- Efficient Generation of BLCL Expressing Foreign Antigen as Antigen-presenting Cells with Recombinant Retroviruses
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Hyun-Il Cho , Soon-Young Paik , Il-Hoan Oh , Kyu-Jung Ahn , Dong-Wook Kim , Jin-Han Kang , Wan-Shik Shin , Chun-Choo Kim , Hoon Han
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J. Microbiol. 2001;39(4):300-304.
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 Abstract
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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 BLCLs are reported to be 1% or less. To generate stable transfectants of BLCLs, we produced high titers of retroviruses encoding pp65 antigen of human cytomegalovirus as foreign antigens and transduced them to BLCLs. The pp65 gene was cloned into the retroviral vector pLXSN. The recombinant retroviral vector was transfected to ecotropic packaging cell line, GP&E86, and this polyclonal recombinant retrovirus was transduced to PA317 that is amphotropic packaging cell line. The titers of cloned PA317 amphotropic retroviruses ranged from 5 to 7 X 10^6 colony forming units (CFU) per ml (CFU/ml). We performed three rounds of consecutive transductions to BLCLs in order to improve the cloning efficiencies. 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.
- Detection of Lymphotropic Herpesviruses by Multiplex Polymerase Chain Reaction
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Sang-Tae Park , Seung-Han Kim , Dong-Gun Lee , Jung-Hyun Choi , Wan-Shik Shin , Tai - Gyu Ki m , Soon-Young Paik , Chun-Choo Kim
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J. Microbiol. 2001;39(3):226-228.
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Abstract
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Human lymphotropic herpesvirus is known to be a major pathogen associated with various diseases in bone marrow transplantation (BMT) recipients. A multiplex nested-polymerase chain reaction (PCR) method was developed for the simultaneous detection of human lymphotropic herpesviruses, including Ebstein-Barr virus (EBV), cytomegalovirus (CMV), and human herpesvirus 6 variants A and B (HHV6-A, HHV6-B). To demonstrate the usefulness of multiplex PCR for the analysis of clinical samples, peripheral blood mononuclear cells and serum from BMT recipients were analysed. The results showed that a clear detection could be made between EBV, HCMV and HHV-6. This multiplex PCR assay is an efficient and cost-effective approach to the analysis of large numbers of samples to determine the epidemiological importance of EBV, HCMV and HHV-6.
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