Detection of Sacbrood Virus Infecting Apis cerana (AcSBV) Using Polyclonal Antibody Raised against Recombinant VP3 Capsid Protein Expressed in Escherichia coli

Abstract

In Korea, sacbrood virus infecting Apis cerana (AcSBV) is a small RNA virus belonging to the family Iflaviridae. AcSBV causes larval death, and even the collapse of entire bee colonies and its viral capsid is composed of four structural proteins (VP1, VP2, VP3 and VP4). In this study, we synthesized VP3 gene based on genomic sequences to insert into a His-tag expression plasmid of pET-28a. The resultant recombinant vector was then transformed into Escherichia coli BL21 (DE3) to express the recombinant VP3 protein (rVP3). The rVP3 protein was strongly expressed in an insoluble fraction, with a molecular size of around 26 kDa. Purification of the rVP3 protein using an affinity Ni-NTA resin, followed by SDS-PAGE separation, resulted in a high purity of the protein and used as an antigen to raise the polyclonal antibodies. Mice were then immunized with the purified rVP3 protein and the antiserum collected at 4SUPth/SUP week and 6SUPth/SUP week was able to detect the rVP3 at a concentration of 500 ng and 250 ng. Furthermore, IgG antibody levels reacted with wild-type larva crude protein extract, AcSBV-infected larva crude protein extracts and rVP3 protein were quantitatively analyzed by ELISA and qualitatively analyzed by western blotting. This is the first report on production of polyclonal antibodies directed against the rVP3 protein of AcSBV and its use for detection and diagnosis of virus from field samples.