[Recombinant expression of Streptococcus pneumoniae ciaH/R genes and their correlation with beta-lactam antibiotic resistance].
To construct prokaryotic expression systems of Streptococcus pneumoniae ciaH and ciaR genes,and to determine their correlation with drug resistance.



The total length of ciaH and ciaR genes was amplified by PCR and their prokaryotic expression systems were established by using routine genetic engineering technique. SDS-PAGE was applied to measure the outputs of target recombinant proteins rCiaH and rCiaR. Rabbits antisera and IgGs against rCiaH and rCiaR were prepared. The resistance to penicillin and cefotaxime of S.pneumoniae strains was examined after CiaH and CiaR were extracellularly and intracellularly blocked by the IgGs.



The homogeneity of nucleotide and amino acid sequences of the cloned ciaH and ciaR genes with the reported sequences was 99.9-100% and 100%, respectively. The recombinant bacteria E.coli BL21DE3pET42a-ciaH and E.coli BL21DE3pET42a-ciaR were able to express the target recombinant proteins rCiaH and rCiaR with efficiency. The outputs of rCiaH and rCiaR were 33% and 45% of the total bacterial proteins, respectively. The double immunodiffusion titers of rCiaH antiserum,rCiaR antiserum,rCiaH-IgG and rCiaR-IgG were 1:4,1:4,1:1 and 1:1, respectively. After CiaH was extracellularly or intracellularly blocked by CiaH-IgG, and CiaR was intracellularly blocked by CiaR-IgG, the penicillin-sensitive or cefotaxime-sensitive strains developed resistance to the two antibiotics; but the blocks did not change that of penicillin-resisting or cefotaxime-resisting strains.



The prokaryotic expression systems of S. pneumoniae ciaH/ciaR genes have been successfully constructed in this study. Both the CiaH and CiaR may be involved in penicillin and cefotaxime resistance of the bacterium.
Sun
Ai-hua
AH
Zhejiang Medical College, and Department of Laboratory Medicine, The Sir Run Run Shaw Hospital, Hangzhou 310053, China.
Fan
Huan
H
Xia
Xiao-ping
XP
Li
Xiang-yang
XY
Yan
Jie
J
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002439:Cefotaxime / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D010403:Penicillin Resistance / Q000235:genetics; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013296:Streptococcus pneumoniae / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics*; D018440:beta-Lactam Resistance / Q000235:genetics*
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D011494:Protein Kinases; C088910:CiaR protein, Streptococcus pneumoniae; D002439:Cefotaxime