Prokaryotic expression of human L-selectin and preparation of polyclonal antibody
- VernacularTitle:人L-选择素的原核表达及多克隆抗体的制备
- Author:
Yanguang GAO
;
Junwei FEI
;
Rui FEI
;
Xianlu ZENG
- Publication Type:Journal Article
- Keywords:
L selectin;
prokaryotic expression;
polyclonal antibody;
Escherichia coli
- From:
Journal of Jilin University(Medicine Edition)
2006;0(01):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct the expression vector and express the recombinant human L-selectin in prokaryotic system, and purify the aimed protein for the preparation of rabbit anti-human L-selectin polyclonal antibody. Methods The partial cDNA of human L-selectin was amplified from the open reading frame (ORF) of N-terminus sequence of L-selectin containing 153 amino acids by PCR, then cloned into the prokaryotic expression vector pQE40 at restriction sites BamH Ⅰ and Hind Ⅲ. The recombinant expression plasmid pQE40-L-selectin was transformed into E. coli M15 for expression. The fusion protein including 6-His-tag was purified by Ni-NTA chromatographic column and analysed by SDS-PAGE. The purified protein was used to immune rabbit for preparing polyclonal antibody. Western blotting analysis and dot immunoblot assay (DIBA) were used to test the titer of the antiserum. Results The expression plasmid pQE40-L-selectin was constructed and confirmed with restriction enzyme digestion. The quantity of the purified protein from E. coli M15 was 600 mg ? L-1. By immuning the rabbit, the polyclonal antibody was successfully prepared. The results of dot immunoblot assay (DIBA) showed that the antiserum had the high titer (1 : 1 000). Conclusion The recombinant human L-seleclin prolein can express with high efficiency in E. coli M15. The prepared polyclonal antibody has a high titer.
