[Recombinant expression of Streptococcus pneumoniae comD/E/C genes and correlation of ComD/C with beta-lactam antibiotic resistance].

OBJECTIVE

To construct prokaryotic expression systems of TCS genes comD/comE/comC of Streptococcus pneumoniae, and to determine the correlation of ComD and ComC with the drug resistance.

METHODS

The entire comD, comE and comC genes were amplified by PCR and their prokaryotic expression systems were established by routine genetic engineering technique. SDS-PAGE and Bio-Rad Agarose Image Analyzor was applied to measure the outputs of target recombinant proteins rComD, rComE and rComC. Rabbits were immunized with these recombinant proteins to prepare antisera. The resistance of S.pneumoniae strains to penicillin and cefotaxime was examined after ComD and ComC were blocked by antisera.

RESULT: Compared with the reported sequences, similarities of nucleotide and amino acid sequences of the cloned comD, comE and comC genes were 98.4% approximately 99.3% and 99.1% approximately 100%, respectively. The constructed engineering bacteria E.coli BL21DE3(pET42a-comD), E.coli BL21DE3(pET42a-comE) and E.coli BL21DE3(pET42a-comC) were able to efficiently express the target recombinant proteins and the outputs of rComD, rComE and rComC were 28%, 25% and 35% of the total bacterial proteins, respectively. The double immunodiffusion titers of rabbit antisera against rComD, rComE or rComC were 1:4, 1:4 and 1:8, respectively. After the ComD and/or ComC were blocked by the antisera, the cefotaxime-sensitive S. pneumoniae strains became to resistant to antibiotics but there were no changes for cefotaxime-resistant strains and resistance to penicillin for all tested strains.

CONCLUSION

The prokaryotic expression systems of S.pneumoniae comD/come/comC genes have been successfully constructed, and the study also indicates that both the ComD and ComC are involved in the drug resistance of S. pneumoniae to cefotaxime.