Construction of anti-lipopolysaccharide single-chain phage antibody library in mice
- VernacularTitle:鼠抗脂多糖单链噬菌体抗体库的构建
- Author:
Ming CHEN
;
Weiling FU
;
Lili YU
;
Xue ZHANG
- Publication Type:Journal Article
- From:Journal of Third Military Medical University
2001;23(4):407-409
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct an anti-LPS single chain phage antibody library in mice for further biomedical works. Methods Total RNA was extracted from splenic cells for reverse transcription after BALB/C mice had been immunized with pure LPS for 4 weeks. The designed primers were used to amplify the variable region genes of both heavy and light chain (VH,VL) with polymerase chain reaction. The VH and VL were then conjugated to form a single chain of variable fragment (ScFv) by a linker. The ScFv was cut by NotⅠ and SfiⅠ and then ligated into a pCANTAB5E phagemid vector which was digested with the same restriction enzymes. The ligated vector was then tranfected into the competent E.coli TG1 cells. Four TG1 clones were randomly selected to detect the exotic DNA. Results The titer of anti-LPS in murine sera was 1∶12 800. The concentration of total RNA was 12.3813 μg/ml. The length of the fragments were 340 bp for VH, 320 bp for VL and 800 bp for ScFv respectively. 1.9×107 clones were grown after transfection and one from the four randomly selected clones was identified to contain the exotic DNA. Conclusion A 4.75×106 murine anti-LPS single chain phage antibody library was successfully constructed.
