Sun Ai-hua, Fan Huan, Xia Xiao-ping, Li Xiang-yang, Yan Jie
Zhejiang Medical College, and Department of Laboratory Medicine, The Sir Run Run Shaw Hospital, Hangzhou 310053, China.
Zhejiang Da Xue Xue Bao Yi Xue Ban. 2008 Nov;37(6):605-11. doi: 10.3785/j.issn.1008-9292.2008.06.011.
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.
构建肺炎链球菌ciaH和ciaR基因的原核表达系统，并确定它们与耐药性的相关性。
采用PCR扩增ciaH和ciaR基因全长，运用常规基因工程技术构建其原核表达系统。应用SDS-PAGE检测目标重组蛋白rCiaH和rCiaR的表达量。制备兔抗rCiaH和rCiaR抗血清及IgG。用IgG细胞外和细胞内阻断CiaH和CiaR后，检测肺炎链球菌菌株对青霉素和头孢噻肟的耐药性。
克隆的ciaH和ciaR基因核苷酸和氨基酸序列与报道序列的同源性分别为99.9% - 100%和100%。重组菌大肠杆菌BL21DE3pET42a-ciaH和大肠杆菌BL21DE3pET42a-ciaR能高效表达目标重组蛋白rCiaH和rCiaR。rCiaH和rCiaR的表达量分别占细菌总蛋白的33%和45%。rCiaH抗血清、rCiaR抗血清、rCiaH-IgG和rCiaR-IgG的双向免疫扩散效价分别为1:4、1:4、1:1和1:1。用CiaH-IgG细胞外或细胞内阻断CiaH，用CiaR-IgG细胞内阻断CiaR后，青霉素敏感或头孢噻肟敏感菌株对这两种抗生素产生耐药性；但阻断对青霉素耐药或头孢噻肟耐药菌株无影响。
本研究成功构建了肺炎链球菌ciaH/ciaR基因的原核表达系统。CiaH和CiaR可能均参与该菌对青霉素和头孢噻肟的耐药过程。
