| Neosporosis is an important economical protozoosis caused by neospora caninum to cattle and other animals, which can cause cow abortion and serious economic losses to the cattle industry. There arent effective drugs and commercialization vaccines to prevent neosporosis currently. With viral vectors vaccines having been extensively studied, adenovirus vector vaccine has also been applied in the field of parasite. In addition, DNA vaccine priming and the virus vector vaccine booster immunization strategy shows good results in the induction of humoral and cellular immune aspects.According to Neospora AMA1gene sequences in GenBank (AB265823.1), two pairs of specific primers were designed and bovine caninum Jilin strain AMAl gene was amplified by RT-PCR method. AMA1was cloned into pMD18-T simple vector, then the correct AMA1gene was subcloned into the eukaryotic expression vector pVAX-1and pCR259adenovirus shuttle vector. The pVAXl-AMAl eukaryotic expression plasmids were transferred into Vero cells, and its expression product was detected by indirect immunofluorescence and western blot. DNA vaccine were prepared by extracting large pVAX1-AMA1plasmids using the kit.The recombinant adenovirus shuttle plasmid pCR259-NcAMA1were transformed into HighQ-1Transpose-AdTM294and HighQ-1Tm competent cells successively to construct and purify caninum AMA1recombinant adenovirus expressing plasmid294-AMA1. Linearized294-AMA1plasmids were transfected into QBI-HEK293cells by liposome to package adenovirus Ad5-NcAMA1. Caninum AMA1gene expression product was detected by IF AT and Western blot methods in QBI-HEK293cells. The Ad5-NcAMA1obtained titer was5.62×109/mL and cultured heavily for preparation adenoviral vector vaccine.According immunization strategy group, Ad5-NcAMA1adenovirus immunization group and pVAXl-AMAl nucleic acid vaccine immunization group, DNA vaccines and adenovirus vaccines were tested on BALB/c mice, setting the PBS control group. Mouse IgG, IgGl and IgG2a antibody levels were measured by indirect ELISA and IFN-y and IL-4cytokine levels were also measured through ELISA kit.Besides, CD4+, CD8+T lymphocyte subsets in mice were analysised by flow cytometry.The results showed that the bovine caninum Jilin strain AMA1gene size amplified is1695bp, which has99.9%nucleotide homology and99.8%amino acid homology with AMA1gene in GenBank (AB265823.1). pVAX1-AMA1recombinant eukaryotic expression plasmid and adenoviral vector plasmid can express caninum AMA1protein, protein molecular weight of which was about68kDa. Ad5-NcAMA1viral titer was5.62×109/ml. Animal experiments showed that IgG antibody levels of Prime-Boost immunization strategy group was significantly higher than other three groups (P<0.01); IgG1levels of Prime-Boost immunization strategy group were significantly higher than Ad5-NcAMA1adenovirus group and pVAX1-AMA1DNA vaccine group (P<0.05), and IgG2a levels were significantly higher than pVAX1-AMA1DNA vaccine group (P<0.05). IL-4cytokine levels of Prime-Boost immunization group was significantly higher than Ad5-NcAMA1immunization group (P<0.05) and pVAX1-AMA1DNA vaccine group (P<0.01), and IFN-y was significantly higher than pVAX1-AMA1DNA vaccine group (P<0.05),and no significant differences with Ad5-NcAMA1immunization group. CD4+levels of Prime-Boost immunization strategy group were significantly higher than Ad5-NcAMA1adenovirus group (P<0.05)and pVAX1-AMA1DNA vaccine group (P <0.05). CD4+/CD8+T of Prime-Boost immunization strategy group was significantly higher than Ad5-NcAMA1adenovirus group(P<0.05) and pVAX1-AMA1DNA vaccine group (P<0.01).CD8+T of Prime-Boost immunization strategy group was significantly higher than PBS group(P<0.05). Compared with Ad5-NcAMA1immunohistochemistry and pVAX1-AMA1DNA vaccine group, Prime-Boost immunization strategy enhanced humoral and cellular immunity in mice in certain extent. The experiment provides a new idea on a new novel vaccines and immunization strategies research to neosporosis. |
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