The recombinant (pKL series) Plasmids showing different levels of HBcAg antigenicity in ELISA were constructed by inserting the HBcAg gene which was randomly deleted at the non-coding region by Bal-31 exonuclease into plasmid vector pKK223-3 containing tac promoter and SD sequence. SDS-PAGE and Western blot experiments indicated that molecular weight of the HBcAg protein generated by these positive clones was 21000D. Three plasmids with high, moderate and low HBcAg expression level were sequenced and the distance between SD sequence and HBcAg gene ATG codon was 12, 13 and 19bp respectively. Computer analysis of secondary structure of the ribosome binding sites on RNA transcripts also revealed energy and structure differences between the low and high level expression plasmids, suggesting the importance of this distance and the mRNA structure to gene expression.