약학회지 (YAKHAK HOEJI)

  • 제49권3호
  • /
  • Pages.217-224
  • /
  • 2005
  • /
  • 0377-9556(pISSN)
  • /
  • 2383-9457(eISSN)
대한약학회 (The Pharmaceutical Society of Korea)
권호 표지

HIV-l 유래 렌티바이러스 벡터의 복제가능 바이러스 검출과 역가측정 분석방법 비교

Comparison of Analysis Methods for Detection of Replication Competent Virus and Functional Titers of HIV-l Based Lentivirus Vector

  • 장석기 (식품의약품안전청 생명공학의약품과) ;
  • 오일웅 (식품의약품안전청 생명공학의약품과) ;
  • 정자영 (식품의약품안전청 생명공학의약품과) ;
  • 안광수 (식품의약품안전청 생명공학의약품과) ;
  • 손여원 (식품의약품안전청 생명공학의약품과)
  • 발행 : 2005.06.01
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초록

Human Immunodeficiency Virus type 1 (HIV-l) based lentivirus vector has demonstrated great potential as gene therapy vectors mediating efficient gene delivery and long-term transgene expression in both dividing and nondividing cells. However, for clinical studies it must be confirmed that vector preparations are safe and not contaminated by replication competent lentivirus (RCL) related to the parental pathogenic virus, HIV-l. In this study, we would like to establish the method for titration and RCL detection of lentivirus vector. The titration was determined by vector expression containing the green fluorescent protein, GFP in transduced cells. The titer was $1{\times}10^7$ Transducing Unit/ml in the GFP expression assay and $8.9{\times}10^7$ molecules/ml in the real-time PCR. Also, for the detection of RCL, we have used a combination method of PCR and p24 antigen detection. First, PBS/psi and VSV-G region in the genomic DNA of transduced cells was detected by PCR assay. Second, transfer and expression of the HIV-1 gag gene was detected by p24 ELISA. In an attempt to amplify any RCL, the transduced cells were cultured for 3 weeks (amplification phase) and the supernatant of amplified transduced cell was used for the second transduction to determine whether a true RCL was present (indicator phase). Analysis of cells and supernatant at day 6 in indicator phase were negative for PBS/psi, VSV-G, and p24 antigen. These results suggest that they are not mobilized and therefore there are no RCL in amplification phase. Thus, real-time PCR is a reliable and sensitive method for titration and RCL detection of lentivirus vector.

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