Objective:To obtain the cDNA encoding Ancylostoma-secreted protein 2 of Ancylostoma duodenale(mAd-ASP-2)and construct the expression system of mAd-ASP-2 in E.coli. Methods:The cDNA encoding of the mature Ancylostoma-secreted protein 2 of Ancylostoma duodenale(mAd-ASP-2)was cloned by RT-PCR. The mAd-ASP-2 was inserted into the pET-22b(+)vector to construct prokaryotic expression plasmid pET-22b-mAd-ASP-2. An optimized codon mAd-ASP-2 gene,designated mAd-ASP-2*,was designed and synthesized based on optimization of synonymous codon bias of E. coli,without modification of amino acid sequence,and inserted into pET-22b(+)to construct prokaryotic expression plasmid pET-22b-mAd-ASP-2*. The two expression plasmids were transformed into E. coli Rosetta-gami-2(DE3)cells. Results:SDS-PAGE analysis showed that the expected recombinant mAd-ASP-2 fusion protein was expressed in E. coli Rosetta-gami-2(DE3)cells induced by isopropyl-[3-thiogalactopyranoside(IPTG),and the mAd-ASP-2* with the optimized codon was more highly expressed than the original mAd-ASP-2 in the soluble fraction of E. coli cell lysates. The recombinant mAd-ASP-2* fusion protein was purified using His Trap HP affinity column. Western blot analysis showed that the recombinant mAd-ASP-2* protein was combined with mouse anti-His-Tag. So the expressed protein was definitely confirmed to be the target protein. Conclusion:The expression system of mAd-ASP-2 in E. coli was constructed successfully,which provided a fundamental basis for further studies on subunit vaccine for prevention of hookworm infections.