| In this study we constructed a novel and universal DNA vaccine vector, pkDNA, based on pVAX1 and pGL3-control and other auxiliary components (immune enhancing CpG motifs, DNA nuclear-targeting sequence, gene asd, and so on) using a method similar to synthetic biology. It as showed that egfp gene can be well expressed at the 15th hour after both pkDNA-egfp and pVAXl-eg/p transiently transfected into COS-7 cells. Protein tgB and UL24 of duck enteritis virus (DEV) were chosen as antigen genes, and either escher-ichia coliheat labile enterotoxin B subunit (LTB) or duck IL-2 gene as a molecular adjuvant were used for constructing DEV DNA vaccine plasmids. c8216△crp/cya (an attenuated S. Typhimurium with deleted crp,cya and asd) was used as carrier for the orally delivery of these DNA vaccine plasmids.20 days old ducks orally immunized two times of c8276 Acrp/cya harboring these vaccine plasmids produced a high level of both mucosal and systemic immune responses, and resist a deadly challenge of DEV. Here are the results:The construction of a novel and universal DNA vaccine vector:A novel and universal DNA vaccine vector, pkDNA, constructed by a method similar to synthetic biology:Firstly, single-gene asd, pUC origin, BGH poly A, CMV promoter/multiple cloning sites gene, SV40 enhancer and SV40 late poly (A) fragment were amplified from the UK-1 genome, pVAXl or pGL-control vector, respectively. Then two fragments were joined by PCR getting three modular segments (1/2DTSII+asd+CpG+pUC), (BGH poly A+CpG+SV40 enhancer) and (CMVMCS+CpG+SV40 poly A+1/2DTSII). Finally pkDNA was got for the assembling of these three fusion fragments by Gibson Assembly(?) Master Mix (New England Biolabs(?)inc). The base sequence of it has been proved correct by a repeat DNA sequencing. Direct fluorescence method showed that EGFP can be well expressed by pkDNA-egfp in COS-7 cells, which indicated that pkDNA could well deliver and express exogenous gene in eukaryotic cell.Construction and expression-ability detection of DEV DNA vaccine plasmids: tgB, UL24, LTB and DuIL2 were amplified respectively. And two fragments UL24-LTB and UL24-DuIL2 were got by overlapping PCR. PCR products of single-genes and fusion genes were digested with restriction enzymes and inserted into pkDNA resulting DEV DNA vaccine plasmids:pkDNA-tgB(451-1650bp), pkDNA-UL24, pkDNA-UL24-LTB and pkDNA-UL24-DuIL2.All the recombinant plasmids have been confirmed by sequencing.Indirect immunofluorescence experiments demonstrated that antigen proteins can be well synthesised in COS-7 cells after 24 hours of transfection, excepting pkDNA and empty control. RT-PCR was also identified that LTB or DuIL2 were transcribed in cells which transfected with pkDNA-UL24-LTB or pkDNA-UL24-DuIL2, but not in other groups.Construction of attenuated S. Typhimurium c8276 △crp/cya and recombinant Salmonella DEV-DNA vaccines:Attenuated S. Ttyphimurium c8276 △crp/cya was constructed on the base of c8276 (S. Typhimurium UK-1 asd gene deletion mutant strain), and it identified correct by PCR and biochemical identification.DEV DNA vaccine plasmids were then extracted and transformed to c8278 by electroporation, respectively, getting recombination attenuated Salmonella DEV-DNA vaccine strains:c8276 △crp/cya (pkDNA-tgB), c8216△crp/cya (pkDNA-UL24), c8276△crp/cya (pkDNA-UL24-LTB), c8276△crp/cya (pkDNA-UL24-DuIL2), c8276△crp/cya(pVAX1-UL24) and c8276 △crp/cya(pkDNA). All the plasmids’ genetics stability were identified good by PCR identification with every five generation until the 20th generation subcultured on LB solid medium, and they all performed avirulence to c8276△crp/cya. Besides, c8276 △crp/cya performed highly secure to ducklings when they orally vaccinated with 1012CFU c8276 △crp/cya (pkDNA).Study on vaccine efficacy of recombination attenuated Salmonella DEV-DNA vaccines:20 days old sheldrakes allotted randomly to 7 groups (each group of 15 duck), marked for groups A-G:c8276 △crp/cya (pkDNA-UL24) (A), c8276 △crp/cya (pkDNA- UL24-LTB) (B), c8276 Acrp/cya (pkDAN-UL24-DuIL2) (C), c8276△crp/cya (pkDNA-tgB +c8276△crp/cya (pkDNA-UL24) (D), c8276△crp/cya (pVAXl-UL24) (E),c8276△crp/cya (pkDNA) (F). Every duck administered at a dose of 1012CFU by a twice immunization with the interval of two weeks. Blood samples of 6 ducks were obtained at day 10,21 and 28 post the first immunization, for serum IgY detection against protein tUL24 and DEV viral particle. And 5 ducks of each group were sacrificed for bile at day 21 post the first immunization for bile IgA detection.All the ducks were challenged by DEV virulent virus two weeks after the second immunization. The result showed that group B induced the highest level of bile IgA (P<0.05), while slightly higher serum IgY titers were induced in ducks of groups B and C compared to the other groups (P<0.05), and a higher serum IgY titers against DEV were detected in group D compared to tUL24 as coating substrate. And all ducks immunized with c8278 strains harboring pkDNA plasmids synthesizing UL24 produced significantly higher antibodys than groups F and G (P<0.01).The percentages of morbidity and mortality in individual groups were monitored and recorded up to 12 days after challenge.The survival ratio was 9/10 in group B,7/10 in group D,6/10 in group C and E,5/10 in group A, which all were significantly greater than group F (2/10) and G (0). |
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