关键词: 原核表达, 多克隆抗体, 大肠杆菌

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 contai ning 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 pre paring 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-selectin protein can express with high efficiency in E.coli M15. The prepared polyclonal antibody ha s a high titer.

Key words: prokaryotic expression, polyclonal antibody, Escherichia coli