| African swine fever is an acute and severe infectious swine disease caused by the African swine fever virus.The disease occurs rapidly after infection in pigs,and the fatality rate is high.ASFV has been popular for more than one hundred years since it was discovered in Kenya in 1921.ASF has spread to Africa,America,Eurasia and other regions,bringing a great threat to the pig industry and food safety.However,the effective commercial vaccine of ASFV has not been successfully developed so far.Live viral vector vaccine can induce better immune protection in pigs,which has become a research hotspot.In this study,replication defective human adenovirus type 5 was used as vector to construct recombinant adenovirus vector carrying multiple ASFV protective antigen genes.Immune experiment was carried out through mouse model,in order to provide theoretical basis for later immunization of pigs and provide basis for live vector vaccine research of ASFV virus.In this study,the target fragments amplified from pUC5-EP153R-B646L?pUC57-A151R-B438L-K205R-A104R?pUC57-EP402R-CP204L-E 183L and pUC57-D 117L-F317L-EP364R vectors were cloned into adenovirus vector to construct pAd-EGFP-P2A-EP153R-B646L?pAd-EGFP-P2A-A 151R-B43 8L-K205R-A104R?pAd-EGFP-P2A-EP402R-CP204L-E183L and pAd-EGFP-P2A-D117L-F317L-EP364R ASFV recombinant adenovirus vectors.Four ASFV recombinant adenoviral plasmids were linearized with PacI endonuclease and then transfected into 293A cells to obtain four recombinant adenoviruses.The results showed that rAd-A151R-B438L-K205R-A104R had the highest titer among the 4 recombinant adenoviruses,and the expression of the fusion target protein could be detected after the 4 recombinant adenoviruses infected cells.In order to detect the immune effect of recombinant adenovirus,we used 4 strains of recombinant adenovirus to immunize mouse for effect evaluation,and at the same time,we constructed a prokaryotic expression vector of ASFV protective antigen,and purified the corresponding ASFV protein for detection in mouse immunization experiments.After the first immunization,a booster immunization was carried out at 2 W and 4 W.On the 14th day after immunization,the specific antibodies in the serum of mouse in the rAd-EP402R-CP204L-E183L experimental group were not significantly higher than that of the control group.The other 3 recombinant adenoviruses the specific antibody of the experimental group is higher than that of the control group,which is extremely different from the control group(P<0.01).After the booster immunization,the antibody levels of the 4 experimental groups increased significantly,and the differences were extremely significant compared with the control group(P<0.01).Splenic lymphocytes were isolated from mouse 42 days after immunization,and the isolated lymphocytes were stimulated in vitro with prokaryotic expressed ASFV recombinant protein.The results of lymphocyte proliferation showed that the proliferation levels of rAd-D117L-F317L-EP364R and rAd-EP153R-B646L were higher than that in the control group,but there were no significant proliferation in the other two groups.The levels of IL-4 and IFN-? in the supernatant after lymphocyte stimulation were detected by ELISA kit.The results were basically consistent with the results of lymphocyte proliferation.This study successfully constructed 4 recombinant adenoviruses by cloning the 12 protective antigen genes of ASFV into an adenovirus vector,and can express ASFV antigen protein.Four recombinant adenovirus strains could induce a good humoral immune response in mouse,especially the specific antibody level induced by rAd-D 117L-F317L-EP364R was the highest.The results of lymphocyte proliferation experiments and cytokine levels showed that the rAd-D117L-F317L-EP364R experimental group and rAd-EP153R-B646L experimental group could induce better cellular immune responses. |
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