Preparation And Immunization Evaluation Of DNA Vaccine Against H9 Subtype Avian Influenza

Avian influenza is a contagious disease caused by avian influenza virus(AIV)mainly displayed with injured respiratory and genital tract,decreased immunity,decreased egg production rate and many other clinical syndromes.Avian influenza virus is a negativestranded RNA virus,belonging to the genus influenza A virus of the family Orthomyxoviridae according to its pathogenicity,and it can be divided into two categories: highly pathogenic avian influenza(HPAI)and low pathogenic avian influenza(LPAI).Avian influenza H9 subtype belongs to LPAI,but it often causes great economic losses because of infected host widely and high infection rate.Hemagglutinin(HA),an important protective antigen,is one of the most important surface proteins of avian influenza virus,and often used for constructing DNA vaccine.In this study,the pVAX1-HA DNA vaccine was constructed by separating the full length HA gene from the H9 subtype of duck-derived strain and cloned to the eukaryotic expression vector pVAX1,and evaluated of immune effects of DNA vaccine on mice,chickens and ducks with live electric shock method,and then explored the efficacy of the combined use of inactivated vaccine.1.The HA gene of the new virus isolated from duck was expanded by RT-PCR.The results of HA gene sequencing and genetic evolution analysis showed that the isolated strain was H9 subtype avian influenza virus,and the amino acid composition of HA protein cleavage sites “PARSSRGL” was consistent with the characteristics of low pathogenic avian influenza virus.The pVAX1-HA eukaryotic expression plasmid was constructed by clone HA gene to eukaryotic expression vetor pVAX1,which was transfected to 293 T cells by transfection reagent PEI and the expression level of HA protein was detected by western blotting.The results proved that pVAX1-HA eukaryotic expression plasmid was successfully constructed,and also the HA protein could be expressed in 293 T cells with the molecular weight of the expected protein.2.BALB /c female mice were randomly divided into 5 groups: DNA vaccine group,DNA vaccine and inactivated vaccine group,inactivated vaccine(immunization once)group,inactivated vaccine(immunization twice)group and PBS control group,11 in each group.The DNA vaccine was immuned by electric shock,with the condition of 60 V 10 ms 6 pulses and immunization dose of 100 ?g per mice;the inactivated vaccine was direct injected to mouse leg muscles with immunization dose 0.2 mL per mice.Strengthen immunization was carried out with the same conditions every 3 weeks.The blood sample was collected after two weeks followed by each immunization,the antibody level was detected by HI test,and the cytokines concentration of IL-4,IL-6 and IFN-? was examined by the ELISA Kit.The results showed that DNA vaccine could induce immune cytokines and stimulate cellular immune response in mice,the antibody level of DNA vaccine group increased gradually,and the antibody level(8.3 log 2)was higher in mice after three times,and in the 8th week,the antibody level(8.8 log 2)was equivalent to that of inactivated vaccine group(9.5 log 2),but the antibody level of the combined group could last for six months.These results showed that the DNA vaccine had good immune effect,and the antibody level can last longer combinated with inactivated vaccine.3.The ducks were randomly divided into 5 groups: Low dose group of DNA vaccine(50 ?g /duck)and high dose group(100 ?g /duck),DNA vaccine and inactivated vaccine group,inactivated vaccine group and blank control group.The immune methods of DNA vaccine and inactivated vaccine were tested with mice.3 weeks with the same conditions of immunization,and 3 consecutive immunization.The results showed that the antibody level of DNA vaccine group reached its peak point at two weeks after the third immunization,the antibody levels were 5.1 log 2(high dose Group)and 4 log 2(low dose Group),then continue to a higher level.The antibody level(10.5 log 2)of the union group in the two weeks after the second immunization was comparable to that of the inactivated vaccine group(11 log 2),however,the antibody level was slightly higher than that in the inactivated vaccine group at 8 weeks after the third immunization,and the inactivated vaccine group declined slightly.The results showed that DNA vaccine alone could stimulate the organism to produce better immune effect with high immune dose and higher antibody level,but could not reach the immune effect of inactivated vaccine.