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In this study were isolated from the domestic IBD super poison, and its successful cultivation, attenuated, revealing vvIBDV from super attenuated virulence to force the conversion process, the main structural protein gene sequence vp2 the change process, and can be obtained on the chick embryo fibroblast proliferation, with a strong super pathogenic strains and have better immunogenic attenuated strains. The powerful poison Vp2 gene was completed Hearst (Pichea) yeast expression system highly expressed protein has good immunogenicity, with its immunogen successfully developed a subunit vaccine. IBD research will provide effective control of advanced, reliable technical support. This study involved China - EU cooperation projects (ERBIC18CT980330): \Press the OIE and EU standards, will flock of Guangxi pathogenesis of IBD IBD wild virus isolated by pathogenicity, antigenic and genetic sequence analysis identified three aspects of the research to determine which is IBD super strains. The strain on the four-week-old SPF chickens lethality rate of 60% or more, antigen test and sequence analysis showed that the drug has a powerful feature, and with the European standards vvIBDV UK661 closer genetic relationship, the main structural protein homology of 99% to 100% amino acid homology. In collaborative research, the EU researchers and Chinese researchers to jointly determine that it is vvIBDV Chinese standard strain, named vvIBDV-GX. IBD and IBDV for our research provides with the international standards. Right vvIBDV-GX SPF chicken strains were quick nurturing and passaged cells induced weak cytotoxicity of various passages performed sequence analysis and biological experiments and found cytotoxic in 7th generations ago, Vp2 gene sequence has not changed; Cell Poison 8th generation, there are individual nucleotides changed, but did not affect the amino acid sequence; cell ninth generation, a super toxic characteristics of the first 222 area alanine and heptapeptide silk amino acids into a third characterized by having attenuated proline and arginine; 10 generations cytotoxic Vp2 attenuated gene sequences with the European standard amino acid sequence homology of 97% or more, then continue to spread to 20 generations, Vp2 gene sequences virtually no recurrence changed. Biology experiments also showed that cells with super toxic poison 5th generation features, inoculated 4-week-old SPF chickens, the mortality rate of 60%; while 20 generations toxic non-pathogenic for chickens, chickens continuous passage in 6th generation does not return Strong. Five generations 20 generations toxic poisoning and can be found in chicken embryo fibroblasts proliferation, drug prices are up more than 2.0 × 10 ~ 8PFU/ml. This study demonstrates that, vvIBDV through cultivation, by the very virulent strain evolution of unstable intermediate strains, and then continue to evolve as a stable attenuated strains. After five generations of drug inactivation, still has good immunogenicity, 10 million PFU / dose of inactivated toxin only vaccinated SPF chickens immunized chickens vvIBDV enable protection rate of 100%. 20 on behalf of non-virulent poison to chickens, there are strong immunogenicity after vaccination, the immune chickens can produce specific antibodies, antibody-positive rate of 100%, and can effectively resist the powerful poison attacks. In this study, five generations of cultivated cells inactivated vaccine toxic species can be used as drugs, 20 on behalf of the drug can be used as attenuated vaccine seed virus. OLIGO software using primers designed to insert vvIBDV-GX Vp2 PUC19 gene as a template, amplified fragment 1.5KB; this fragment was subcloned into the yeast expression vector (PpiczA), and The constructed vector (PpiczA-Vp2) with Sacll linearized GS115 cells transfected yeast; identified by PCR, phenotypic identification, screening recombinant yeast, SDS-PAGE electrophoresis, expressed in 41KD protein band appears at; induced by small-scale methanol, filter out high expression strain , adjusted to optimize the induction time, temperature, shaking speed, methanol content, and other technical parameters, temperature 28 ℃, speed 220rpm, methanol content of 0.5% under the conditions inducing 120 hours, the expressed protein agar diffusion test the PhD thesis of infectious bursal disease virulent strain of nurturing China attenuated and subunit vaccines are specific precipitation line, D. Bu * u8A iKI \u0026 specific color reaction occurs, the expression of 26%, are highly expressed. Expression of proteins in the ratio of 1:1.5 white oil emulsion adjuvant, made subunit vaccine immunized SPF chickens, can produce specific antibodies, 0.3mgi dose only after immunization and antibody titer in the 1:1000 or more, and can protect the immune chicken resist vvlnDV lethal attacks, the protection rate of 90%.
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