[The construction and application of adenovirus vector coexpressing the heterodimer of human IL-12].
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.


CONCLUSIONS
Prepared hIL-12 recombinant adenovirus vector can express biologically active hIL-12 and can be potentially used in cancer gene therapy.
Department of Immunology, Second Military Medical University, Shanghai.
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Zhang
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Cao
D000260:Adenoviruses, Human / Q000235:genetics*; D005347:Fibroblasts; D005822:Genetic Vectors; D006801:Humans; D018664:Interleukin-12 / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D012867:Skin / Q000166:cytology; D014162:Transfection
D011994:Recombinant Proteins; D018664:Interleukin-12