Journal of Microbiology

The Microbiological Society of Korea (한국미생물학회)
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Efficient Generation of BLCL Expressing Foreign Antigen as Antigen-presenting Cells with Recombinant Retroviruses

  • Hyun-Il Cho (Department of Microbiology and Immunology, Clinical Institute of Sr, Mary′s Hospital) ;
  • Soon-Young Pail (Department of Microbiology and Immunology, Clinical Institute of Sr, Mary′s Hospital) ;
  • Il-Hoan OH (Department of Microbiology and Immunology, Clinical Institute of Sr, Mary′s Hospital) ;
  • Kyun-Jung Ahn (Department of Microbiology and Immunology, Clinical Institute of Sr, Mary′s Hospital) ;
  • Dong-Wook Kim (Depratment of Internal Medicine, College of Medicine, The Catholic University of Korea)
  • Published : 2001.12.01
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Abstract

Epstein-Barr Virus(EBV)-transformed lymphoblastoid B cell lines, BLCL which expresse antigens, are potential antigen-presenting cells(APCs) for the induction of CTL in vitro. However transfection of BLCLs with subsequent selection by antibiotics is notoriously difficult because plating efficiencies of BLCLsare reported to be 1% or less. To generated stable transfectants of BLCLs we produced high titers of retroviruess encoding pp 65 antigen of human cytomegalovirus of foreign antigens and trans-duced them of BLCLs. The pp 65 gene was cloned into the retroviral vector pLXSN. The recombinant retroviral vector was transfected to ecotropic packaging cell line, CP&E86, and this polyclonal recom-binant retrovirus was transduced to PA317 that is amphotropic pakaging cell line. The titers of colned PA317 amphotropic retroviruses ranged from 5 to $\times$10$^{6}$ colony forming units (CFU)per ml (CFU/ml) We performed three rounds of consecutive transductions to BLCLs in order to improve the clon-ing effieiencies. The expression of recombinant HCMV-pp65 antigen was more than 20% after the final transduction. THe third-transduced BLCLs were easily selected in optimal concentration of G418. BLCLs expressing foreign antigens could be used as target cells for CTL assay and/or as APCs for induction of in vitro CTL responses specific for viral and tumor antigens.

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