Prokaryotic expression and immune effect evaluation of VP2 protein of a novel variant infectious bursal disease virus strain

【Objective】 This paper constructed the prokaryotic expression plasmid of VP2 protein of a novel variant of infectious bursal disease virus（vaIBDV），and clarified the immunogenicity of VP2 protein，so as to provide technical support for the development of vaIBDV subunit vaccine.【Method】 The VP2 gene fragment was amplified from vaIBDV isolate（BZ strain）and cloned into vector pET-28a （+）to recombine prokaryotic expression plasmid， then transformed into the Escherichia coli BL21（DE3）competent cell，and induced by IPTG. The expressed products were identified by SDSPAGE and Western blotting，and the titer was determined by vaIBDV-positive serum through agar-gel precipitation （AGP）. After purification by Ni-NTA affinity chromatography and proper concentration，the emulsified vaccine was prepared and used to immunize 21-day-old SPF chickens. The immunogenicity of the fusion protein VP2 was further verified by safety test，antibody titer determination and immune challenge experiment.【Result】 The fusion protein VP2 was mainly expressed in soluble form at 25 ℃ induced by IPTG，and the size was about 40 kD. The fusion protein VP2 in the expression supernatant could specifically react with vaIBDV positive serum，and its agar diffusion titer could reach 1∶32. After purification by Ni-NTA affinity chromatography and appropriate concentration，the agar diffusion titer of the fusion protein VP2 was as high as 1∶256. The subunit vaccine prepared with the fusion protein VP2 as the antigen had good stability，high safety，and no adverse reactions caused by immunization. No obvious damage，local inflammation，and poor vaccine absorption were found at the inoculation site. After 21 d of immunization，the average agar diffusion titer of chicken serum was 1∶76.8，and the serum antibody titer was above 1∶20000，indicating that the vaccine prepared with the fusion protein VP2 could stimulate the body to produce a strong humoral immune response，and the antibody positive rate was 100%. The pathological changes of bursa of Fabricius in chickens were observed 7 d after challenging with IBDV BZ strain. The results showed that none of the chickens in the immunized group had disease，and there was no obvious atrophy of bursa of Fabricius，which meant that the immune protection rate was 100%.【Conclusion】The soluble expression of vaIBDV VP2 protein is successfully achieved by prokaryotic expression system，and the obtained fusion protein VP2 has good immunogenicity and can provide 100% immune protection against vaIBDV，which provides technical support for the development of vaIBDV subunit vaccine.