W Zhang1, X Cao. 1. Department of Immunology, Second Military Medical University, Shanghai.
Abstract
OBJECTIVE: To construct the recombinant adenovirus vector co-expressing the heterodimer of human interkeukin-12. METHODS: The full-length cDNA encoding human IL-12 subunits p40 or p35 was cloned by RT-PCR separately. The cDNA was ligated with encephalomyocarditis internal ribosome entry site (IRES), placed under the control of CMV promoter, and inserted into E1-substituted adenovirus vector pAx1cw to produce the bicistronic coexpression vector. Subsequently, the hIL-12 recombinant adenovirus vector was cotransfected into 293 cells together with EcoT22I-digested Ad5 DNA-TPC, and the replication-deficient hIL-12 recombinant adenoviruses was generated efficiently by homologous recombination. RESULTS: The human IL-12 recombinant adenoviruses were obtained with the titers of 2.1 x 10(9) pfu/ml. 48 hours after the infection of the 293 cells, HepG2 cells and human primary skin fibroblasts with hIL-12 recombinant adenoviruses, the hIL-12 expressions were detected by ELISA (30-50 ng/10(6) cells/24 h). The expressed hIL-12 could stimulate the in vitro proliferation and IFN-gamma production of human PMNC. CONCLUSION: Prepared hIL-12 recombinant adenovirus vector can express biologically active hIL-12 and can be potentially used in cancer gene therapy.
OBJECTIVE: To construct the recombinant adenovirus vector co-expressing the heterodimer of human interkeukin-12. METHODS: The full-length cDNA encoding human IL-12 subunits p40 or p35 was cloned by RT-PCR separately. The cDNA was ligated with encephalomyocarditis internal ribosome entry site (IRES), placed under the control of CMV promoter, and inserted into E1-substituted adenovirus vector pAx1cw to produce the bicistronic coexpression vector. Subsequently, the hIL-12 recombinant adenovirus vector was cotransfected into 293 cells together with EcoT22I-digested Ad5 DNA-TPC, and the replication-deficient hIL-12 recombinant adenoviruses was generated efficiently by homologous recombination. RESULTS: The human IL-12 recombinant adenoviruses were obtained with the titers of 2.1 x 10(9) pfu/ml. 48 hours after the infection of the 293 cells, HepG2 cells and human primary skin fibroblasts with hIL-12 recombinant adenoviruses, the hIL-12 expressions were detected by ELISA (30-50 ng/10(6) cells/24 h). The expressed hIL-12 could stimulate the in vitro proliferation and IFN-gamma production of human PMNC. CONCLUSION: Prepared hIL-12 recombinant adenovirus vector can express biologically active hIL-12 and can be potentially used in cancer gene therapy.