Generation of Recombinant Porcine Parvovirus Virus-Like Particles inSaccharomyces cerevisiaeand Development of Virus-Specific Monoclonal Antibodies

Abstract

Porcine parvovirus (PPV) is a widespread infectious virus that causes serious reproductive diseases of swine and death of piglets. The gene coding for the major capsid protein VP2 of PPV was amplified using viral nucleic acid extract from swine serum and inserted into yeast Saccharomyces cerevisiae expression plasmid. Recombinant PPV VP2 protein was efficiently expressed in yeast and purified using density gradient centrifugation. Electron microscopy analysis of purified PPV VP2 protein revealed the self-assembly of virus-like particles (VLPs). Nine monoclonal antibodies (MAbs) against the recombinant PPV VP2 protein were generated. The specificity of the newly generated MAbs was proven by immunofluorescence analysis of PPV-infected cells. Indirect IgG ELISA based on the recombinant VLPs for detection of PPV-specific antibodies in swine sera was developed and evaluated. The sensitivity and specificity of the new assay were found to be 93.4% and 97.4%, respectively. In conclusion, yeast S. cerevisiae represents a promising expression system for generating recombinant PPV VP2 protein VLPs of diagnostic relevance.

Investigation of Kluyveromyces marxianus as a novel host for large‐scale production of porcine parvovirus virus‐like particles

“…Previous studies revealed that the recombinant VP2 expressed in insect, eukaryotic or prokaryotic cells formed virus-like particles (VLPs) [ 16 , 18 , 19 , 41 , 42 ]. To test whether VP2 proteins were spontaneously assembled into VLPs in K. marxianus , cell lysates of KM-PPV-VP2 was scanned by TEM, and results confirmed that VP2 proteins were assembled into VLPs with a diameter of approximately 20 nm in K. marxianus (Fig.…”

“…Attribute to high growth rate and facile high biomass, the recombinant K. marxianus strain produced 2.5 g/L PPV VLPs after just a 48 h fermentation. As a comparison, the production level of VLPs in Pichia pastoris , E. coli , S. cerevisiae is 595.76 mg/L [ 19 ], 15 mg/L [ 35 ], and 8–9 mg/L [ 18 ] respectively. Moreover, K. marxianus has a notable advantage over other strains, such as S. cerevisiae and E. coli , on high production of VLPs that there is no additional inducement during its fermentation since glucose serves as the carbon resources to support cell growth as well as an inducer for its inulinase promoter to obtain high expression of heterologous proteins [ 25 ].…”

“…As to yeast cells, there is no additional treatment to remove endotoxins, but other downstream operations, such as disrupting the rigid wall of the yeast cells to release VLPs, clarification of cell lysates, and purification processes, are the main factors that significantly increased the production costs overall [ 44 ]. In previous studies, the PPV VLPs were commonly purified by sucrose or cesium chloride gradient centrifugation [ 18 , 45 ], or Ni-NTA affinity chromatography [ 20 , 41 ], which are not conducive to large-scale production. Aiming to create a simple and low-cost purification process, we assessed the ion-exchange chromatography for purification of PPV VLPs comprehensively.…”

New Monoclonal Antibodies for a Selective Detection of Membrane-Associated and Soluble Forms of Carbonic Anhydrase IX in Human Cell Lines and Biological Samples

“…Flow cytometry data were analyzed with FlowJo (FlowJo, LLC, Ashland, Oregon, USA). As an isotype control, irrelevant MAbs of IgG1 subtype prepared in-house against porcine parvovirus (PPV) were used [42].…”

Inhibitory Monoclonal Antibodies and Their Recombinant Derivatives Targeting Surface-Exposed Carbonic Anhydrase XII on Cancer Cells

“…The MAbs 1B10 and 14D6 reactive with cellular CA XII and the non-reactive MAb 9B9 are shown. Isotype control—the in house produced MAb of the relevant isotype raised against Porcine Parvovirus [ 32 ]. Secondary antibody: Alexa Fluor 488-conjugated Goat anti-Mouse IgG (H + L) (Thermo Fisher Scientific, A–11029).…”