Infectious Bursal Disease Virus (ibdv) Of Vp2 In Yeast Cells And Efficient Expression And Immunogenicity Of The Determination

Infectious bursal disease is an acute, highly contagious viral disease mainly imperiling young chickens. Infectious bursal disease virus can not only cause high mortality in 3-6week susceptible chickens, but more seriously induce immunosuppression in young chickens, and subsequent vaccination failures for important chicken infectious diseases, such as New Castle抯 disease, Marek抯 disease infectious bronchitis etc. In this study, the major antigen gene VP2 of very virulent infectious bursal disease virus(vvIBDV) was high-efficiency expressed in pichea yeast expression system. The oligo primers P5-P6 was designed to amplify IBDV VP2 from previously cloned vvIBDV-VP2 in Puc 119, of which the upstream primer was added the yeast consensus sequence. The amplified 1.5kb VP2 gene fragment was ligated into the SinaI site of yeast plasmid PpiczA.The recombinant transfer plasmid inserted correctly by VP2 was identified by enzyme restriction analysis and nucleotide sequencing. According to the protocol provided by the company, the transfer plasmid was linearized by Sad and transfected into GS 115. The yeast recombinant was isolated and identified by polymerase chain reaction and Mut phenotype. After induction by Methanol, yeast cells were disrupted by mini glass beads method to release cellular proteins. Expression of VP2 was examined by SDS-PAGE, 3 Western-blot, AGID, and Dot-ELISA. PAGE revealed one distinct specific 41 kD band in the recombiant yeast lysate, which was compatible with the deduced VP2 of IBDV. Western blot analysis showed the 41 kd band could react with the positive serum, which confirmed that the expressed 4lkD protein is VP2. In AGID test, the lysate of recombinant yeast showed one thick precipitate line, similar to the positive control of condensed infectious bursal disease virus, versus there were not any precipitation for the negative GS 115 control. In Dot-ELISA, recombinant yeast showed distinct color reaction as the condensed virus positive control. All these tests showed that IBDV Vp2 was remarkably expressed and remain the similar biochemical and immunological activities. Expression efficiency of the recombinant yeast was examined by gel scanning analysis, the expressed Vp2 was up to OO.6282gIL, i.e. 16% of the total proteins of yeast cells. The recombinant yeast strain with highest expression efficiency was large-scale expressed, then sonicated and emulsified to immunize SPF chickens. It was revealed that recombinant yeast can induce precipitation antibody, and protect the chickens from lethal attack by highly virulent vvIBDV. The protection ratio of recombinant yeast is valuable, highlighting a good basis for the production of IBDV subunit vaccine.