Construction and screening of phage display single chain antibody library against Bursaphelenchus xylophilus cellulase.
- Author:
Wang TIAN
1
;
Qi ZHANG
;
Wen-Bo YANG
;
Gang BAI
Author Information
1. College of Life Sciences, Nankai University, Tianjin 300071, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Cellulase;
genetics;
immunology;
Cloning, Molecular;
Electroporation;
Escherichia coli;
genetics;
metabolism;
Female;
Helminth Proteins;
genetics;
immunology;
Immunoglobulin Variable Region;
genetics;
Mice;
Mice, Inbred BALB C;
Nematoda;
enzymology;
Peptide Library;
Pinus;
parasitology;
Plant Diseases;
parasitology;
Recombinant Proteins;
biosynthesis;
genetics
- From:
Chinese Journal of Biotechnology
2005;21(6):900-905
- CountryChina
- Language:Chinese
-
Abstract:
A phage display single-chain variable fragment (scFv) library against Bursaphelenchus xylophilus cellulase (BXC) was constructed and used to screen the specific antibodies binding to BXC. The total RNA was extracted from fresh spleens of BALB/C mice immunized with BXC. Gene fragments encoding VH and VL were amplified by RT-PCR and assembled into a single chain by overlapping PCR with a linker DNA encoding the peptide (Gly4Ser)3. The recombinant fragments were cloned into the phagemids (pCANTABSE) and electroporated into E. coli TG1. The recombinant phagemids were rescued by reinfection of helper phage M13K07. The repertoire of the phage display antibody was about 5 x 10(4). The specific antibodies against BXC were obtained after five rounds of affinity selection. The positive phage clone was used to infect E. coli HB2151. SDS-PAGE and western blot analysis showed that the soluble scFv antibodies expressed bound specifically to BXC. The studies laid foundation for quarantine and pathological study of Bursaphelenchus xylophilu.
