Terminal Protein-specific scFv Production by Phage Display.
- Author:
Myung Shin LEE
1
;
Myung Hee KWON
;
Sun PARK
;
Ho Joon SHIN
;
Hyung Il KIM
Author Information
- Publication Type:Original Article
- Keywords: HBV; HBV DNA polymerase; phage display; scFv; TP (terminal protein)
- MeSH: Animals; Bacteriophages*; Base Sequence; Clone Cells; DNA; DNA Polymerase I; Hepatitis B virus; Hepatitis B, Chronic; Humans; Mice; Prognosis; Protein S; Reverse Transcription; RNA, Messenger; Spleen; Tyrosine
- From:Immune Network 2003;3(2):126-135
- CountryRepublic of Korea
- Language:Korean
- Abstract: BACKGROUND: One of the important factors in the prognosis of chronic hepatitis B patient is the degree of replication of hepatitis B virus (HBV). It has been known that HBV DNA polymerase plays the essential role in the replication of HBV. HBV DNA polymerase is composed of four domains, TP (Terminal protein), spacer, RT (Reverse transcriptase) and RNaseH. Among these domains, tyrosine, the 65th residue of TP is an important residue in protein-priming reaction that initiates reverse transcription. If monoclonal antibody that recognizes around tyrosine residue were selected, it could be applied to further study of HBV replication. METHODS: To produce TP-specific scFv (single-chain Fv) by phage display, mice were immunized using synthetic TP-peptide contains 57~80th amino acid residues of TP domain. After isolation of mRNA of heavy-variable region (VH) and light-chain variable region (VL) from the spleen of the immunized mouse, DNA of VH and VL were obtained by RT-PCR and joined by a DNA linker encoding peptide (Gly4Ser)3 as a scFv DNA fragments. ScFv DNA fragments were cloned into a phagemid vector. ScFv was expressed in E.coli TG1 as a fusion protein with E tag and phage gIII. To select the scFv that has specific affinity to TP-peptide from the phage-antibody library, we used two cycles of panning and colony lift assay. RESULTS: The TP-peptide-specific scFv was isolated by selection process using TP-peptide as an antigen. Selected scFv had 30 kDa of protein size and its nucleotide sequences were analyzed. Indirect- and competitive-ELISA revealed that the selected scFv specifically recognized both TP-peptide and the HBV DNA polymerase. CONCLUSION: The scFv that recognizes the TP domain of the HBV DNA polymerase was isolated by phage display.
