that induces neutralizing antibodies and stimulates protective immune responses in the body.It is the best choice for studying bovine viral diarrhea virus subunit vaccines.In this experiment,the E2 gene of BVDV-1 GS5 strain was selected as a candidate gene,and the intracellular region of E2 protein was expressed by the baculovirus insect cell expression system.After purification by nickel column,a higher purity E2 protein was obtained,which was compatible with ISA 206 immune adjuvant.The vaccine was prepared into subunit vaccine and immunized calves,and the immunization effect of recombinant E2 protein was evaluated by indirect ELISA and neutralization test.The specific test results are as follows:1.Find part of the E2 gene sequence of BVDV-1 from Gen Bank,construct a gene evolutionary tree and conduct homology analysis with the molecular evolution genetic analysis software Mega7.0,and use DNAstar for antigenicity,hydrophilicity and surface accessibility analysis,using TMHMM-2.0 and Signal P-5.0 to predict the transmembrane region and signal peptide of the E2 gene.Finally,the intracellular region of the E2 gene of the BVDV-1 GS5 strain was selected as the recombinant expression gene,and bee venom(HBM)signal peptide and 8histidine tags(8his)were added to the amino terminus and carboxyl terminus of the E2 intracellular region of the GS5 strain,respectively,and the constructed HBM-E2-8his fusion sequence was codon-optimized and synthesized by a bio company.2.The p Bac PAK9-HBM-E2-8his recombinant vector was constructed by the method of homologous recombination,and the recombinant baculovirus BVDV-1-E2 was obtained after cotransfection with the linearized baculovirus into Sf9 insect cells.SDS-PAGE and Western blot analysis confirmed that E2 protein could be secreted and expressed.Through the groping of the docking dose and expression time,it was determined that the optimal dose of recombinant baculovirus BVDV-1-E2 to express E2 protein was 1.5 MOI,and the optimal expression time was96 hours.Through nickel column purification,recombinant E2 protein with higher purity can be obtained.3.Prepare subunit vaccine with ISA 206 adjuvant,immunize 2-6 month old calves with recombinant E2 protein 50 μg/head and 100 μg/head and collect serum regularly.Indirect ELISA test results showed that both groups of subunit vaccines could induce BVDV-specific antibodies in calves.BVDV-1-specific antibodies could be detected 14 days after the first immunization,and the antibody level reached the highest value at 21 days after the second immunization.The results of the neutralization test showed that 21 days after the second immunization,the average neutralizing antibody titer of the calf serum in the low-dose immunization group was 1:223,and the highest neutralizing antibody titer was 1:512.The neutralizing antibody titer is 1:676,and the highest neutralizing antibody titer can reach 1:1024.In conclusion,the E2 protein was successfully expressed by the baculovirus insect cell expression system using the E2 gene sequence of the GS5 strain,the dominant domestic isolate of BVDV-1,as the target gene.E2 protein subunit vaccine immunization test of calves showed that recombinant E2 protein had good immunogenicity and could induce high levels of neutralizing antibodies in calves.The results provide experimental evidence for the development of subunit vaccines for bovine viral diarrhea virus. |
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