Generation of genetically engineered M13 bacteriophage for delivery of heterologous DNA and protein in fish

Abstract

The M13 bacteriophage is a virus that explicitly infects Escherichia coli which exhibits an F pilus. It is composed of a simple structure with five different coat proteins surrounding its genome. Since the development of phage display technology that enables the display of foreign peptides/proteins on phage surfaces, a variety of applications ranging from medicine to nanomaterials have attempted to utilize phages. Especially, studies about using phage as a delivery tool for drugs and protective antigens have been getting much attention recently. In this study, a recombinant phage was generated to deliver foreign DNA and proteins via genetic engineering. The foreign DNA delivered via the phage was CpG-ODN 1668, which was chosen because of its protective efficacy against pathogens including scuticociliate and Edwardsiella tarda reported in olive flounder. The phage was administered by intraperitoneal injection, and the head kidney of the olive flounder was removed 48 h after injection to confirm the activated level of the head kidney phagocytes via chemiluminescence assay. The stimulatory CpG motif harboring phage administered group showed the highest CL intensity compared to other groups. The delivery of foreign antigens via phage surfaces is based on the phage display technology. With the existing system, the size of the molecule that can be expressed on the major coat protein of the M13 phage is limited to 15aa. By applying the SpyCatcher/SpyTag system, which enables the decoration of virus-like particles with antigens of diverse sizes, we aimed to display foreign antigens that cannot be displayed on the major coat protein with the existing system. The conjugation reaction between SpyTag displaying phage on the major coat protein and SpyCatcher-fused protein was confirmed by SDS-PAGE and Western blotting, and the protein displaying phage administered group showed a higher antibody titer than other groups in ELISA. Unlike subunit vaccines, which require the addition of an adjuvant to induce a sufficient immune response, phages do not require the addition of an adjuvant as a self-adjuvanting agent. However, to use phages as an antigen delivery tool in vaccine development, it is necessary to confirm the relationship between antibody production and protection efficacy. If so, we can expect the use of phages as versatile delivery tools.