Chracterization Of The Immunogenicity Of Recombinent Knob-S Derived From Egg Drop Syndrome Virus And Its Adjuvanted By Recombinant Chicken IL-2and IL-18

Egg drop syndrome(EDS-76) was a virulent infections disease caused by EDS virus with the main characteristics of decreasing egg laying and it was caused enormous economy lose to egg-laying hens industry. Virus antigen of traditional inactivated vaccine propagated in duck egg embryos. Such egg can be a source for other potential biosecurity risk factors. Industrial manufacturing of EDS vaccine urgently neeed a safety and efficacy vaccine to substitute the traditional duck egg derived vaccine.The prokaryotic expression of recombinant Knob-S in pET-32(a). One pairs of oligoprimer sequence were selected by primer premer5.0based on the previously reports and the Knob-S gene sequence of AV-127EDS virus strain in GenBank. Knob-S fragment was amplified by PCR and cloned into pET-32(a) vector. The recombinant vector was reconfirmed by PCR, restriction enzyme digestion and sequencing. The expected recombinant vector was transformed into E.Coli BL21stain. The culture conditions including IPTG concentration and expression time were optimized. The expression products were in forms of inclusion. The inclusion renaturation products were positive in Western-blot test and negative in hemaglutinin test. The results indicated immunogenicity of the prokaryotic expression products.The eukaryotic expression of recombinant Knob-S in Bac-to-Bac baculovirus expression system. Reference to the prokaryotic expression system, the Knob-S gene was cloned into pFastBacl vector and transformed to DH10Bac and got the recombinant Bacmid. The purified Bacmid was transfected in Sf9cell. The recombinant baculovirus expressed in Sf9cell with titer of106.625/0.1mL.The expression products were positive in indirect immunofluorescence test, agarose gel precipitation test and Western-blot. Combination all these results indicated immunogenicity of the baculovirus expression procucts.The prokaryotic expression of recombinant chicken interleukin-2(cIL-2) and interleuin-18(cIL-18) in pET-32(a). cIL-2and cIL-18were selected as immunopotentiator to add in vaccine and test its immune enhancement efficacy. Based on the sequence available in GenBank, a pair of primers were designed to amplify cIL-2or cIL-18gene, respectively. The chicken peripheral blood mononuclear cells stimulated by ConA were used as templates to amplify both cIL-2and cIL-18gene via reverse-transcriptase PCR. Nucleotide sequence of cIL-2and cIL-18gene shared more than99%homology with that published in GenBank and have no diversity in amino acid sequence. The cIL-2and cIL-18gene were cloned into pET-32(a) vector, sebsequently transformed into E.coli BL21, respectively. Both recombinant protein expressed in forms of inclusion which analysised by SDS-PAGE. The inclusions were renatured and purified with niccloum in a kit, which provided bio-material for next step of vaccination in animal experiment.Characterization the immunogenicity of the recombinant Knob-S and immuno enhancement efficacy of cIL-2and cIL-18. Seven candidate vaccines were prepared from the expressed products, vaccine antigen including inclusions forms of Knob-S expressed by E.coli, purification forms of Knob-S expressed by E.coli, purification forms of Knob-S expressed by E.coli and combination with cIL-2, purification forms of Knob-S expressed by E.colr and combination with cIL-18. Knob-S expressed by Sf9cell, Knob-S expressed by Sf9cell, and combination with cIL-2, Knob-S expressed by Sf9cell, and combination with cIL-18, resectively. E.coli with empty vector or Sf9with empty vector were made of vaccine as control. Vaccine containing the dose of each recombinant protein were described as follows:50μg per dose for purification forms of E.coli expressed Knob-S,300μg per dose for inclusion forms of E.coli expressed Knob-S,50μg per dose for cIL-2or cIL-2, respectively.1.5x106TCID50per dose for Sf9expressed Knob-S. The other two control vaccination groups were included commercial inactivated EDS vaccine immunized group and unvaccinated group. Total of11groups of ten six-week old chicken were included in this trial. Blood were collected at two-week, three-week, four-week and five-week post vaccination. Serum antibody against EDSV were tested by hemagglutinin inhibition. All birds injection with the vaccine containing the recombinant Knob-S components were elicited antibody against EDSV. The immuno-efficacy of Sf9expressed Knob-S slightly better than that of E.coli expressed Knob-S, particularly, the Sf9expressed Knob-S combination with cIL-2showed the best immune efficacy among all recombinant Knob-S vaccination groups. Antibody response was negative in both empty vector vaccination groups and unvaccinated group. Results indicated that recombinant Knob-S proteins and recombinant cIL-2and cIL-18showed good immunogenicity in chickens, recombinant cIL-2and cIL-18also possessed immune enhancement efficacy.Combination of all results of this study indcated that all recombinant Knob-S proteins expressed by prokaryotic or eukaryotic expression system displayed good immunogenicity. Immune efficacy of these recombinant Knob-S proteins were elevated when combined utilization with cIL-2and cIL-18as immunopotentiator, Immunoenhancement efficacy of cIL-2surpass that of cIL-18. Achievement of this study will provid bio-materials in developing EDS subunit vaccine.