| Newcastle disease(ND),commonly known as pseudo-chicken plague,has been considered to be must report infectious diseases in World Organisation for Animal Health(OIE),which is characterized by the typical mucosal bleeding of the respiratory tract and digestive tract.Currently,prevention and control of the disease is mainly based on the vaccine.Traditional inactivated vaccines and attenuated vaccines can play a role in preventing and controlling diseases,but can not completely control the occurrence of the disease.Recently,the genotype ? NDV has been popular in China,whereas the traditional vaccine does not work well against this kind of NDV infection,therefore,it is of great significance to research and create new types of vaccines.DNA vaccines are safe,stable,and free from interference from maternal antibodies,the use of attenuated Salmonella as a vector for novel DNA vaccines has good research potential and application prospects,regulated delayed lysis Salmonella have unmatched advantages with other types of Salmonella,it is a kind of bacterial vector dependent on the presence of arabinose,without the presence of antibiotic selective marker which inhibit the possibility to transfer antibiotic resistant genes to other organisms.DNA vaccine against NDV type ? NDV gene based on pre-laboratory work,in this study,we used the fusion PCR method to link the major protective antigen F and HN protein genes together and employed the regulated delayed lysis Salmonella as a vector.The newly constructed F and HN dual antigen DNA vaccine vectors were evaluated for their preliminary immune effects.The specific research contents and results were as follows:(1)Construction and antigenicity analysis of recombinant DNA vector pYA4545-pUC-F-HN-opt-FlagIn this experiment,the recombinant plasmid pYA4545-pUC-F-HN-opt-Flag was constructed and validated by fusion PCR,enzyme digestion,ligation,transformation and colony PCR;The extracted plasmid was then transfected to HEK 293 T cells usingLipofectamine 3000,followed by western blotting and indirect immunofluorescence assays to confirm the synthesized F and HN proteins.The plasmid was then transformed to ?11218 strain for further animal study.(2)Evaluation of the immune effects of recombinant Salmonella?11218 pYA4545-pUC-F-HN-opt-Flag)In this study,newly hatched chicks were divided into 4 groups of 20 in each group.The primary immuniz(ation and booster immunization of the chicks were carried out by oral administration of ?11218(pYA4545-pUC-F-HN-opt-Flag)and ?11218(pYA4545),respectively.Meanwhile,the inactivated vaccine and saline were also included as positive and negative controls.The primary immunization was performed on the fourth day,and the booster immunization was performed twice every 14 days thereafter.Samples were taken 14 days after the first booster immunization.Flow cytometer and lymphocyte proliferation assay were used to detect the activation and proliferation of T lymphocytes and B lymphocytes in the peripheral blood of chicks from each group,and the peripheral blood of chicks in each group was collected for further serum IgY antibody determination.Intestinal lavage fluid was used to detect specific IgA levelsby indirect ELISA.Two weeks after the second booster immunization,the chicks were infected with leathal dose of NDV via nasal drops.After the challenge,the chicks were observed for their mental status and their weight changes were recorded.On the 10 th day after the challenge,the chicks were slaughtered to remove the lungs and thymus,pathological sections were prepared,and the pathological changes of the lungs and thymus were evaluated by HE staining.The experimental results showed that the number of T lymphocytes and B lymphocytes in the peripheral blood of chicks after oral administration of recombinant Salmonella?11218(pYA4545-pUC-F-HN-opt-Flag)was significantly higher than that of the blank control group,showing significant differences(P<0.05).There was a significant difference in the expression level of specific antibodies between serum and intestinal lavage(P<0.001).Observing and comparing the mental status of the chicks infected with the virus showed that the chicks in each group showed signs of apathy,unstable standing,and reduced feed intake.However,after a period of time,the other groups except the normal saline group were present.Weighing the body weight results showed that the inactivated vaccine group and the ?11218(pYA4545-pUC-F-HN-opt-Flag)group continued to decline at the initial stage,and remained stable at the lowest level around the 5th day,with a slight increase.? 11218(pYA4545)group and saline group showed a continuous downward trend,? 11218(pYA4545)group was slightly better than the saline group.The survival rates indicated that the recombinant strains caused about 50% survival,compared with 90% of inactivated vaccine and 20% of negative control groups.The results of pathological sections of lung and thymus in each group of chicks showed that the pathological symptoms of the pYA4545-pUC-F-HN-opt-Flag group and the inactivated vaccine group were mild,and all other groups had severe pathological changes.The above results indicate that the recombinant Salmonella strain?11218(pYA4545-pUC-F-HN-opt-Flag)has a significant protection effect against the Newcastle disease virus and lays the foundation for the further development of the Newcastle disease virus DNA vaccine. |
PDF Full Text Request