Studies On Bacterial Ghost Loading Suicidal DNA Vaccine Of NDV F Gene And ChIL-18 Gene

Newcastle Disease(ND),caused by Newcastle disease virus(NDV),is a kind of hightly contiguous infection disease.It can infect many kinds of poultry such as chicken,turkeys,ducks, pigeons,parrots and so on.It has made large economic loses in the poultry industry.The Office International Des Epizooties(OIE)determined ND as class A of animal infectious disease,our country regarded it as classâ… of animal infectious disease.Vaccination is one of the main measures to control ND.DNA vaccine as a novel vaccine has became a research focus in the vaccine field for its high specificity,non-pathogenity,easy to manufacture.however,it also has some disadvantage,such as,less immunogenic and integration between foreign plasmid DNA and host genome.In order to increase the efficiency and securityof DNA vaccine,researchers hope to using adjuvants and developing new delivery systems to increase the efficiency and securityof DNA vaccine.1.Construction of the suicidal DNA vaccine pSFV-F-ChIL18In this study,the NDV F gene of Lasota strain was amplified from NDV RNA by RT-PCR, using specific primers with BamHâ… /site in the forward and Salâ… site in the downward,designed by the NDV F gene sequence published in GenneBank,and the BamHâ… /Salâ… -digested NDV F fragment was inserted into the BamHâ… /Salâ… site of plasmid pMD18-T to generate the plasmid pMD18-T-F by using standard cloning procedures.At the same time,the ChIL-18 gene with upstrom Salâ… site and down-strom Hindâ…¢/BamHâ… /site was amplified,and the PCR fragment of ChIL-18 gene digested by Salâ… and Hindâ…¢was inserted into the Salâ… /Hindâ…¢site of the plasmid pMD18-T-F'to generate the fusion gene F-ChIL-18.At last,the BamHâ… -digested F-ChIL-18 fragment was inserted into to pSFV construct a suicidal plasmid which was named as pSFV-F-ChIL-18.And also,another NDV F gene with BamHâ… site was obtained by RT-PCR,using the primers with BamHâ… site in both the forward and downward,then it was inserted into the pSFV construct a suicidal plasmid named pSFV -F.2.Preparation of bacterial ghost loading suicidal DNA vaccine pSFV-F-ChIL18E.coli DH5a harboring the lysis plasmid pHH43 was grown at 28℃with agitation(200rpm). When the growing culture reached an optical density at 600 nm(OD₆₀₀)of 0.4-0.6,the incubation temperature was shifted up to 42℃to induce the gene E-mediated lysis process.After another 4h of incubation at 42℃,the culture was harvested.The CFU counting showed that 99.98%E.coli DH5a was killed efficiently.Electron microscopic studies showed that the cytoplasmic content of the bacteria was expelled leaving an empty internal space and that the protein E-specific transmembrane tunnel structure,which permeabilized the bacterium was predominantly,in the middle of the cell or at polar sites.Except for the lysis hole,the morphology of the bacteria,including all cell surface structures and appendices,was not affected by the lysis event.Bacterial ghosts were resuspended in PBS buffer saline(pH7.4)containing pSFV-F-ChIL18 and pSFV-F,and after CaCl₂ supplemention(final concentration 25mM),they were incubated at 37℃,150rpm for 30min.Then the culture product were centrifuged at 8000 rpm for10 min.to separate the bacterial ghosts from PBS.And then determinating the OD260 and OD280 of supernatant to count the content of DNA vaccine.3.Immunity test of bacterial ghost loading suicidal DNA vaccine pSFV-F-ChIL18One hundred and fifity chickens were randomly divided into 5 groups.,respectively at 7 day-old.and 21 day-old,â… group was injected with bacterial ghost loading pSFV-F-ChIL18,â…¡group was injected with pSFV-F-ChIL18,â…¢group was immunized with bacterial ghost loading pSFV-F,â…£group was immunized with NDV Lasota vaccine by eye-dropping,â…¤group was the control group without any treatment.The ELISA test showed that the content of IL-2,INF-γofâ… andâ…¡group was significantly higher thanâ…¢group(P＜0.01).Thelevels of antibody inâ…£group had risen obviously,which much higher than the other group immunized with DNA vaccine(P＜0.01).â… andâ…¢group were higher when compared withâ…¡group(P＜0.05).The protective immunization score were 60%forâ… group,50%forâ…¡group,40%forâ…¢group,100%â…£group and 0%forâ…¤group.