IMMUNE NETWORK

  • 제4권1호
  • /
  • Pages.16-22
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  • 2004
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  • 1598-2629(pISSN)
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  • 2092-6685(eISSN)
대한면역학회 (The Korean Association of Immunobiologists)
권호 표지

Phage Display 기법을 이용한 B형 간염 바이러스 Polymerase의 RNase H 활성을 억제하는 인간 단세포군 항체의 개발

Development of Human Antibody Inhibiting RNase H Activity of Polymerase of Hepatitis B Virus Using Phage Display Technique

  • 이성락 (인제대학교 의과대학 미생물학교실) ;
  • 송은경 (인제대학교 의과대학 미생물학교실) ;
  • 정영주 (인제대학교 의과대학 미생물학교실) ;
  • 이영이 (동국대학교 의과대학 미생물학교실) ;
  • 김익중 (동국대학교 의과대학 미생물학교실) ;
  • 최인학 (인제대학교 의과대학 미생물학교실) ;
  • 박세광 (인제대학교 의과대학 미생물학교실, 인제대학교 백인제기념임상의학연구소 세포유전자치료연구부)
  • Lee, Seong-Rak (Department of Microbiology, College of Medicine, Inje University) ;
  • Song, Eun-Kyoung (Department of Microbiology, College of Medicine, Inje University) ;
  • Jeong, Young-Joo (Department of Microbiology, College of Medicine, Inje University) ;
  • Lee Young-Yi (Department of Microbiology, College of Medicine, Dongguk University) ;
  • Kim, Ik-Jung (Department of Microbiology, College of Medicine, Dongguk University) ;
  • Choi, In-Hak (Department of Microbiology, College of Medicine, Inje University) ;
  • Park, Sae-Gwang (Department of Microbiology, College of Medicine, Inje University, Department of Cell and Gene Therapy, Paik Institute for Clinical Research, Inje University)
  • 발행 : 2004.03.31
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초록

Background: To develop a novel treatment strategy for hepatitis B virus infection, a major cause of liver chirosis and cancer, we aimed to make human monoclonal antibodies inhibiting RNase H activity of P protein playing in important role in HBV replication. In this regard, phage display technology was employed and demonstrated as an efficient cloning method for human monoclonal antibody. So this study analysed the usability of human monoclonal antibody as protein based gene therapy. Methods: RNase H of HBV was expressed as fusion protein with maltose binding protein and purified with amylose resin column. Single chain Fv (scFv) phage antibody library was constructed by PCR cloning using total RNAs of PBMC from 50 healthy volunteers. Binders to RNase H were selected with BIAcore 2000 from the constructed library, and purified as soluble antibody fragment. The affinity and sequences of selected antibody fragments were analyzed with BIAcore and ABI automatic sequencer, respectively. And finally RNase H activity inhibiting assay was carried out. Results: Recombinant RNase H expressed in E. coli exhibited an proper enzyme activity. Naive library of $4.46{\times}10^9cfu$ was screened by BIAcore 2000. Two clones, RN41 and RN56, showed affinity of $4.5{\times}10^{-7}M$ and $1.9{\times}10^{-7}M$, respectively. But RNase H inhibiting activity of RN41 was higher than that of RN56. Conclusion: We cloned human monoclonal antibodies inhibiting RNase H activity of P protein of HBV. These antibodies can be expected to be a good candidate for protein-based antiviral therapy by preventing a replication of HBV if they can be expressed intracellularly in HBV-infected hepatocytes.

키워드

과제정보

연구 과제 주관 기관 : 한국학술진흥재단

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