Screening Of Novel Nucleic Acid Vaccine Adjuvants And Enhancement Of Immune Efficacy In Livestock And Poultry Vaccines

There are some short nucleotide sequences(dinucleotides made up of cytosine,guanine,and phosphodiester bonds connecting cytosine and guanine)in bacteria,which can be used as a dangerous signal for simulating bacteria and viruses to be recognized by the immune system.And they directly stimulate the proliferation and differentiation of B lymphocytes,macrophages and dendritic cells,upregulate the expression of immune stimulatory molecules,regulate the immune response,and induce the secretion of IL-6,IL-12,IFN-γand TNF-α and other.In addition,these nucleotide sequences can also indirectly activate natural killer cells and T lymphocytes,produce Th1-type cellular immunity,and induce a more balanced and strong immune response in the immune system.In addition,these nucleotide sequences have the advantages of high efficiency,low toxic and side effects,and can be used as a new type of nucleic acid adjuvant.They are the research hotspots for novel immunostimulants in livestock and poultry.A nucleotide sequence with immunostimulatory activity on livestock and poultry was designed and cloned into plasmid pc DNA3.0 to construct oligoglycosides with different copy numbers(15,20,30,40,45,50 and 65).A total of seven recombinant plasmids were designated as pc DNA-1,pc DNA-2,pc DNA-3,pc DNA-4,pc DNA-5,pc DNA-6,and pc DNA-7.To verify the genetic stability of the constructed recombinant plasmids,the transformants carrying the recombinant plasmids were subcultured on LB solid plates containing ampicillin resistance for 30 consecutive passages,and were identified by colony PCR,restriction enzyme digestion,and sequencing of the desired gene sequences.The results showed that within 30 generations,the length and sequence of the inserted nucleotide fragments of the seven recombinant plasmids could be stably inherited.In order to explore the immunostimulatory effect of the constructed recombinantplasmid,different doses(2.5 and 5.0 and 7.5 μg,respectively)of recombinant plasmids pc DNA-1,pc DNA-2,pc DNA-3,pc DNA-4,pc DNA-5,pc DNA-6,and pc DNA-7 were respectively used to stimulate chicken peripheral blood lymphocytes,and Con A stimulation was set up as positive control group.Lymphocytes transformation experiments in vitro were performed to test the proliferative capacity and metabolic intensity of peripheral blood lymphocytes.The results showed that the recombinant plasmids pc DNA-1,pc DNA-2,pc DNA-3,pc DNA-4,pc DNA-5,pc DNA-6 and pc DNA-7 all have different stimulating effects on peripheral blood lymphocytes.The same plasmid had the strongest stimulatory effect at 7.5 μg.The stimulation index SI values of pc DNA-6 and pc DNA-7were higher than other recombinant plasmid groups under the same stimulation amount of each recombinant plasmid,and the stimulation index was over 5.0 at a stimulation dose of7.5 μg.The research used the constructed seven kinds of recombinant plasmids pc DNA-1,pc DNA-2,pc DNA-3,pc DNA-4,pc DNA-5,pc DNA-6,pc DNA-7 and inactivated vaccine with porcine circovirus type 2 baculovirus vector,respectively to immunize the SPF BACB/c mice by subcutaneous injection.After immunization,the blood was collected on the 7th,14 th,28th and 35 th days.The serum was separated and the serum cap protein antibody was detected by ELISA.At the same time,a separate vaccine-immunized group and saline-experimental group were established.The results showed that compared with the vaccine-alone vaccine group,the recombinant plasmids and the porcine circovirus type 2baculovirus vector inactivated vaccine had higher antibody levels,and the antibody levels of recombinant plasmids pc DNA-6 and pc DNA-7 groups were all higher than that of other recombinant plasmid groups.It was indicated that the recombinant plasmid can increase the antibody level of cap protein of porcine circovirus type 2 in mice.In order to explore the enhanced effect of the recombinant plasmid in avian influenza inactivated vaccine,the research used the recombinant plasmids pc DNA-6 and pc DNA-7and the avian influenza inactivated vaccine to simultaneously immunize 20-day-old healthy chicks.Blood was collected on the 7th,14 th,28th,and 35 th days after immunization,serum was separated,and the HI titer of avian influenza in the serum was measured.At the same time,a vaccine-alone and normal saline blank experimental group wasestablished.The results showed that the HI titers of the recombinant plasmids pc DNA-6 and pc DNA-7 and the avian influenza inactivated vaccine were higher than those of the vaccine alone group,and the HI titer of the recombinant plasmid pc DNA-6 group was higher than that of the pc DNA-7 group.The HI titer of pc DNA-6 group was 1.5 log2 higher than that of the vaccine control group.It was demonstrated that the constructed recombinant plasmids pc DNA-6 and pc DNA-7 in combination with avian influenza can significantly increase the antibody levels of bird flu in chicks.In summary,seven kinds of nucleotide sequences with immunopotentiation were successfully cloned into plasmids and the gentic stability of recominant plasmids was verified.The seven constructed recombinant plasmids had immune stimulatory effects on peripheral blood lymphocytes of chickens.The combination of recombinant plasmids with porcine circovirus type 2 baculovirus vector vaccine and avian influenza inactivated vaccine can increase antibody levels.Among them,pc DNA-6 has the most prominent immunopotentiation effect,so it can be used as a nucleic acid immunopotentiator in the production of vaccines,laying the foundation for the development and application of nucleic acid immunopotentiators.