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KMID : 0614620070490020085
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Korean Journal of Gastroenterology
2007 Volume.49 No. 2 p.85 ~ p.92
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Human Monoclonal Antibody Inhibiting Reverse Transcriptase Activity of Hepatitis B Virus Polymerase Protein
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Park Sung-Jae
Seol Sang-Yong
Jee Sam-Ryong
Park Eun-Taek
Lee Youn-Jae
Lee Sang-Hyuk
Chung Jung-Myung
Cho Hyun-Dae
Jeong Young-Ju
Choi In-Hak
Park Sae-Gwang
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Abstract
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Background/Aims: To develop a novel treatment method for hepatitis B virus (HBV) infection, we aimed to make a human monoclonal antibody inhibiting reverse transcriptase (RT) activity of P protein which was important in HBV replication by using phage display technique. Therefore, we analysed the usability of human monoclonal antibody as a protein based gene therapy.
Methods: Reverse transcriptase/polymerase (RT/POL) functional motif of P protein of HBV was cloned in pMAL-c vector and expressed as maltose binding fusion protein form. The RT/POL recombinant protein (pMRT/POL) was purified by amylose resin column. Using human single chain Fv phage antibody library with 1.1×10(10) size, human antibody against pMRT/POL was selected with BIAcore panning. Selected antibody fragments were analyzed for the activity of RT inhibition. Finally, they were analyzed for the affinity with BIAcore and the complementarity determining regions with nucleotide sequencing.
Results: pMRT/POL recombinant protein expressed in E. coli showed RT activity, 1㎍ of recombinant protein had an activity equivalent to 5 unit of MMLV RT. By BIAcore panning, we could select 3 clones; POL-A5, POL-B8 and POL-B12. Each clone`s RT inhibiting activity were 52-82%, affinity against antigen were 8.15×10(-8) M to 1.75×10(-6) M.
Conclusion: Human monoclonal antibodies produced in this study showed low affinity, but efficiently inhibited the activity of RT in vitro. If POL-A5, POL-B8, and POL-B12 can be converted to intracellular antibody form, it can be used for protein-based gene therapy by inhibiting the replication through the neutralization of polymerase protein of HBV. (Korean J Gastroenterol 2007;49:85-92)
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KEYWORD
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Hepatitis B virus, Reverse transcriptase, Phage display, Human monoclonal antibody
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