Construction And Immunogenicity Of Nucleic Acid Vaccine Of Canine Distemper Virus

Canine distemper(CD)is an acute and highly contagious disease,which resulted from canine distemper virus(CDV).CD poses a serious threat to the development of animal husbandry(like dog industry,fur animal husbandry,and other economic animal breeding industry)and the stability of natural ecosystems(like threat to wild animals),and causes incalculable losses.Most of the CD vaccines currently available in the market are uesd dogs,only two vaccines are used in fur animals(mink and fox).Vaccines can effectively control CD in dogs,but the safety and immune efficacy of endangered species(such as Siberian tiger,south China tiger and red panda)are still highly controversial.Emerging genetic engineering vaccines in recent years not only have the safety of inactivated vaccines or subunit vaccines,but also have the characteristics that attenuated vaccines can induce cellular immune responses.Therefore,it provides a new idea for the study of CD vaccine and points out a new direction.This study mainly sequenced and analyzed the H gene and F gene of CDV isolated from the Siberian tiger and red panda.To construct recombinant eukaryotic expression plasmids pcDNA-F and pcDNA-H,the H gene and F gene of tiger-origin virus were inserted into eukaryotic expression vector pcDNA3.1(+),and the immunogenicity of recombinant plasmids were evaluated.Sequence analysis of the results: four CDV H genes 1824 bp in length,encoding 607 amino acids,contain nine potential N-glycosylation sites,with the characteristics of 549 Y.Similarity analysis revealed a high degree of nucleotide similarity(99%)with giant panda-origin CDV strain(AF178038)and tiger-origin strain(KM386683).Compared with the standard strain A75/17,the similarity was 96.5%~96.9%.Moreover,compared with the traditional vaccine strain(Onderstepoort strain and Recombinant Snyder Hill strain),the similarity was 92.2%~92.6% and 91.9%~92.3%,respectively.Similarity analysis showed that there was no significant difference among the canine-origin strain(GZ0803,KC667066),the canine-origin strain(GZ0804,KC667068),the fox-origin strain [HeB(07)1,EU327874],the raccoon-origin strain [HeB(07)2,KP064126] and mink-origin strains(KC427278)with a high degree of nucleotide similarity(99%).Compared with the standard strain A75/17,the similarity was 93.9%.In addition,compared with the Onderstepoort vaccine strain and the traditional vaccine strain CDV3,the similarity was 90.4%~90.5% and 90.4%~90.6%,respectively.In this study,two pairs of specific primers were designed to amplify the F gene and H gene of tiger-origin CDV.The sites of Xho? and Xba? were inserted at the 5' end of the upstream primer and the 3' end of the downstream primer,respectively.And the FLAG tag sequence was added after the upstream primer start codon ATG.The target fragments were amplified and the eukaryotic expression vector pcDNA3.1(+)by endonuclease digestion with Xho? and Xba?,and the recombinant expression plasmids pcDNA-F and pcDNA-H were obtained after the treatment of T4 DNA ligase.The immunogenicity of pcDNA-F and pcDNA-H was co-verified by the experiments in vivo and in vitro.The experiments in vitro transfected BHK-21 cells with pcDNA-F and pcDNA-H eukaryotic expression plasmids using liposome-mediated methods,indirect immunofluorescence and Western blot were used to detect the expression of recombinant plasmids in vitro;the test in vivo was carried out by immunizing BALB/c mice with muscle and detecting the expression of the recombinant plasmid in vivo before and after four-time immunization.The results showed that the eukaryotic expression vectors pcDNA-F and pcDNA-H were successfully constructed,and all of them were immunogenic,but the induction of antibody titers remained to be further improved.