| Avian influenza is the avian disease caused by infection with avian influenza type A viruses.It has resulted in severe economic loss in poultry ind ustry and threatened human health.It was the first cases reported that the H7N9 avian influenza infected human in Shanghai in March,2013.In January,2017,the highly pathogenicity H7N9 avian influenza virus from Guangdong infected both human and poultry.The H7N9 avian influenza virus infected 1508 people including 577 died and caused a great number of poultry death till May 17,2017.In recent years,DNA vaccine of avian influenza has attracted more and more attention.In spite of the safety and respons iveness of DN A vaccine,the low transfection efficiency and immunogenicity of that are problems,the proper adjuvant can improve the immune efficacy of DNA vaccine.To develop H7 subtype avian influenza DNA vaccine,we amplificated HA gene of H7 avian influenza virus A/chicken/Guangdong/OY1/2013(H7N9)by RT-PCR firstly.The HA gene was optimized according to the chicken codon bias and it was named op HA7.The optimized and non-optimized HA gene of H7 avian influenza virus were cloned into the eukaryotic expression vector p CK,respectively.So these recombinant plasmids(p CKHA7 and p CKop HA7)were constructed.We transfected these recombinant plasmids into 293 T cells.The western blotting results showed that these recombinant plasmids(p CKop HA7 and p CKHA7)could express the HA protein efficiently,but the protein expression level of the optimized plasmid was higher than that of the non-optimized,and the relative molecular mass of these protein were about 70 k Da.In order to construct the eukaryotic expressed plasmids of chicken and duck IFN-? and IL-2 as DNA vaccine adjuvants,we optimized the genes sequence of chicken and duck IFN-? and IL-2 published in the NCBI Gen Bank according to the codon bias of chicken and duck,respectively.These optimized genes was na med op C h IFN-??op Ch IL-2?op Du IFN-? ? op Du IL-2,respectively.These optimized duck IFN-? and IL-2 genes(op Du IFN-??op Du IL-2)were added the HIS tag at the 3' end of them by PCR.After that,these gene(op C h IFN-?,op C h IL-2,op Du IFN-?+HIS,op Du IL-2+HIS)were cloned into eukaryotic expression vector p CK,respectively.They were named p CKop Ch IFN-?,p CKop Ch IL-2,p CKop Du IFN-?+HIS,p CKop Du IL-2+HIS,respectively.We transfected these recombinant plasmids into 293 T cells.The western blotting results showed that these recombinant plasmids could express target protein efficiently,and the relative molecular mass of these protein were between 15-25 k Da.To evaluate the immune efficacy of DNA vaccine adjuvants,30 SPF chickens of 5-weeks-old were divided into 4 groups.12 chickens were injected with p CK plasmid as the control group,6 chickens of the group p CKop HA7 were injected with p CKop HA7 plasmid,6 chickens of the group p CKop HA7 and p CKop Ch IFN-? were injected with p CKop HA7 and p CKop Ch IFN-? plasmids,6 chickens of the gro up p CKop HA7 and p CKop Ch IL-2 were injected with p CKop HA7 and p CKop Ch IL-2 plasmids.All chickens were immunized by multi-point injection in legs muscle.Second immunization was performed on the fourteenth day of the first immunization.All chickens were attacked with 100 ?L 106 EID50 A/chicken/Guangdong/Q1/2016(H7N9)virus on the fourteenth day of the second immunization by intranasal inoculation.The results showed the average value of serum antibody in the p CK control group was 0log2 and all chickens of the group died within 7 days after attacked.The average value of serum antibody in the group p CKop HA7 increased from 0.6log2 on the fourteenth day of the first immunization to 4.8log2 on the fourteenth day of the second immunization.There were no death and the rate of virus shedding was 50%,the protection rate was 50%.The average value of serum antibody in the group p CKop HA7 and p CKop Ch IFN-? increased from 3.7log2 on the fourteenth day of the first immunization to 6.2log2 on the fourteenth day of the seco nd immunization,which was 1.4log2 higher than the group p CKop HA7.There were no death and the rate of virus shedding was 33.33%,the protection rate was 66.67%.The average value of serum antibody in the group p CKop HA7 and p CKop Ch IL-2 increased from 2.4lo g2 on the fourteenth day of the first immunization to 6.2log2 on the fourteenth day of the second immunization,which was 1.4log2 higher than the group p CKop HA7.There were no death and the rate of virus shedding was 33.33%,the protection rate was 66.67%.To sum up,the SPF chickens immunized H7 avian influenza DN A vaccine p CKop HA7 could produce a high antibody levels to against the H7N9 avian influenza virus attacks.In addition,these DNA vaccine adjuvants(p CKop Ch IFN-? plasmid and p CKop Ch IL-2 plasmid)could enhance the immune efficacy of H7 avian influenza DNA vaccine. |
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