Construction And Immune Efficacy Evaluation Of Co-expressed Gene Type ? Newcastle Disease F Protein And Chicken IL18 Salmonella

Newcastle disease(ND)is one of the infectious diseases that seriously harm the poultry industry.Office International Des Epizooties(OIE)has listed this as an infectious disease.At present,the major approach to control ND is vaccination,however,the current vaccines on the market has poor performance against genotype ? NDV,which is the most popular strain in China currently.Compared with the first generation vaccine(microbial vaccine)and the second generation vaccine(subunit vaccine),the so called “third generation of vaccine” DNA vaccine,can induce both humoral and cellular immune response.In this study,the codon-optimized F gene of genotype ? NDV was ligated into the eukaryotic expression vector pYA4545 to construct plasmid pYA4545-pUC-F-opt-Flag(pYA4545-F).In order to enhance the immune effect,the chicken interferon 18(cIL-18)gene under the control of prokaryotic promoter Ptrc was inserted into pYA4545-F,yielding a dual promoter plasmid pYA4545-pUC-F-opt-Ptrc-cIL18-TT-Flag(pYA4545-F-IL18).The pYA4545-F and pYA4545-F-IL18 plasmids were then electroporated into the delayed-lysed Salmonella strain ?11218,and the immune effect was evaluated after oral immunization of chicks.The specific research content and results are as follows:(1)Preparation and reactivity analysis of recombinant Salmonella ?11218(pYA4545-F)and ?11218(pYA4545-F-IL18)Firstly,pUC-F-opt-Flag with Flag tag was constructed by using pUC-F-opt as template,and then it was ligated with eukaryotic expression vector pYA4545 to construct pYA4545-F.Next,primers were designed using the plasmid pYA3342-cIL-18 as template and the fragment Ptrc-cIL18-TT including the prokaryotic promoter Ptrc,cIL-18 fragment,and the terminator TT was amplified.The pYA4545-pUC-F-opt-Flag plasmid was ligated to construct pYA4545-F-IL18 plasmid containing dual expression promoters(CMV eukaryotic promoter and Ptrc prokaryotic promoter).Then the expression of F protein was confirmed by Western Blot and indirect immunofluorescence.The cIL18 protein was successfully expressed.The plasmid pYA4545-F and pYA4545-F-IL18 were electroporated into the delayed lysed Salmonella vector to obtain the recombinant Salmonella ?11218(pYA4545-F)and ?11218(pYA4545-F-IL18).(2)Comparison and evaluation of immunoprotective effects of recombinant Salmonella ?11218(pYA4545-F)and ?11218(pYA4545-F-IL18)The immune protective effect of recombinant Salmonella was evaluated by immunoprotection test.100 1-day-old chicks were divided into 5 groups with 20 animals in each group.On the 7th and 21 st day,the groups were orally immunized with ?11218(pYA4545),?11218(pYA4545-F)and ?11218(pYA4545-F-IL18,saline control group was given an equal volume of normal saline,and the NDV inactivated vaccine group was immunized subcutaneously in the neck only on the 14 th day.At the age of 35 days,chicks of each group were subjected to experimental analysis and flow cytometry was used to detect changes in the number of T lymphocytes and B lymphocytes in the spleen and peripheral blood.The results showed that recombinant bacteria could increase T and B lymphocytes.MTT assay was performed to determine the proliferation ability of immunized chicken bursal B lymphocytes.The results showed that the B lymphocytes in the ?11218(pYA4545-F)group and the ?11218(pYA4545-F-IL18)group were significantly responsive to PMA stimulation.ELISA was used to detect the serum IgG antibody,intestinal IgA antibody,and the production of IL-4 and IL-18.The results showed that the recombinant Salmonella ?11218(pYA4545-F)and ?11218(pYA4545-F-IL18)could increase serum NDV antibody,IL-4,IL-18 and intestinal lavage IgA content.At the same time,the results of immunoprotection tests showed that the recombinant Salmonella strains ?11218(pYA4545-F)and ?11218(pYA4545-F-IL18)had certain immunoprotective effects on gene ? ND.The results of pathological section showed that the ?11218(pYA4545-F)and ?11218(pYA4545-F-IL18)was less effective to protect challenge compared with inactivated vaccine,however,the thymic tissue and lung tissue pathological changes performed better than saline group and the ?11218(pYA4545)group.The above results show that the recombinant Salmonellas have a certain protective effect,which provides a theoretical basis for taking use of regulated delayed lysis Salmonella as a live vector to design novel ND vaccine,and provides a new approach for the development of ND DNA vaccine.