EUKARYOTIC EXPRESSION AND CHARACTERIZATION OF PORCINE PARVOVIRUS VP2 PROTEIN AS POTENTIAL DIAGNOSTIC CANDIDATE
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Date
2018
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Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana
Abstract
PPV, a ubiquitous pathogen in swine prevalent throughout the world, causes endemic infection leading to considerable economic losses to farmers in terms of stillbirth, mummified fetuses, early embryonic and fetal death, delayed return to estrus, and infertility (SMEDI). Recombinant protein based diagnostics have more advantages over the conventional methods for large scale screening of animals. This study aimed at eukaryotic expression and characterization of PPV VP2, a major immunogenic capsid protein, as a potential diagnostic candidate. PPV VP2 gene was codon optimized for better eukaryotic expression and then the full length as well as N- and C- terminal fragments of the protein were expressed in the High Five insect cells following transfection of the recombinant plasmids. Subsequently expression vector pIZ-V5-His and pIZ-GLPS were used to clone and express the VP2 gene in High Five insect cells for getting the recombinant proteins in cytoplasmic and secreted forms, respectively. The High Five cells could successfully express the full length and the C terminal fragment of the PPV VP2 protein (VP2-Ctm) as secreted proteins with product size of ~64KDa and ~42KDa, respectively. The purified, secreted VP2 proteins (both full and C terminal fragment) showed good reactivity with anti-PPV sera as well as anti-His antibody in Dot blot and Western blot assays. Transmission electron microscopy (TEM) of the recombinant full length VP2 revealed self-assembly and formation of virus like particles (VLPs). The complete recombinant VP2 protein when used as coating antigen in an Indirect ELISA showed better reactivity with antiPPV pig sera as compared to VP2-Ctm recombinant protein. These results clearly showed the capability of the recombinant VP2 capsid proteins as a potential diagnostic/vaccine candidate.
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