Construction And Immune Efficacy Of DNA Vaccine Of Modified HN Gene NDV F₄₈E₉ Strain

To seek a better way to prevent and control NDV efficiently, we constructed the DNA vaccine of modified NDV F48E9 strain HN gene and evaluated the efficiency of the DNA vaccine according to experiments in vitro and vivo.We modified the NDV F48E9 strain HN gene according to optimizing the condon usage and inserting thesecretary leader sequence [A/Goose/Guangdong/1/96 (H5N1) HA gene, Accession No: AF 144305] in our study. The HN modified genes and the HN genes that signal peptide was knocked off named SoptiHN and optiHN. The three sequence: SoptiHN, optiHN and the HN gene of NDV F48E9 strain were inserted into the vector pVAX1 and vector pVAX1-CpG including CpG-ODN sequence respectively, then we got six recombinant plasmids: pV-SoptiHN,pVC-SoptiHN, pV-optiHN,pVC-optiHN and pV-HN,pVC-HN, Which were them to transfect 293T cells and detected in West-blotting and indirect immunofluorescence test after 48 hours. Subsequently, Six groups of three-week-old SPF chickens were injected with plasmids of pVAX1, pVAX1-CpG, pV-HN, pVC-HN, pV-SoptiHN and pVC-SoptiHN on the quadriceps repectively, 200ug per chicken, the control group was injected with PBS as a placebo. After three weeks of primary vaccination, boostered with the same dose. Chickens were challenged intramuscularly with 1000 EID50 NDV F48E9 strain after second immunization. The morbidity, mortality and virus shedding were calculated , Sera and PBMC were collected every week before and after immmunization and challenge.The final result in vitro showed that the expression levels of HN gene modified in transiently transfected 293T cells and detected in West-blotting were higher from the codon-optimized gene and the counterpart, moreover, the two ways of optimization of condon usage and addition of signal peptide could all elevate the level expression of HN gene. In addition, results according to vaccinating SPF chickens showed that HI titer of the groups of modified HN gene were ahead of 24 only, groups of pV-SoptiHN and pVC-SoptiHN reached 5.33±0.72 and 5.17±1.17 respectively, rest of groups all could not reach 24. In challenge experiment, immune groups of modified HN gene produced higher protection rate (71%) than unmodified HN (50%), while groups of plasmids and control were 0%. We got the results from kinetic change of IFN-γand IL-18, immune groups of pV-SoptiHN and pVC-SoptiHN could induce higher expression level of cytokine than the rest groups. Except for control groups, expression level of cytokine in all of groups started to rising after one week of primary immunization, and the level reached highest pink after two weeks of booster. Then, they got down quickly and rised after challenge, but the pinks were not higher than ones of two weeks of booster. In this procession, we found that plasmid with synthesied CpG-ODN sequence could induce higher expression of cytokines than that without synthesied CpG-ODN sequence.