Expression Of VP2 Gene Of IBDV NB Strain In Pichia Pastoris

Infectious bursal disease(IBD) is chicken infected with infectious bursal disease virus(IBDV),Which mainly infringes chicken lymphoid tissue, especially the central immune organ bursa and causes immuse suppression. IBDV variants and virulent strains bring more challenge to current vaccination strategy against IBDV. Hence, the epidemiology of IBDV infection becomes more complicated. The major viral structural protein is VP2 protein, including the main protective antigen. Therefore, the express of recombinant VP2 protein can provide useful technical support and theoretical basis for the prevention and treatment of IBDV as well as the development of new IBDV subunit vaccine.The primers were designed according to the IBDV VP2 sequence in GenBank, the cleavage site of VP2 protein during maturation and the analysis of the antigen clusters, and then the gene of maturity VP2 protein was cloned by PCR from IBDV cDNA. The PCR products were subcloned to pMD18-T simple vector and transformed into DH5α. The recombinant cloning plasmid was identified by PCR, enzyme digestion and sequencing analysis. pMD-VP2 and eukaryotic expression vector were digested by Eco R I and Kpn I. The digestive products were connected with T4 DNA ligase enzyme and transformed into DH5α. After correction identification, the recombinant plasmid pPICZαC-VP2 was transformed into Pichia pastoris X-33 by electroporation, which was linearized by Pme I restriction enzymes. The positive transformants(pPICZαC-VP2-X33) were screened by ZeocinTM. The transformants were inoculated into culture medium and induced with methanol. The result was identified by SDS-PAGE and Western blotting.Results showed that the VP2 genes were successfuly amplified and cloned into expression vector of pPICZαC. The vectors were transformed into Pichia pastoris X-33 and induced by methanol. The expression products were analyzed by SDS-PAGE and showed that the products of specific expression in form of secretory expression with a relative molecular weight of 60.0 KDa, which were more than the theoretical molecular weight. The expression products of VP2 Combining with IBDV positive serum specific were dentified by Western blotting. The result showed that the recombinant protein of IBDV VP2 mature protein was succeessfully obtained.