Abstract: Cytotoxic enterotoxin and outer membrane protein have been proved to be protective antigens against Aeromonas hydrophila. Primers P1,P2,P3 and P4 were designed based on the sequences of cytotoxic enterotoxin gene (act) and outer membrane gene (OmpTS) of Aeromonas hydrophila in Genbank (primers P1,P2 for act, primers P3,P4 for ompTS). The genomic DNA of a strain of Aeromonas hydrophila isolated in Guangdong Province was extracted and used as PCR template. Then the partial act fragmetnt and ompTS fragment without the signal sequence were amplified separately and purified by DNA agarose gel purification Kit. With these two fragments mixed together as the template, one target fragment about 2.1kb was amplified with primer P1 and P4 after the second step PCR amplification. A linker, (Gly4Ser)3, was inserted between these two genes. The down stream primer of act overlapped the upstream primer of OmpTS in 21bp and the linker (Gly4Ser)3 was encoded by this two primers together. The 2.1 kb fragment was digested by BamHⅠ and HindⅢ, ligated into BamHⅠ/HindⅢ linearized pQE-30 plasmid(Qiagen Co.). pQE30/act-GS-OmpTS,an expression vector with the fusion fragment was then constructed. After transformed into E. coli M15 (pREP4) and induced with IPTG, pQE30/act-GS-OmpTS was hyper-expressed. To optimize the expression of the recombinant fusion protein, expression conditions ranging in salinity, pH value, IPTG concentration, induced time, induced temperature and medium type were tested. The optimal condition was proved to be pH7, 0、0.2% NaCl,LB,30℃and 0.2mmol/L IPTG for 3-5h. The recombinant fusion protein (Act-GS-OmpTS) exhibited a molecular weight of about 81.0 kDa in 12% SDS-PAGE, which was identical to what had been anticipated. Scanned by CS-950 spot scanning densitometer (SHIMADZUTM),the band of the recombinant fusion protein showed about 42% of total E. coli proteins. Western blot analysis shown that rabbit polyantibody to Act and OmpTS all reacted with Act-GS-OmpTS, which indicated the fusion protein may have similar epitopes to those of the natural proteins.