Abstract:

This study aimed to construct a recombinant adenovirus expressing the structural protein VP1 of bovine enterovirus (BEV), and to perform preliminary verification of its immunogenicity and safety. The BEV VP1 gene was amplified by PCR and cloned into the adenoviral vector pDC316-CMV-copGFP. The recombinant plasmid pDC316-VP1-copGFP obtained was co-transfected with pBHGlox-△E1, 3cre into 293 cells, and then virus titer assay and RT-PCR were carried out to identify the virus; the construction of pET-32a-BEV-VP1 prokaryotic expression plasmid was constructed and BEV VP1 protein was purified after optimising the induction time and temperature; negative control adenovirus rAdv-copGFP, recombinant adenovirus rAdv-VP1, and blank control PBS were used to immunise BALB/c mice by myocardial injection to observe the clinical symptoms of mice; mice were infected with BEV after 21 days of immunisation and liver, spleen, lung, spleen and blood samples were collected from mice on day 0, 5, 10, and 15. The samples were collected from mice on day 0, 5, 10 and 15. After 21 days of BEV infection, the liver, lung, spleen, kidney and small intestine were collected on day 0, 5, 10 and 15. Tissue RNA was extracted and pathological sections were made, and the viral load in each tissue was detected and the histopathological changes were observed; the serum of adenovirus-immunised mice was tested for the response to the prokaryotic expression of the VP1 protein by Western blot; and the levels of total IgG antibody, specific antibody, and cytokine IFN-γ, IL-4 in the immunised serum were detected by ELISA. The adenoviral vector pDC316-VP1-copGFP was successfully constructed; the optimal conditions for VP1 prokaryotic expression were induced at 37 ℃ for 8 h, and pET-32a-BEV-VP1 protein was successfully purified; the serum of adenoviral-immunised mice was able to recognise the purified VP1 protein; the recombinant adenoviral immunisation was able to make the mice produce specific antibodies, which increased the levels of humoral immunity and cellular immunity. The recombinant adenovirus immunisation can make mice produce specific antibodies, thus improving humoral and cellular immunity. The recombinant adenovirus expressing BEV VP1 protein was successfully constructed in this study, and it could induce humoral and cellular immune responses in mice after immunisation, thus exerting a certain degree of protection against BEV infection, and laying a foundation for the further development of a new BEV vaccine.

Key words: bovine enterovirus, recombinant adenovirus, structural protein VP1, cellular immunity