The different construction of mRNA 5' non-coding region may affect gene expression. To improve gene expression levels, we constructed recombinant plasmids with different nucleotide composition of the 5' non-coding region which direct the synthesis of human toxicogenic E. coli heat-labile enterotoxin B subunit coded by LT-B gene under the control of the PRPL tandem promoter of vector pBV220. These recombinants were expressed in E. coli HB101 and DH5 alpha, respectively. The results show that the expression levels of LT-B gene with two tandem SD sequences upstream from the initiation codon are lower than those with only one SD sequence and translation coupling can improve the expression levels; the different SD sequences can affect the expression of LT-B gene a little. The SD sequence of LT-B gene itself may be better for expression than that of the vector pBV220; the length of 5' non-coding region in the recombinants harboring only one SD sequence has no effect on the expression of LT-B gene; the expression of LT-B gene in HB101 is higher than that in DH5 alpha.