Prokaryotic expression of human L-selectin and preparation of polyclonal antibody

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 dotimmunoblot 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-selectin protein can express with high efficiency in E. coli M15. The prepared polyclonal antibody has a high titer.