Production of Mouse Single Chain Fv Antibody to Surface Protein of Hepatitis B virus using Antibody Phage Display Library.
- Author:
In Hak CHOI
;
Hee Sun KIM
;
Ik Jung KIM
;
Jun Ho CHUNG
;
Se Kwang PARK
- Publication Type:Original Article
- MeSH:
Animals;
Antibodies;
B-Lymphocytes;
Bacteriophages*;
Blotting, Western;
Cell Surface Display Techniques;
Clone Cells;
Culture Media;
Digestion;
DNA;
DNA, Complementary;
Enzyme-Linked Immunosorbent Assay;
Hepatitis B virus*;
Hepatitis B*;
Hepatitis*;
Isopropyl Thiogalactoside;
Mice*;
Periplasm;
Polymerase Chain Reaction;
Recombination, Genetic;
Single-Chain Antibodies*
- From:Journal of the Korean Society for Microbiology
1997;32(4):447-454
- CountryRepublic of Korea
- Language:Korean
-
Abstract:
In this study, we are to produce the single chain variable fragment (scFv) antibodies against surface protein of hepatitis B virus (HBV) using antibody phage display technique. Balb/c mice were immunized with preS1 and cDNAs of heavy and light chains of splenic B cells from immunized mice were prepared using RT-PCR. Two cDNAs were linked with (64S) linker DNA under recombination PCR to produce single chain Fv DNA. After digestion of scFv DNA with Sp 1 and Not 1, the digested DNA was ligated into pCANTAB 5E and electroporated into E. coli XL1-Blue to prepare scFv-library. The size of library was 1 * 10' pfu/ml. Phage antibodies (phabs) against preS1 were rescued with M13K07 helper phages, and preS1-binders were selected through 3 times of panning using 96 well microtitre plates. Phage antibody clones were assayed directly for the ability to bind preS1 by ELISA. And then 7 phage antibody clones had high ELISA signals against preS1. Phabs from preS1-specific pMsc-17 had the strongest ELISA signal to preS1. Phabs from pMsc-17 were used for Western blot to preS1 and the results revealed that it was specific to preS1. To prepare the soluble scFv antibody, phabs from pMsc-17 were transfected into non-suppressor E. coli HB2151, and grown under 1 mM IPTG. Soluble scFv antibody was mainly accumulated in the periplasmic space, but small amount of antibody was secreted into culture media.
