The Development Of H5 Subtype Avian Influenza DNA Vaccine And Poultry Immunopotentiator

Avian Influenza（AI）is a disease syndrome caused by type A influenza virus in many birds.In the past two years,the outbreaks of H5 AI have increased significantly,and cause huge economic losses to the poultry industry in China.The traditional inactivated vaccine of AI was used in birds So that it was difficult to eliminate diseases,so it is still necessary to develop new vaccines（such as DNA vaccines）.In addition,because of low antibody level of poultry by immunized DNA vaccine,it is necessary to develop new immunopotentiators.In this study,we first amplified the HA gene of the H5 subtype avian influenza virus A/Goose/Guangdong/P45/2016（H5N6）belonged to clade 2.3.4.4 by RT-PCR.The HA gene was cloned into the eukaryotic expression vector pCK to obtain the recombinant plasmid pCKHA5.After the recombinant plasmid was correct by sequencing,we transfected it into 293T cells.The western blot result showed that the recombinant plasmid pCKHA5 could express HA protein specifically and the relative molecular mass of this protein was about 64 kDa.In order to study whether eukaryotic expression plasmid of the duck RIG I can be used as a immunopotentiator of DNA vaccine,the duck RIG I gene and the chicken MDA5 gene from NCBI were optimized according to the chicken codon bias,the optimized gene were synthesized with HIS labe,which named opCH-DKRIG I-HIS and opCK-MDA5-HIS,respectively.Duck RIG I gene was optimized according to the duck codon bias,and the optimized gene was synthesized with HIS labe named the optimized gene opDK-DKRIG I-HIS.Then these optimized genes were cloned into the eukaryotic expression vector pCK to obtain these recombinant plasmids of pCKopCH-DKRIG I-HIS,pCKopCK-MDA5-HIS and pCKopDK-DKRIG I-HIS.These recombinant plasmids of pCKopCH-DKRIG I-HIS and pCKopDK-DKRIG I-HIS were transfected into 293T cells.The western blotting results showed that these recombinant plasmids could express the RIG I protein efficiently,which were both approximately 106 kDa protein.To evaluate immune effect of the eukaryotic expression plasmid pCKHA5 as a DNA vaccine in chickens and whether the optimized eukaryotic expression plasmid pCKopCH-DKRIG I-HIS could enhance immune effect of DNA vaccine,44 3–week-old SPF chickens were alone immunized or co-immunized with pCKHA5 and pCKopCH-DKRIG I-HIS.These chickens were randomly divided into 4 groups,11 in each group.11 chickens were injected with PBS as the control group with 200μL per chicken.The chickens of other groups were immunized with pCKHA5 plasmid,pCKopCH-DKRIG I-HIS plasmid,pCKHA5 plasmid and pCKopCH-DKRIG I plasmid with 200μL per chicken respectively.All chickens were immunized by multi-point injection in legs muscle.The second immunization was performed on the fourteenth day after first immunization.The results showed the average HI value of serum antibody was 0.08log2 in chickens by immunized the plasmid pCKHA5 on the seventh day after first immunization,the average HI value of serum antibody was 1log2 on the fourteenth day after first immunization,the average HI value of the serum antibody was 2.83log2 on the seventh day after second immunization,and the average HI value of serum antibody was 3log2 on the fourteenth day after second immunization.The results showed the average HI value of serum antibody was0.08log2inchickensbyco-immunizedtheplasmidspCKHA5and pCKopCH-DKRIGI-HIS on the seventh day after first immunization,the average HI value of the serum antibody was 1.25log2 on the fourteenth day after first immunization.the average HI value of the serum antibody was 3.25log2 on the seventh day after second immunization,the average HI value of serum antibody was 3.58log2 on the fourteenth day after second immunization.Thus,the chickens in the co-immunized pCKHA5 and pCKopCH-DKRIG I-HIS groups had slightly higher antibody levels than in the immunized pCKHA5 group.All chickens in the PBS control group and the immunized group were challenged with100μL of 10⁶EID₅₀0 A/Goose/Guangdong/P45/2016（H5N6）virus belonged to clade 2.3.4.4on the fourteenth day after second immunization by intranasal inoculation.All the chickens died within 2 days in the PBS control group and the immunized pCKopCH-DKRIG I-HIS group.In the immunized pCKHA5 group,and the co-immunized pCKHA5 and pCKopCH-DKRIG I-HIS groups,all chickens survived within 14 days,but some chickens could shed virus.Their shedding-virus rates were 27.27%and 18.18%,respectively and the protection rates were 72.73%and 81.82%,respectively.Thus,the chickens immunized by plasmid pCKHA5 produce enough antibodies against the clade 2.3.4.4 H5 subtype avian influenza virus,but some immunized chickens could shed virus.