| African classical swine fever(ASF)is an acute,severe,and highly contagious disease caused by African classical swine fever virus(ASFV)infection,which has caused substantial economic losses to the global pig industry.So far,due to the particularity of the African classical swine fever virus,no vaccine and effective therapeutic drugs have been produced to provide adequate protection,which has brought significant challenges to the prevention and control of African classical swine fever.Therefore,in this study,E.coli heat-labile enterotoxin B subunit(LTB)was used as an adjuvant to communicate with African swine fever virus CD2v protein and P49protein,construct recombinant nucleic acid vaccine and subunit vaccine,and evaluate its immunogenicity to provide a way for the research and development of African swine fever vaccine.1.Recombinant nucleic acid vaccineThe amplified gene fragments CD2v,P49 and LTB were ligated into pc DNA3.1(-)expression vector to construct pc DNA3.1-CD2v,pc DNA3.1-p49,pc DNA3.1-CD2v+LTB and pc DNA3.1-p49+LTB 4 recombinant nucleic acid vaccines.Firstly,the construction of the recombinant plasmid was verified by double endonuclease digestion and sequencing.In order to verify whether the recombinant eukaryotic expression vector can be expressed and transcribed,the mammalian cells were transfected in vitro,the His tags were detected by western blot,and the specific bands in accordance with the expected protein size were observed.The non-endotoxic plasmid was extracted and inoculated into mice,and the titer was evaluated by detecting the peripheral blood of mice.The level of antibody Ig G in the pc DNA3.1-CD2v group,pc DNA3.1-p49 group,pc DNA3.1-CD2v+LTB group,and pc DNA3.1-p49+LTB group increased significantly after 28 days and 42 days,and there was a significant difference between pc DNA3.1-CD2v and pc DNA3.1-CD2v+LTB groups at 28 days.CD2v protein and P49 protein were used as antigens to coat the ELISA plate,and the titer of the antibody was evaluated.It was found that the titer of mouse serum injected with pc DNA3.1-CD2v plasmid was about 1:12800,and the titer of mouse serum of plasmid pc DNA3.1 was about 1:12800.The titer of mouse serum of plasmid pc DNA3.1 was about 1:12800.The stimulating effect of the recombinant nucleic acid vaccine on T lymphocytes was detected.The results showed that the CD4~+/CD8~+ratios of pc DNA3.1-CD2v,pc DNA3.1-p49,pc DNA3.1-CD2v+LTB and pc DNA3.1p49+LTB were 2.619,2.487,5.567 and 3.42,respectively,and those of empty vector pc DNA3.1(-)and PBS group were 1.976 and 1.780,respectively.To detect the effect of recombinant plasmid on cytokine level in mice INF-γ,and IL-2levels,compared with the control group,the experimental group pc DNA3.1-CD2v、pc DNA3.1-p49、pc DNA3.1-CD2v+LTB、pc DNA3.1-p49+LTB showed a high level of growth,and pc DNA3.1-CD2v+LTB group compared with pc DNA3.1-CD2v group,INF-γand IL-2 increased significantly(P<0.05),pc DNA3.1-p49+LTB group compared with pc DNA3.1-p49 group,the level of IL-2 increased significantly(P<0.05).In the detection of IL-4 level,compared with the control group,pc DNA3.1-CD2v and pc DNA3.1-p49+LTB group increased significantly(P<0.05),pc DNA3.1-CD2v+LTB group increased significantly(P<0.01).However,there was no significant difference between pc DNA3.1-CD2v,pc DNA3.1-CD2v+LTB group and pc DNA3.1-p49,pc DNA3.1-p49+LTB group.2.Recombinant subunit vaccine.The target genes P49 and LTB were cloned into p PIC9K plasmid to construct recombinant yeast expression vectors p PIC9K-p49,and p PIC9K-p49+LTB.Firstly,the construction of the recombinant plasmid was verified by double enzyme digestion and sequencing.The recombinant plasmid was transferred into methanol-induced Pichia pastoris by electroporation.The genome of the positive strain was extracted and verified by plasmid PCR.After the positive strains were selected and expressed,the added His tags were detected by the western blot method,and the strains with correct protein were chosen to expand and induce.The target protein was obtained by mass fermentation and purified by His tags.The purified recombinant proteins P49,p49+LTB,and LTB were injected intraperitoneally with Freund’s adjuvant and Freund’s incomplete adjuvant.After 14days,28 days,and 42 days after immunization,the level of antibody Ig G in the P49group and p49+LTB group was significantly higher than that in the control group(P<0.01).There was significant difference between p49 and p49+LTB groups(P<0.01).P49 protein was used as the detection antigen to coat the Elisa plate,and the antibody titer was evaluated.It was found that the serum titer of mice inoculated with P49 protein was about 1:25600,and that of mice inoculated with p49+LTB protein was about 1:102400.The stimulating effect of recombinant subunit vaccine on T lymphocytes was detected.The results showed that the CD4~+/CD8~+ratio of p49,p49+LTB,and LTB groups was 3.191、5.288、2.487,respectively,which was significantly higher than that of the PBS group(1.700).The results showed that the proportion of CD4~+lymphocytes in peripheral blood of mice increased after vaccination.Further,detect whether the level of cytokines changed after the recombinant protein was inoculated into mice.42 days after immunization,the serum of mice was taken for the ELISA test.The levels of IL-2,IL-4,and INFγin the experimental group were higher than those in the control group(P<0.01).Compared with the p49+LTB group,the levels of INF-γand IL-2 in the P49group increased significantly,but the difference in IL-4 level was not significant.The above results show that the recombinant nucleic acid vaccine and recombinant subunit vaccine constructed in this study have good immunogenicity,stimulating the body to produce specific antibodies and activating the corresponding cellular and humoral immune responses.At the same time,LTB as an adjuvant can significantly improve the body’s immune,which provides a technical idea for the research and development of the African classical swine fever vaccine. |
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