Expression of VP1 protein of bovine nebovirus by recombinant adenovirus and evaluation of its immune effect
The aim of this study was to construct the recombinant human adenovirus rAd5-BNeV_VP1 that expresses the VP1 protein of bovine nebovirus(BNeV)and evaluate its immune effect.The recombination shuttle plasmid pDC316-VP1 was constructed using the optimized gene se-quence of BNeV VP1.The recombinant adenovirus rAd5-BNeV_VP1 was packaged by AdMax ade-novirus packaging system.The immune effect was evaluated in mice.BNeV VP1 sequence with a size of 1 650 bp of Bo/LN-13/18/CH strain was optimized and synthesized according to codon preference of HEK293 cells by comparing the amino acid sequence of BNeV VP1 in China.Shuttle plasmid pDC316-VP1 and backbone vector pBHGIox_E1,3Cre were co-transfected into HEK293 cells to package recombinant adenovirus rAd5-BNeV_VP1.BNeV VP1 gene band with a size of a-bout 1 650 bp was amplified by RT-PCR.Western blot and indirect immunofluorescence assay con-firmed that rAd5-BNeV_VP1 successfully expressed VP1 protein with a size of about 57.5 kDa.The results showed that rAd5-BNeV_VP1 was successfully packaged and VP1 gene was stably ex-pressed in the recombinant adenovirus.TCID50 was 10-5.34/0.1 mL.A high level of specific antibody was detected in mice after immunized by intramuscular and intranasal routes.The highest antibody titer was 1∶105 in the group of intramuscular and 1∶104 in the group of intranasal on 7 d after the second immunization.The group of intranasal can produce two times of INF-γ and IL-2 than the group of PBS(P＜0.05).The recombinant adenovirus rAd5-BNeV_VP1 expressing BNeV VP1 was successfully constructed.It has a good immunogenicity and can stimulate mice to rapidly pro-duce specific antibodies against BNeV VP1,which provides a basis for the vaccine development for bovine nebovirus infection.
recombinant adenovirusbovine nebovirusimmunogenicityVP1 protein
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