Immune Netw.  2004 Mar;4(1):16-22. 10.4110/in.2004.4.1.16.

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

• Lee SR
• Song EK
• Jeong YJ
• Lee YY
• Kim IJ
• Choi IH
• Park SG

• KMID: 2054239
• DOI: http://doi.org/10.4110/in.2004.4.1.16

Abstract

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 X 10(9) cfu was screened by BIAcore 2000. Two clones, RN41 and RN56, showed affinity of 4.5 X 10(-7) M and 1.9 X 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.

Keyword

Hepatitis B virus; RNase H; phage display; human monoclonal antibody

MeSH Terms

Amylose
Antibodies
Antibodies, Monoclonal
Bacteriophages*
Cell Surface Display Techniques*
Clone Cells
Cloning, Organism
Genetic Therapy
Healthy Volunteers
Hepatitis B virus*
Hepatitis B*
Hepatitis*
Hepatocytes
Humans*
Immunoglobulin Fragments
Liver
Maltose-Binding Proteins
Polymerase Chain Reaction
Ribonuclease H*
Ribonucleases*
RNA
Single-Chain Antibodies
Amylose
Antibodies
Antibodies, Monoclonal
Immunoglobulin Fragments
Maltose-Binding Proteins
RNA
Ribonuclease H
Ribonucleases
Single-Chain Antibodies