Figure 3. Cloning and characterization of pVAX14.

A. Schematic diagram showing the cloning of the 824 bp insert into the BamHI site of the multiple cloning site (MCS) of the pVAX1 plasmid vector, generating the pVAX14 clone, the sequence of the insert comprising of the kanamycin resistance gene showing the GC-rich regions in green italics, the single consensus human specific CpG is in blue. The kanamycin resistance region is rotated 180° and the antisense reversed strand is coding, effectively flipped, hence the name Flipper. The 6 Open reading frames (ORFs) are depicted with start codons ATG in bold and stop codons TGA are in italics. The nucleotide numbers are boxed to coincide with the numbers on Supplemental Figure S1A; B. Expression of mRNA transcripts from the predicted ORFs. Expression was performed in COS and Phoenix cells. Gene expression was calculated for each probe set using the ΔΔCt method of empty vector (pVAX1) or pVAX14 against the untransfected RNA, all normalized against the ABL gene. Normalized pVAX1 was subtracted from normalized pVAX14 and plotted on a log scale. The ORF 4 is too small (29bp) to assay. Data are represented as mean +/− SEM of triplicate reactions; C. Identification of immunogenic peptides predicted from the inserted sequence (Supplementary Table S3). Four synthetic peptides are immunogenic in FVB/N mice as measured by the Ig production (Optical density); Open reading frames (ORFs) 1, 2 and 5 are immunogenic; inset shows protocol for induction of immune responses with the synthetic peptides. FVB/N mice (mouse no.1-3), n = 3 for each peptide (100 μg) were injected subcutaneously (SC) with Complement Freund's Adjuvant (CFA) on day 1 and the peptides were injected intraperitoneally (IP) (D1, D8, D21 and D30). Sera were collected before immunization and at day 35. Ig production was measured by measuring the optical density at a wavelength of 495 nm. Control mice were injected with PBS.