The Research Of MUC1-VNTR DNA Vaccine For Pancreatic Cancer

The research of MUC1-VNTR DNA vaccinefor pancreatic cancerThe incidence of adenocarcinoma of the pancreas has risen steadily over the past four decades. The disease is characterized by its aggressive nature. The diagnosis of pancreatic cancer is usually established at an advanced stage, and a lack of effective therapies leads to an extremely poor prognosis. Despite advances in our understanding of the molecular biology of pancreatic cancer, the systemic treatment of this disease remains unsatisfactory. Conventional radiotherapy and chemotherapy has not produced dramatic improvements in response rates or patient survival. On the other hand, current new adjuvant radiotherapy and chemotherapy have shown only modest benefits. New treatment strategies are clearly needed. And immunotherapy is a novel potential alternative systemic treatment, because it can act specifically against the tumor, without causing damage to normal tissues. Vaccines, especially DNA vaccines are one form of immunotherapy that can also provide active immunization, which allows amplification of the immune response.MUC1 mucin is aberrantly expressed in many epithelial malignancies and is a promising tumor antigen for target-directed immunotherapy against human pancreatic cancer. Therefore, we constructed a DNA vaccine encoding human MUC1 VNTR core protein to study its induction of humoral and cellular immunity to VNTR. Part OneThe construction and cell transfection assay of MUC1-VNTR DNA vaccine for pancreatic cancerObjective To construct MUC1-VNTR DNA vaccine for pancreatic cancer and to investigate whether VNTR is expressed by COS7 cells after transfection. Methods A signal sequence from human MCP-1 was inserted before the VNTR sequence, one tandem repeat sequence from human MUC1. And a KOZAK sequence was inserted before the ATG start codon. The recombinant gene of VNTR was synthesized and cloned into MCS in the pcDNA3.1/Myc-his(+) A vector. Then the recombinant plasmid pcDNA3.1-VNTR/Myc-his(+) A was transfected into COS7 cells. 48 hours after transfection, Western blot analysis using MUC1 monoclonal antibody recognizing VNTR was accomplished to assay the expression of VNTR by the transfected COS7 cells. ResultsThe recombinant plasmid pcDNA3.1-VNTR/Myc-his(+) A encode the whole exact translation frame region of the pcDNA3.1/Myc-his(+) A vector and the recombinant gene of VNTR. Transfection assay in vitro showed that the transtected cells expressed transgene products at 48 hours after transfection, implying that the recombinant gene of VNTR cloned into the expression vector was correctly transcripted, translated and partly secreted. For the expressed VNTR polypeptide was fused with epitopes of signal peptide of human MCP-1 and Myc and His, its molecular is bigger than the synthesized VNTR polypeptide.ConclusionsThe recombinant plasmid pcDNA3.1-VNTR/Myc-his(+) A is successfully constructed by ourselves could exactly express VNTR polypeptide in the transfected mammalian cells.Part TwoVNTR-specific immune responses in mice immunized with MUC1-VNTR DNA vaccine for pancreatic cancerObjective To investigate the VNTR specific cytotoxic T lymphocytes and antibodies inducted in mice immunized with DNA vaccine encoding human MUC1 VNTR core protein.MethodsWe injected a total of 100 ug palsmid DNA of pcDNA3.1-VNTR/Myc-his(+) A in 100 ul of 0.9% NaCl distributed into the anterior tibialis muscle of C57BL/6(H-2b)female mice (V group, n=15) 3 days after the injection of 100 ul bupivacaine (0.25%) as a facilitator compound. Mice inoculated with either the empty plasmid pcDNA3.1/Myc-his(+) A vector (D group, n=15) or 0.9% NaCl (NS group, n=15) were used as control. Four weeks later, all mice were inoculated again with the same solution as before. Two weeks after the last immunization, spleen cells were obtained and analyzed for cytotoxic activity 6 days after in vitro stimulation by the synthesized VNTR polypeptide (Calcein AM assay). Blood was collected for the ELISA assay of anti-VNTR antib...