When the experiment was finished mice were decoded and results were analyzed

When the experiment was finished mice were decoded and results were analyzed. 2.5. al., 2009, Brinkman et al., 2005). In contrast, information about the capacity of HaPyV VP1 based chimeric VLPs to induce effective epitope-specific CTL immune response is still missing. In this study we have investigated the capacity of HaPyV VP1-based chimeric VLP to trigger the development of effective CTL response by using well established model system dependent on CTL immunity based protection against computer virus and tumors and were used to study GP33-specific CTL response in mice Rabbit polyclonal to ADRA1C and and selection of positions #1 and #4 in the primary structure of VP1 (corresponding to aa residues between 80C89 and 288C295) for introduction of foreign sequences were explained previously (Gedvilaite et al., 2000, Sasnauskas et al., 1999). An oligonucleotide duplex encoding the 9 amino acid long LCMV GP33 CTL peptide (Fig. 1 A) was inserted into the VP1-encoding sequence into positions #1 and #4 (Fig. 1B) cloned into the expression cassette of the yeast vector pFX7 (Sasnauskas et al., 1999). Chimeric VP1-GP33 protein-encoding sequences were confirmed by DNA sequencing. Open in a separate window Fig. 1 Generation and purification of HaPyV VP1 based VLPs. (A) Nucleotide and amino acid sequences of LCMV GP33 epitope. (B) Schematic physique of VP1, VP1-GP33-1 and VP1-GP33-4 proteins (aa positions of insertion sites #1 and #4 are indicated) and their identification in SDS-PAGE and Western blot analysis (WB) with anti-HaPyV VP1 mAbs 6D11 (C). In lanes: 1 C purified VP1 protein; 2 C lysate of yeast cells expressing VP1-GP33-1 VLPs; 3 C lysate of yeast cells expressing VP1-GP33-4 VLPs; 4 C purified VP1-GP33-1 protein; 5 C purified VP1-GP33-4 protein; 6 C PageRuler prestained protein ladder (#SM0671 Fermentas, Lithuania). (D) Electron microscopy pictures of VLPs, stained with 2% aqueous uranyl acetate answer and examined by Morgagni electron microscope. The procedure utilized for VLP generation in the strain AH22 derivative 214 (a for 48?h on CsCl gradient (1.23C1.38?g/mL). Fractions made up of purified proteins were collected, pooled, dialyzed against PBS, lyophilized and stored at ?20?C until further use. All purified VLPs were examined for nucleic acid contaminations according to a protocol explained previously (Sasnauskas et al., 1999) and DNA/RNA was not detected. 2.2. SDS-PAGE, Western blot analysis and electron microscopy Preparations of protein samples, SDS-PAGE, and Western blot analysis were performed according to methods explained previously (Gedvilaite et al., 2004). As main antibody anti-HaPyV-VP1 mAb 6D11 (Dr. A. Zvirbliene, Vilnius, Lithuania) or mice serum (1:300) were used. To confirm VLP assembly purified proteins were placed on 400-mesh carbon coated palladium grids. The samples were stained with 2% aqueous uranyl acetate answer and examined by Morgagni electron microscope (FEI, Oregon, USA). Dynamin inhibitory peptide 2.3. Peptide and computer virus GP33 synthetic peptide (KAVYNFATM) corresponding to an H-2Db-restricted CTL epitope (amino acid residuals 33C41) from LCMV surface glycoprotein, was purchased from Biosyntan Dynamin inhibitory peptide GmbH (Berlin, Germany). The original cysteine at the anchor position 41 in the LCMV GP33 peptide was replaced by methionine (Aichele et al., 1994). The LCMV-WE isolate used in this study was originally obtained from R. Zinkernagel (Zurich, Switzerland). LCMV-WE was cultivated in MC57 (H-2Db) fibrosarcoma cell collection using DMEM supplemented with 2?mM l-glutamine, Dynamin inhibitory peptide penicillin and streptomycin, 10% FCS. Mice were infected i.v. with 200?pfu and viral titers were determined using MC57 cell collection as described in Battegay et al. (1991). Statistical significance of virus challenge experiments was calculated using Wilcoxon MannCWhitney test. 2.4. Mice and tumors C57BL/6 (B6) mice were obtained from breeding stock of the Friedrich-Loeffler Institute (FLI, Germany). Transgenic H-2Db (P14) mice, collection 327, transporting the TCR specific for the amino acids 33C41 epitope of LCMV glycoprotein GP33 (Aichele et al., 1994) were purchased from H.P. Pircher (Freiburg, Germany). 8C16-week-old mice of both sexes were used and were kept under SPF conditions in the animal facility of the FLI. All animal experiments were carried out in accordance with institutional guidelines Dynamin inhibitory peptide and permission of the national government bodies. For tumor experiments MCA102 and MCA102-GP33 fibrosarcoma tumor cells were used (Blohm et al., 2002). MCA102-GP33 cells were derived from parental MCA102 tumor cells by gene transfection using the LCMV GP33 minigene (Prevost-Blondel et al., 1998). Both MCA102 cells were cultured in DMEM high glucose supplemented with 10% FCS, 2?mM l-glutamine, penicillin and streptomycin (PAA Laboratories GmbH, Austria), transfected MCA102-GP33 cells additionally under 600?g/mL G418 selection (Gibco, Invitrogen, UK). In tumor animal trials mice were encoded by ear tagging to eliminate subjective bias and injected with 106 of MCA102-GP33 tumor cells in 100?L PBS into the.