| Bovine viral diarrhea-mucosal disease（BVD/MD） is an infectious disease caused bybovine viral diarrhea-mucosal disease virus（BVDV）, which can infect many kinds of animalssuch as cattle, sheep, deer, pigs and others. It is one of the serious diseases which harm to thehealthy development of animal breeding industry for a long time, which has been classified at Bclass infectious diseases by Office International des Epi-zooties. In view of the currentepidemic situation and the prevention and control of BVD, the safe and effective BVDVvaccine is urgently to development. E0and E2protein are two major protective antigens ofBVDV, which can be used for researching genetically engineered vaccine. In recent years,scholars have done a lot of beneficial work in research and development of new BVDVvaccines by using of different genes, expression systems and vaccine forms. In this study, E0and E2gene were selected to construct recombinant BCG, and then carried out immunogenicityresearch of BVD vaccine.In this research, E0gene of C24V standard strain was artificially synthesized by using ofcenter template method after truncated and carried out codon optimization, which was fusedwith E2truncated gene of changchun184specific strain splicing by overlapping extention, andwere subcloned into the expression vector pMV261and pMV361respectively to constructrecombinant expression plasmids. Then the constructed recombinant plasmids wereelectro transformed into bacillus calmette-guerin Vaccine（BCG）, which was identified byacid-fast staining and PCR of bacteria. The total protein of each recombinant BCG（rBCG）wereexpressed by heat-induced method under45℃, and analyzed by SDS-PAGE and Westernblotting. Finally, the mice were immunized with each rBCG and the rBCG-pMV361-E2whichhad been constructed in laboratory, and the specific antibody levels in serum of mice weredetected by indirect ELISA, the lymphocyte proliferations in spleen of mice were analyzed withCCK-8, the IFN-γ, IL-4, IL-10and IL-12levels in serum of mice were measured respectivelyby biotin double-antibody sandwich ELISA, the percentages of CD4+and CD8+T lymphocytesubsets in spleen of mice were detected by flow cytometry. Then the immune efficacies of eachrBCG were evaluation comprehensively.The results showed that the mE0gene and E0-E2fusion gene were amplified successfully,the pMV261-mE0, pMV261-E2, pMV261-E0-E2, pMV361-mE0and pMV361-E0-E2 recombinant expression plasmids were constructed successfully; The target proteins with size of18kDa,43.9kDa and66.5kDa were expressed in BCG successfully, and each one hadreactogenicity; After the mice were immunized with rBCG, the rBCG could stimulate mice toproduce anti-BVDV antibodies in varying degrees, the lymphocyte levels of mice had increasedafter stimulated by antigen, the change of IFN-γ, IL-4, IL-10and IL-12levels during the periodof immune showed that the rBCG could stimulate the Th1and Th2type reaction, and tend toTh2type reactions, the percentages of CD4+and CD8+T lymphocyte subsets had changed afterthe third time immune, which indicated that each rBCG could induce mice to generate humoraland cellular immune responses in a certain extent and more inclined to the former. In general,the immune effect of rBCG-pMV261-E0-E2was stronger than the rest of the group.This study confirmed that the E0-E2fusion protein can improve the immunogenicity of thesingle antigen, can be used as a candidate vaccine of BVD. The successful construction ofrBCG-pMV261-E0-E2strain lays a foundation for developing bivalent genetic engineeringvaccine which can prevent bovine viral diarrhea-mucosal disease and tuberculosis at the sametime. |
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