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Construction of the dicistronic adenovirus vector expressing bioactive human interleukin-12

  • Published:
Chinese Journal of Cancer Research

Abstract

The full-length cDNA encoding the subunits p40 and p35 of human interleukin-12(hIL-12) were cloned separately by RT-PCR, linked together by internal ribosomal entry site (IRES) of encephalomyocarditis virus which initiates cap-independent translation to form a dicistronic gene fragment. The dicistronic fragment was placed between the cytomegalovirus (CMV) promoter and SV40 polyA signal to form a dicistronic expression cassette. Subsequently, the dicistronic expression cassette was inserted into El region of Ad5 genome in cosmid vector pAxlcw of El-substitution type. By homologous recombination with EcoT22I-digested Ad5 DNA-TPC in 293 cells, the replication-deficient recombinant adenoviruses of ML-12 were generated efficiently. After infected with hIL-12 recombinant adenoviruses in vitro, 293 cells, human hepatocellular carcinoma cells HepG2, and primary human skin fibroblasts expressed and secreted hIL-12 at comparable levels (30⊃60ng/106cells/24hr), which could stimulate the proliferation and IKN-γ production of human lymphoblasts. These suggest that the dicistronic adenovirus vector of hIL-12 could effectively mediate the expression of bioactive hIL-12 and might be used in cancer gene therapy.

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Authors and Affiliations

  1. Department of Immunology, Second Military Medical University, 200433, Shanghai, P.R. China

    Zhang Weiping, Cao Xuetao & Hirofumi Hamada

  2. Department of Molecular Biotherapy Research, Japanese Foundation of Cancer Research, Tokyo, Japan

    Hirofumi Hamada

Authors
  1. Zhang Weiping
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  2. Cao Xuetao
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  3. Hirofumi Hamada
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Additional information

This work was supported by grants from National Natural Science Foundation of China (No. 39421009).

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Zhang, W., Cao, X. & Hirofumi, H. Construction of the dicistronic adenovirus vector expressing bioactive human interleukin-12. Chin J Cancer Res 9, 299–303 (1997). https://doi.org/10.1007/BF02974979

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  • DOI: https://doi.org/10.1007/BF02974979

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