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KMID : 0364820150510020115
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Korean Journal of Microbiology
2015 Volume.51 No. 2 p.115 ~ p.125
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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
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Kim Cheol-Hong
Han Seung-Hee
Kim Hyeong-Min
Han Jae-Yong
Lim Myeong-Woon
Kim Jin-Kyu
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Abstract
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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 VH and VL cDNA of AP64 IgM-secreting hybridoma by RT-PCR. The two variable regions were joined with a single 15 amino acid linker [G₄S]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.
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KEYWORD
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human acute nonlymphocytic leukemia, HL60, IgM, Ni+-NTA-agarose, single-chain Fv, westernblot
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