Cloning and Expression of Gumboro VP2 Antigen in Aspergillus niger

Abstract

Background: Infectious Bursal Disease Virus (IBDV) causes a highly immunosuppressive disease in chickens and is a pathogen of major economic importance to the poultry industry worldwide. The VP2 protein is the major host-protective immunogen of IBDV and has been considered as a potential subunit vaccine against the disease. VP2 coding sequence was cloned in an inducible fungal vector and the protein was expressed in Aspergillus niger (A. niger).

Methods: Aiming at a high level of expression, a multicopy AMA1-pyrG-based episomal construct driven by a strong inducible promoter, glaA, was prepared and used in transformation of A. niger pyrG-protoplasts. SDS-PAGE and western blot analysis was carried out to confirm the expression of the protein.

Results: A number of pyrG (+) positive transformants were isolated and the presence of expression cassette was confirmed. Western blot analysis of one of these recombinant strains using monospecific anti-VP2 antibodies demonstrated the successful expression of the protein. The recombinant protein was also detected by serum obtained from immunized chicken.

Conclusion: In the present study, we have generated a recombinant A. niger strain expressing VP2 protein intracellulary. This recombinant strain of A. niger may have potential applications in oral vaccination against IBDV in poultry industry.

Keywords: Aspergillus niger; Recombinant proteins; VP2 protein.

Figure 1

A) Schematic representation of expression cassette construction. Vp2 final fragment was cloned into NotI site of pRG3-AMA1; B) Restriction analysis of pAMA_vp2. M: Size marker. Lane 2: Undigested pRG3-AMA1-NotI (plasmid backbone). Lane 3: NotI linearized pRG3-AMA1-NotI (∼ 10 kb). Lane 3: undigested pAMA_vp2. Lane 4: NotI digested pAMA_vp2. The backbone plasmid (∼ 10 kb) and the glaA-vp2 fragment (∼ 3kb) are present

Figure 2

Western blot analysis on A. niger cell lysates using anti-VP2 antibodies. A) Detection of VP2 using polyclonal serum obtained from the immunized chickens. Lane 1: wild type strain; lane 2: vp2 transformant 24 hr sample; lane3: vp2 transformant 48 hr sample; B) Detection of VP2 using anti-vp2 monoclonal antibody. Lanes’ order is the same as A