A Eukaryotic Expression Plasmid Carrying Chicken Interleukin-12 With Electroporation Technology Enances The F Gene DNA Vaccine Against Newcastle Disease Virus

Newcastle disease(ND) is a highly contagious and highly mortality viral disease of poultry caused by Newcastle disease virus(NDV), which can cause severe losses in poultry. With the popular of genotype Ⅶ strains in recent years, the inactivated vaccine and attenuated vaccine is unable to provide good immune effect on genotype Ⅶ strains. Therefore, it is of great significance to develop a new genetic engineering vaccine to deal with the gene type Ⅶ NDV. The DNA vaccine is the third generation of vaccines, which can stimulate both humoral and cellular immue response and has relatively long immunity duration, so it is considered as one of the most potential genetic engineering vaccines. In order to further study the DNA vaccine of Newcastle disease, this research successfully constructed eukaryotic expression plasmid p CAG-F and p CAG-IL12 by using the plasmid p CAGGS which contains beta actin promoter and CMV enhancer. Two approaches were used to enhance the immunogenicity. Electroporation(EP) and plasmid expressing chicken IL-12 were used to immprove the immunogenicity of p CAG-F.Firstly, NDV F gene of CHICKEN/CH/GD/GM-03(GM) was cloned into the vector p CAGGS and adding Kozak sequence at the upstream. SPF chickens were immunized with p CAG-F by Electroporation(EP) or intramuscular injection(IM). The EP effects on the immunogenicity of Newcastle disease virus DNA vaccine was evaluated by detecting the humoral and cellular immune response level and the protection rate of chickens against the NDV. The result of microneutralization assay showed that EP can enhance the antibody responses. T cell response testing showed that cellular immunity level of p CAG-F plasmid vaccinated by EP was higher when compared with those chickens immunized p CAG-F plasmid by IM. Four weeks later, all chickens were challenged with a leathal dose of NDV GM strain, 90% chickens vaccinated by IM of p CAG-F plasmid survived. However, chickens vaccinated by EP were completely protected from leathal dose. These results showed that EP immunization can enhance the protection effects of chickens from a leathal dose challenge compared with those immunized by IM. Oral and cloacal swabs virus isolation results indicated that the SPF chickens vaccinated by EP have low rate and short time in virus shedding.Secondly, Interleukin-12 was cloned into the vector p CAGGS and adding Kozak sequence at the upstream for investigate the immunopotentiation effects of molecular adjuvant chicken IL-12 to NDV F gene DNA vaccine on the basis of EP immunization. The result of microneutralization assay showed that EP combined with IL-12 resulted in marked enhancement of F-specific antibody responses after the third immunization. Moreover, T cell response testing showed that cellular immunity level of p CAG-F plasmid vaccinated by EP combined with IL-12 was higher when compared with those chickens immunized p CAG-F plasmid by EP alone. Both chickens immunized p CAG-F alone or with p CAG-IL12 by EP were completely protected from a leathal dose.These results showed the humoral immunity and cellular immunity of chickens vaccinated with p CAG-IL12 are better than p CAG-F alone and could effectively inhibit the copy of the virus in body and short time in virus shedding.In summary, it is concluded that EP and p CAG-IL12 can significantly improve the immumogenicity of p CAG-F DNA vaccine, which provides new ideas and direction for the development of a new generation of Newcastle disease virus DNA vaccine.