Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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The development of murine recombinant single-chain variable domain fragment (ScFv) specific to acute non-lymphocytic leukemia (ANLL) cell line HL60

인간의 급성 비임파성 백혈암세포(HL60)의 표면항원에 결합하는 재조합 single-chain Fv (ScFv)의 개발

  • Kim, Cheol Hong (Department of Microbiology, College of Natural Sciences, Changwon National University) ;
  • Han, Seung Hee (Department of Microbiology, College of Natural Sciences, Changwon National University) ;
  • Kim, Hyeong Min (Department of Microbiology, College of Natural Sciences, Changwon National University) ;
  • Han, Jae Yong (Department of Agricultural Biotechnology, Seoul National University) ;
  • Lim, Myeong Woon (Joongkyeom: Antibody Therapy Inc.) ;
  • Kim, Jin-Kyoo (Department of Microbiology, College of Natural Sciences, Changwon National University)
  • 김철홍 (창원대학교 자연과학대학 미생물학과) ;
  • 한승희 (창원대학교 자연과학대학 미생물학과) ;
  • 김형민 (창원대학교 자연과학대학 미생물학과) ;
  • 한재용 (서울대학교 농업생명과학대학 농생명공학부) ;
  • 임명운 ((주)중겸 생명공학연구소) ;
  • 김진규 (창원대학교 자연과학대학 미생물학과)
  • Received : 2015.04.21
  • Accepted : 2015.05.29
  • Published : 2015.06.30
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Abstract

A monoclonal antibody AP64 IgM binds to human acute nonlymphocytic leukemia (ANLL) cell line HL60 and also cross-reacts with the homologous antigen in a rat ANLL cell. This antibody mediated by complement, has leukemia a suppression effect. In this study, we generated a recombinant single-chain variable domain fragment (ScFv) which were derived from $V_H$ and $V_L$ cDNA of AP64 IgM-secreting hybridoma by RT-PCR. The two variable regions were joined with a single 15 amino acid linker $(G_4S)_3$. This recombinant ScFv was expressed as a single polypeptide chain from Escherichia coli BMH 71-18. The recombinant ScFv was purified by applying the periplasmic extract to $Ni^+$-NTA-agarose affinity column and detected with westernblot. The purified recombinant ScFv recognized a surface antigen (about 30 kDa) of HL60 cell line which is the same antigen detected by parental AP64 IgM. But the affinity of ScFv for a surface antigen of HL60 was lower than that of the parental AP64 IgM, which needs to be further improved. Overall, the recombinant ScFv specific to HL60 might be a useful bioreagent for either diagnostic or therapeutic purposes.

단일클론항체 AP64 IgM은 인간의 급성 비임파성 골수암(ANLL) 세포주 HL60에 결합하며 쥐의 ANLL 세포에도 교차결합(cross-react)한다. 또한 complement에 의해 매개되어지면 골수암 억제효과를 나타낸다. 본 연구에서는 RT-PCR에 의해 AP64 IgM을 분비하는 하이브리도마의 $V_H$ 및 $V_L$ cDNA로부터 유래된 재조합 single-chain variable domain fragment (ScFv)를 제조하였다. $V_H$ 및 $V_L$은 15개 아미노산으로 구성된 linker $(G_4S)_3$으로 연결되었다. 재조합 ScFv는 Escherichia coli BMH 71-18에서 single polypeptide chain으로 발현되었다. Periplasmic extract를 $Ni^+$-NTA-agarose affinity column에 가하여 발현된 재조합 ScFv를 정제하였으며 westernblot으로 정제된 단백질을 탐지하였다. 정제된 재조합 ScFv는 AP64 IgM 모항체가 탐지하는 항원과 같은 HL60 세포의 표면항원(약 30 kDa)을 인지하였다. 그러나 HL60의 표면항원에 대한 ScFv의 결합력은 AP64 IgM 모항체보다 낮아서 추후 이에 대한 개선이 필요하다. 종합하여 볼 때 HL60 세포주에 특이적인 재조합 ScFv는 진단 또는 치료목적으로 유용한 생물학적 제재가 될 수 있을 것이다.

Keywords

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