Production of goose parvovirus (GPV) VP2 based recombinant subunit protein

Goose parvovirus (GPV), first identified as Derzsy disease in Europe in the 1960s, causes great economic losses and high mortality in the waterfowl industry. For this reason, it is very important to establish prevention/control targets at the point of fighting the disease. The GPV genome is approximately 5.1 kilobases (kb) long and contains two open reading frames (ORFs). The structural proteins of the virus, VP1, VP2 and VP3, are formed by alternative splicing mechanisms of the same ORF. Of these, the VP2 protein is the most expressed protein, and it is the protein that most strongly stimulates the host immune response. For this reason, in thesis the aim was to produce the VP2 protein in the Baculovirus expression vector system (BEVS), intended for use as a candidate antigen in the development of a recombinant vaccine or rapid test kit against GPV. For this purpose, the VP2 gene was first amplified by PCR using specific primers for GPV. After the obtained VP2 amplicon was purified, it was cloned into the commercial pENTR™/TEV/D TOPO™ entry vector. Then, the target gene contained in the pENTR™/TEV/D-TOPO™ vector was transferred to linear N-Term BaculoDirect™ DNA via LR Recombination. This created structure was transfected into Spodoptera frugiperda (Sf9) insect cells and baculoviral virions were obtained. In order to confirm whether the VP2 gene was transferred to BaculoDirect™ Linear DNA, PCR was performed using external primer sets for both the VP2 gene and the expression vector. As a result of amplification, a band of approximately 600 bp in size was observed. To verify the production of the recombinant protein, Western Blot analysis was performed using the V5 epitope located at the N terminus of the produced protein. As a result of the analysis, a band with the expected molecular weight (~65 kDa) belonging to the VP2 gene was detected. IFA analysis was performed using the same epitope to demonstrate protein expression in Sf9 cells infected with recombinant baculovirus. After the expression was confirmed, the protein was purified with the Ni-NTA nickel chelating resin kit by utilizing the 6×His tag in the protein. Finally, the fusion-tagged recombinant protein was cut with AcTEV protease and purified from His and V5 epitopes. This thesis research aimed to obtain recombinant protein closest to indigenous by using the BEVS technique. It is believed that the production of this protein could pave the way for rapid serology-based detection kits or recombinant subunit particles suitable for use as vaccines.