中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2009年
9期
801-806
,共6页
汪浙炯%夏肖萍%楼宏强%孙爱华%严杰
汪浙炯%夏肖萍%樓宏彊%孫愛華%嚴傑
왕절형%하초평%루굉강%손애화%엄걸
铜绿假单胞菌%phoQ/phoP基因%基因敲除%氨基糖苷类抗生素
銅綠假單胞菌%phoQ/phoP基因%基因敲除%氨基糖苷類抗生素
동록가단포균%phoQ/phoP기인%기인고제%안기당감류항생소
Pseudomonas aeruginosa%phoQ/phoP genes%Gene knock out%Aminoglycoside anti-biotics
目的 分析氨基糖苷类抗生素敏感或耐药铜绿假单胞菌菌株二元信号系统PhoQ/PhoP编码基因序列并确定该系统与耐约性关系.方法 采用PCR获得铜绿假单胞菌菌株全长phoQ和phoP基因片段,T-A克隆后测序.构建phoQ和phoP基因原核表达系统,Ni-NTA亲和层析法提纯目的 重组表达产物rPhoQ和rPhoP,皮内注射免疫法制备兔抗血清,免疫双扩散法测定抗血清效价.采用Red重组系统敲除氨基糖苷类抗生素耐药铜绿假单胞菌菌株phoQ基因,采用PCR、测序和Western blot对phoQ突变株进行鉴定.采用试管稀释法测定各铜绿假单胞菌野生株和突变株对4种氨基糖苷类抗生素的最低抑菌浓度(MIC).结果 与GenBank中相关序列比较,所克隆的phoP和phoQ基因核苷酸和氨基酸相似性分别为98.7%～99.6%和98.7～100%、98.4%～99.8%和99.1%～100%.采用pET-42a和E. coli BL21DE3系统成功地表达了rPhoQ和rPhoP.rPhoQ和rPhoP兔抗血清免疫双扩效价分别为1:4和1:8抗血清.经PCR、测序和Western blot鉴定,两株phoQ突变株phoQ基因及产物均缺失.上述phoQ突变株对4种氨基糖苷类抗生素的MIC值分别为其野生株的1/512～1/2048.结论 PhoQ/PhoP是序列保守的铜绿假单胞菌二元信号转导系统,该系统介导细菌对氨基精苷类抗生素的耐药性.
目的 分析氨基糖苷類抗生素敏感或耐藥銅綠假單胞菌菌株二元信號繫統PhoQ/PhoP編碼基因序列併確定該繫統與耐約性關繫.方法 採用PCR穫得銅綠假單胞菌菌株全長phoQ和phoP基因片段,T-A剋隆後測序.構建phoQ和phoP基因原覈錶達繫統,Ni-NTA親和層析法提純目的 重組錶達產物rPhoQ和rPhoP,皮內註射免疫法製備兔抗血清,免疫雙擴散法測定抗血清效價.採用Red重組繫統敲除氨基糖苷類抗生素耐藥銅綠假單胞菌菌株phoQ基因,採用PCR、測序和Western blot對phoQ突變株進行鑒定.採用試管稀釋法測定各銅綠假單胞菌野生株和突變株對4種氨基糖苷類抗生素的最低抑菌濃度(MIC).結果 與GenBank中相關序列比較,所剋隆的phoP和phoQ基因覈苷痠和氨基痠相似性分彆為98.7%～99.6%和98.7～100%、98.4%～99.8%和99.1%～100%.採用pET-42a和E. coli BL21DE3繫統成功地錶達瞭rPhoQ和rPhoP.rPhoQ和rPhoP兔抗血清免疫雙擴效價分彆為1:4和1:8抗血清.經PCR、測序和Western blot鑒定,兩株phoQ突變株phoQ基因及產物均缺失.上述phoQ突變株對4種氨基糖苷類抗生素的MIC值分彆為其野生株的1/512～1/2048.結論 PhoQ/PhoP是序列保守的銅綠假單胞菌二元信號轉導繫統,該繫統介導細菌對氨基精苷類抗生素的耐藥性.
목적 분석안기당감류항생소민감혹내약동록가단포균균주이원신호계통PhoQ/PhoP편마기인서렬병학정해계통여내약성관계.방법 채용PCR획득동록가단포균균주전장phoQ화phoP기인편단,T-A극륭후측서.구건phoQ화phoP기인원핵표체계통,Ni-NTA친화층석법제순목적 중조표체산물rPhoQ화rPhoP,피내주사면역법제비토항혈청,면역쌍확산법측정항혈청효개.채용Red중조계통고제안기당감류항생소내약동록가단포균균주phoQ기인,채용PCR、측서화Western blot대phoQ돌변주진행감정.채용시관희석법측정각동록가단포균야생주화돌변주대4충안기당감류항생소적최저억균농도(MIC).결과 여GenBank중상관서렬비교,소극륭적phoP화phoQ기인핵감산화안기산상사성분별위98.7%～99.6%화98.7～100%、98.4%～99.8%화99.1%～100%.채용pET-42a화E. coli BL21DE3계통성공지표체료rPhoQ화rPhoP.rPhoQ화rPhoP토항혈청면역쌍확효개분별위1:4화1:8항혈청.경PCR、측서화Western blot감정,량주phoQ돌변주phoQ기인급산물균결실.상술phoQ돌변주대4충안기당감류항생소적MIC치분별위기야생주적1/512～1/2048.결론 PhoQ/PhoP시서렬보수적동록가단포균이원신호전도계통,해계통개도세균대안기정감류항생소적내약성.
Objective To analyze the sequences of two component signaling system PhoP/PhoQ encoding genes of Pseudomonas aeruginosa strains sensitive or resistant to aminoglycoside antibiotics and to determine the correlation between the PhoQ/PhoP and the resistance. Methods The segments of entire pimQ and phoP genes of P. aeruginosa were obtained by PCR and then sequenced after T-A cloning. Two prokaryotic expression systems of phoQ and phoP genes were constructed and the target recombinant expres-sion products rPhoQ and rPhoP were extracted by Ni-NTA chromatography. Rabbits were intracutaneoualy immunized with rPhoQ and rPhoP to obtain antisera and double immunodiffusion test was used to detect the titers of antisera. The phoQ genes of aminloglycoside antibiotics-resistant P. aeruginosa strains were knocked out by using Red recombination system, and phoQ mutants were identified by PCR plus sequencing and Western blot assay. Tube dilution method was applied to determine MIC values of wild and mutant strains of P. aeruginosa to four different aminoglycoside antibiotics. Results In comparison with the corresponding sequences in GenBank, the similarities of nueleotide and putative amino acid sequences of the cloned phop and phoQ genes were 98.7%-99.6% and 98.7%-100% , and 98.4%-99.8% and 99.1%-100%, respec-tively. Both rPhoQ and rPhoP were successfully expressed using pET-42a and E. coil BL21 DE3 system, and their rabbit antisera with 1 : 4 and 1 : 8 double immunodiffusion titers were also obtained. The deletion of phoQ genes and absence of the products in the two phoQ mutants were confirmed by PCR, sequencing and West-ern blot assay. MIC values of the four different aminoglycoside antibiotics to the two mutants were 1/512-1/2048 as those of their wild strains. Conclusion PboQ/PhoP is a sequence conserved two component sig-naling system of P. aeruginosa, and this system mediates resistance of the microbe to aminoglycoside antibiotics.
