Cell Lines and Animals The K562 cell line was obtained from the Institute of Hematology, Jinan University College of Medicine, and it was cultured in RPMI-1640 medium (Gibico-BRL, USA) supplemented with 10% fetal bovine serum (Gibico-BRL, USA) at 37C in a 5% CO2 atmosphere. Male BALB/c mice, 6 to 8 8 weeks of age, were purchased from the Guangdong Provincial Medical Experimental Animal Center (animal certificate no. Materials and Methods 2.1. Cell Lines and Animals The K562 cell line was obtained from the Institute of Hematology, Jinan University College of Medicine, and it was cultured in RPMI-1640 medium (Gibico-BRL, USA) supplemented with 10% fetal bovine serum (Gibico-BRL, USA) at 37C in a 5% CO2 atmosphere. Male BALB/c mice, 6 to 8 8 weeks Rabbit polyclonal to COPE of age, were purchased from the Guangdong Provincial Medical Experimental Animal Center (animal certificate no. SCXK2008-0002), and they were bred at the Experimental Animal Center of Jinan University College of Medicine under controlled conditions and received standard laboratory chow and water according to institutional guidelines. Upon delivery, the mice were allowed to acclimatize for one to two weeks before the start of the experiment. The experiments were approved by the local Animal Ethics Committee and followed international ethical standards of conduct. 2.2. DNA Vaccine Preparation The 487?bp hIL-2 fragment, which contains four exons expressing the hIL-2 core structure, was amplified as previously described [24]. Amplification of the BCR/ABL fusion gene segment was performed in cDNA from K562 cells by RT-PCR. The primers used in the present study were listed in Table 1. The following is the RT-PCR program file used: 30 cycles at 95C for 4?min, at 94C for 1?min, at 60C for 1?min, and at 72C for 1?min, one cycle at 72C for 10?min. Briefly, the BCR/ABL segment with was designed to cover the fusion point of BCR and ABL gene corresponding to p210BCR/ABL(b3a2), including part of exon 14 from BCR gene and part of exon 3 and exon 2 from ABL gene (GenBank accession numbers “type”:”entrez-nucleotide”,”attrs”:”text”:”AJ131466.1″,”term_id”:”4033554″AJ131466.1). The p210BCR/ABL junction region selected contained 110 amino acids (GLYGFLNVIV HSATGFKQSS Lemildipine KALQRPVASD FEPQGLSEAA RWNSKENLLA GPSENDPNLF VALYDFVASG DNTLSITKGE KLRVLGYNHN GEWCEAQTKN GQGWVPSNYI), with lysine of the fusion point in the middle, 20 adjacent amino acids (aa) from the C-terminal of the BCR protein fragment, and 90 adjacent aa from the N-terminal of the ABL protein fragment. Table 1 Sequences of primers used in PCR. Sequencesand restriction sites, and the hIL-2 segment was inserted into the MCS B site of the pIRES eukaryotic expression vector (BD Biosciences Clontech, Palo Alto, CA, USA) using andNot Irestriction sites. The resulting plasmid was named BCR/ABL-pIRES-hIL-2. The plasmid pIRES-hIL-2, which contained only the hIL-2 gene, and the plasmid BCR/ABL-pIRES, which contained only the BCR/ABL gene, were also prepared. The nucleotide sequence of the insert was verified by sequencing. Plasmids were maintained and propagated in transformed Top10 bacteria (Pubo Biotech, Beijing, China) in the presence of ampicillin. Large-scale plasmid production was performed using an EndoFree plasmid Giga kit (Pubo Biotech, Beijing, China) according to the protocol of the manufacturer. Plasmid purification was conducted using PureLink (Invitrogen, Paisley, UK) according to the instructions of the manufacturer, and it was assessed by spectrophotometer (OD260/OD280). All samples were tested by the limulus amebocyte lysate assay (Sigma Chemical Co., St. Louis, MO, USA) to ensure that they were free of endotoxin contamination. 2.3. Immunization Mice were separated Lemildipine into the following administration groups by random allocation. Five mice per group were used: (1) saline control, (2) pIRES, (3) BCR/ABL-pIRES, (4) BCR/ABL-pIRES-hIL-2, and (5) pIRES-hIL-2. One day before immunization, procaine was injected in the injection sites to enhance vaccine absorption. All mice were intramuscularly administered on one side of a hind leg with 200?in serum samples. The IFN-and IL-4 sample concentrations were determined by commercial sandwich ELISA kits (Pubo Biotech Co., Ltd., Beijing, China) following the protocol of the manufacturer. The 450?nm optical density was measured with a BIO-RAD model 450 (BIO-RAD, Hercules, CA, USA) ELISA plate reader. A standard curve was created by plotting the Lemildipine mean absorbance of each standard versus the IFN-and IL-4 concentrations. The results were expressed in nanograms per milliliter by reading directly from the standard curve. 2.5. Flow Cytometry Lymphocyte subset analysis (CD4+/CD8+ ratio) was conducted by flow cytometry (FCM, Epics Elite ESP, Coulter) for dual-color flow cytometric analysis. Cells collected from mice spleens were immunostained with the FITC-conjugated anti-CD4 and PE-conjugated anti-CD8 antibodies (Pubo Biotech Co., Ltd., Beijing, China). Briefly, 5? 0.05 was considered statistically significant. 3. Results 3.1. Construction of DNA Vaccines The hIL-2 gene fragment was inserted into site B of the pIRES and the BCR/ABL-pIRES vectors to make the plasmids pIRES-hIL-2 and BCR/ABL-pIRES-hIL-2. All constructs were confirmed to be identical Lemildipine to published genomic sequences by sequencing. 3.2. IFN-and IL-4 Secretion The mean units of IFN-and IL-4 were detected in the sera of five groups of mice immunized with different DNA.