The expression unit of heat-stable α-amylase (amy) gene including the promoter, signal peptide and amylase gene was amplified by PCR and then digested and ligated into cloning vector pHY300PLK. The anti-ampcillin gene of the recombinant plasmid was deleted by inverse PCR and homologous recombination for high expression of auto-protein and heterogeneous protein using the promotor and signal peptide of α-amy gene. The result indicated that the pHY300PLK with anti-ampcillin gene, namely, plasmid pAMY, could express secretory α-amylase in host strain while the plasmid pAMY without anti-ampcillin gene, namely, plasmid pAMY1, could express the secretory α-amylase more efficiently. Then the cellulase gene was inserted into the vector pAMY1 at the position immediately after the promoter and signal peptide of α-amy gene, the resulting recombinant plasmid, named pCEL could express secretory cellulase in host strain. These results indicated the promoter and signal peptide of α-amy gene could express not only α-amylase but also foreign protein as well. According to the growth curve of recombinant strains and the plasmid copy number of pAMY and pAMY1, deletion of the anti-ampcillin showed no influence on the growth of the recombinant strain while improved the replication efficiency of the plasmid, leading to higher expression of protein by pAMY1. The above results suggested the cloning vector pHY300PLK was modified into expressing vector and could be used further in the expression of other heterogeneous proteins.