The fractions containing proteins of 42 and 45kDa corresponding to VP1 and p16INK4Afused with VP2 (VP2-p16INK4A), respectively, were pooled and diluted in buffer DB 150 (150mM NaCl, 1mM CaCl2and 0.001% Trition X-100 in 10mM Tris/HCl-buffer pH 7.2). pseudotype VLPs induced in immunized mice a strong immune response against the target antigen. The antisera raised against pseudotype VLPs showed specific immunostaining Dulaglutide of p16INK4Aprotein in malignant cervical tissue. Spleen cells of the immunized Dulaglutide mice were used to generate monoclonal antibodies against p16INK4Aprotein. The specificity of antibodies was confirmed by the immunostaining of HPV-transformed cells. In conclusion, the current study demonstrates the potential of pseudotype VLPs with inserted target antigen as a new type of immunogens to generate antibodies of high diagnostic value. == 1. Introduction == Gene and protein engineering provides an opportunity to generate novel chimeric proteins with desired features, such as enhanced immunogenicity. Structural proteins originating from human and animal viruses, for example, papilloma, hepatitis B, and parvo- and rotaviruses with their intrinsic capacity to selfassemble to highly organized structuresvirus-like particles (VLPs)have been shown to possess high immunogenicity and therefore exploited as potential vaccines [13]. Moreover, recombinant VLPs can be employed as carriers for non immunogenic proteins or peptides in order to enhance their immunogenicity. Previous studies exhibited that insertions/fusions of foreign protein segments at certain sites of VLP carriers derived from papilloma-, polyoma-, hepadna-, parvo-, and retroviruses did not influence protein folding and assembly of chimeric VLPs. The immunogenicity of foreign sequences presented on the surface of chimeric VLPs is usually enhanced making these VLPs promising vaccine candidates [47]. Recently, we have exhibited that hamster polyomavirus (HaPyV) major capsid protein VP1-derived VLPs are highly immunogenic and tolerate inserts of different size and origin at certain surface-exposed positions. The chimeric HaPyV-VP1 VLPs have been shown to activate efficiently the antigen-presenting cells and induce strong insert-specific B- and T-cell responses in mice [8,9]. These studies exhibited that chimeric VLPs represent promising novel immunogens to generate Dulaglutide monoclonal antibodies (MAbs) of the desired epitope-specificity. The main advantage of chimeric VLPs over tradicional immunogens such as synthetic peptides chemically coupled to carrier proteins is the exposure of the target sequence on the surface of VLPs thus allowing its accessibility to the B cells [9]. Although Dulaglutide chimeric VLPs tolerate inserts up to 120 amino acid (aa) residues, the insertion of longer protein sequences generally affects proper folding and self-assembly of VLPs (our unpublished observation). Therefore, new approaches for enhancing the immunogenicity of long protein segments or full-length proteins are needed. This is especially important for human cellular proteins that may be tolerogenic in mice because of high homology with murine proteins. Strong immunogens presenting the target protein sequence on a suitable carrier may break the tolerance barrier and increase the immunogenicity of non-immunogenic proteins or protein segments. In the current study, we designed novel recombinant immunogens based on pseudotype VLPs consisting of two HaPyV-derived capsid proteinsan intact VP1 protein and altered VP2 protein harbouring the target protein sequence at VP2 N terminus. As a target sequence, we have used full-length cellular protein of high diagnostic relevance p16INK4Athat is considered to be a potential marker for cells transformed by high-risk human papillomavirus (HPV). We have exhibited that pseudotype VLPs consisting of an intact VP1 protein and VP2 protein fused with the p16INK4Aantigen at its N terminus induced a strong antibody response against the target sequence which allowed generation of p16INK4A-specific MAbs. == 2. Materials and Methods == == 2.1. Production of Pseudotype VLPs Harbouring Full-Length p16INK4AProtein == All DNA manipulations were carried out according to standard procedures [10]. Enzymes and kits for DNA manipulations were purchased from Thermo Scientific Fermentas (Vilnius, Lithuania). Recombinant plasmids were screened inE.coliDH10B cells. The synthetic gene encoding the full length p16INK4Aprotein (synthesized by Integrated DNA Technologies, BVBA, Leuven, Belgium) was fused to hamster polyomavirus (HaPyV) VP2 gene altered at its N terminus in the plasmid pFGG3-VP1/VP2Bg. This plasmid was constructed by inserting HaPyV VP1 gene into GAL 7 expression cassette and altered HaPyV VP2 gene under GAL10-PYK1 hybrid promoter into yeast expression vector pFGG3 [11]. To construct the altered HaPyV VP2 gene, the sequence encoding 1100 aa was deleted and GSS linker coding sequence and the BglII restriction site were introduced at Rabbit polyclonal to AFF2 its N terminus for a fusion with p16INK4Acoding sequence. The resulting plasmid pFGG3-VP1/VP2-p16 was used for the transformation of theSaccharomyces cerevisiaestrain AH22-214 (a, leu2-3, 112, his4-519). Dulaglutide Transformed yeast cells were grown in.