整合子中氨基糖苷-3″-腺苷酰基转移酶多克隆抗体的制备与初步应用
정합자중안기당감-3″-선감선기전이매다극륭항체적제비여초보응용
Preparation and primary application of antiserum to aminoacy1-3″-adenylyltransferase in integron
  • 万方数据
    中华检验医学杂志 중화검험의학잡지
    CHINESE JOURNAL OF LABORATORY MEDICINE
    2012年 3期 227-232 ,共6页

    魏取好%蒋晓飞%李敏%胡庆丰%吕火祥%周永列%吕元

    위취호%장효비%리민%호경봉%려화상%주영렬%려원

    核苷酸酰基转移酶类%整合子类%启动区(遗传学)%抗药性,细菌 覈苷痠酰基轉移酶類%整閤子類%啟動區(遺傳學)%抗藥性,細菌
    핵감산선기전이매류%정합자류%계동구(유전학)%항약성,세균
    Nucleotidyltransferases%Integrons%Promoter regions ( genetics )%Drug resistance,bacterial
    目的 制备氨基糖苷-3″-腺苷酰基转移酶[AAD(3″)]特异性抗血清,并探讨其在检测第1类整合子8种不同可变区启动子(PcS、PcH2、PcH1、PcW、PcS-P2、PcH2-P2、PcH1 -P2和PcW-P2)下游基因盒中aadA2基因表达水平的应用价值.方法 用聚合酶链反应(PCR)扩增aadA2基因,并克隆至表达载体pET19b中,诱导表达并纯化带组氨酸标签的重组AAD(3″)蛋白,免疫大耳白兔制备抗AAD(3″)特异性抗血清.以制备的抗AAD(3″)抗血清为一抗,用免疫印迹法(WB)检测不同可变区启动子下游基因盒中aadA2基因的翻译水平,用微量肉汤稀释法检测链霉素对含不同可变区启动子及其下游aadA2基因盒的大肠埃希菌JM109的最低抑菌浓度(MIC).结果 成功构建重组的AAD(3″)蛋白表达质粒pET1 9b-aadA2,经测序确认转化大肠埃希菌BL21( DE3)获得1株高表达可溶性重组AAD(3″)蛋白的工程菌株,发酵后用镍柱纯化获得纯度＞90％的重组AAD(3″)蛋白,免疫大耳白兔获得效价＞ 1∶100 000的抗AAD(3″)特异性抗血清.WB检测8种不同可变区启动子下游aadA2基因翻译产物AAD(3″)蛋白的表达水平,设定启动子PcW下游AAD(3″)蛋白的相对表达水平为1,重复3次测得启动子PcS、PcH2、PcH1、PcS-P2、PcH2 -P2、PcH1-P2及PcW-P2下游AAD(3″)蛋白相对表达水平依次为12.9±2.3、9.1±1.0、2.0±0.4、16.0±1.3、14.1±1.3、10.5±0.7、8.9±1.7,且不同可变区启动子下游AAD(3″)蛋白的相对表达水平差异具有统计学意义(F=32.421,P＜0.01).微量肉汤稀释法重复3次测得链霉素对含启动子PcS、PeH2、PcH1、PcS-P2、PcH2-P2、PcH1 P2、PcW、PcW-P2及其下游aadA2基因重组质粒的大肠杆菌JM109的MIC平均值依次为256、256、64、128、32、128、4、64 mg/L,表明不同可变区启动子下游aadA2基因的表达水平不同可赋予宿主细菌对链霉素产生不同水平的耐药.结论 成功纯化出可溶性重组AAD(3″)蛋白,免疫大耳白兔获得高效价的抗AAD(3″)特异性抗血清,为进一步研究整合子中整合酶基因与可变区基因盒表达之间的相互关系奠定基础.不同可变区启动子下游耐药基因盒的表达水平明显不同,赋予宿主细菌对相应抗菌药物的耐药水平明显不同,因此在对临床菌株进行第1类整合子分子流行病学调查时,应重视可变区启动子分类方面的研究.
    목적 제비안기당감-3″-선감선기전이매[AAD(3″)]특이성항혈청,병탐토기재검측제1류정합자8충불동가변구계동자(PcS、PcH2、PcH1、PcW、PcS-P2、PcH2-P2、PcH1 -P2화PcW-P2)하유기인합중aadA2기인표체수평적응용개치.방법 용취합매련반응(PCR)확증aadA2기인,병극륭지표체재체pET19b중,유도표체병순화대조안산표첨적중조AAD(3″)단백,면역대이백토제비항AAD(3″)특이성항혈청.이제비적항AAD(3″)항혈청위일항,용면역인적법(WB)검측불동가변구계동자하유기인합중aadA2기인적번역수평,용미량육탕희석법검측련매소대함불동가변구계동자급기하유aadA2기인합적대장애희균JM109적최저억균농도(MIC).결과 성공구건중조적AAD(3″)단백표체질립pET1 9b-aadA2,경측서학인전화대장애희균BL21( DE3)획득1주고표체가용성중조AAD(3″)단백적공정균주,발효후용얼주순화획득순도＞90％적중조AAD(3″)단백,면역대이백토획득효개＞ 1∶100 000적항AAD(3″)특이성항혈청.WB검측8충불동가변구계동자하유aadA2기인번역산물AAD(3″)단백적표체수평,설정계동자PcW하유AAD(3″)단백적상대표체수평위1,중복3차측득계동자PcS、PcH2、PcH1、PcS-P2、PcH2 -P2、PcH1-P2급PcW-P2하유AAD(3″)단백상대표체수평의차위12.9±2.3、9.1±1.0、2.0±0.4、16.0±1.3、14.1±1.3、10.5±0.7、8.9±1.7,차불동가변구계동자하유AAD(3″)단백적상대표체수평차이구유통계학의의(F=32.421,P＜0.01).미량육탕희석법중복3차측득련매소대함계동자PcS、PeH2、PcH1、PcS-P2、PcH2-P2、PcH1 P2、PcW、PcW-P2급기하유aadA2기인중조질립적대장간균JM109적MIC평균치의차위256、256、64、128、32、128、4、64 mg/L,표명불동가변구계동자하유aadA2기인적표체수평불동가부여숙주세균대련매소산생불동수평적내약.결론 성공순화출가용성중조AAD(3″)단백,면역대이백토획득고효개적항AAD(3″)특이성항혈청,위진일보연구정합자중정합매기인여가변구기인합표체지간적상호관계전정기출.불동가변구계동자하유내약기인합적표체수평명현불동,부여숙주세균대상응항균약물적내약수평명현불동,인차재대림상균주진행제1류정합자분자류행병학조사시,응중시가변구계동자분류방면적연구.
    Objective To prepare antiserum specific to aminoacy1-3″-adenylyltransferase [ AAD (3″) ],and to explore the application value of the prepared antiserum in detecting the expression levels of aadA2 gene that downstream of 8 different promoters (PcS,PcH2,PcH1,PcW,PcS-P2,PcH2-P2,PcH1P2 and PcW-P2 ) of variable regions in class 1 integron.Methods aadA2 gene was amplified by polymerase chain reaction(PCR) and cloned into the expression plasmid pET19b.After inducing,the recombined aminoacy1-3″-adenylyltransferase[ AAD(3″)] with His-tag was expressed,purified and immunized rabbits to get anti- AAD(3″) specific serum.The prepared antiserum was used to detect the translation levels of aadA2 gene that downstream of different promoters of variable regions in class 1 integron by Western blotting (WB).Broth microdilution method was used to detect the minimal inhibitory concentrations (MIC) to streptomycin in Escherichia coli JM109 with aadA2 gene downstream of different promoters of variable regions.Results Recombined AAD (3″) expression plasmid pET19b-aadA2 was constructed successfully and was verified by sequence analysis.After transformed into E.coli BL21 ( DE3 ),a resoluble recombined AAD(3″) high expression strain was obtained.After fermentation,recombined AAD(3″) was purified and immunized rabbits.The anti- AAD(3″) specific serum was obtained with titer ＞ 1∶100 000.WB was used to detect the expression levels of AAD (3″),the translation product of aadA2 gene,that downstream of 8 different promoters of variable regions.The relative expression level of AAD (3″) that downstream of PcW was assumed to be 1,then the relative expression levels of AAD(3″),which all were detected 3 times independently,that downstream of PcS,PcH2,PcH1,PcS-P2,PcH2-P2,PcH1-P2 and PcW-P2 were 12.9±2.3,9.1±1.0,2.0±0.4,16.0±1.3,14.1 ±1.3,10.5±0.7 and 8.9 ±1.7 respective.Very different expression levels of AAD (3″) that downstream of different promoters of variable regions were obtained( F =32.421,P ＜ 0.01 ).The mean values of MIC,which all were detected 3 times independently,to streptomycin in E.coli JM109 with aadA2 gene downstream of PcS,PcH2,PcH1,PcW,PcS-P2,PcH2P2,PcH1-P2 and PcW-P2 were 256,256,64,128,32,128,4 and 64 mg/L respective.These results indicated the different expression levels of aadA2 gene that downstream of different promoters of variable regions can confer their host bacteria different resistance levels to streptomycin.Conclusions Resoluble recombined AAD(3″) is purified successfully and high titer anti- AAD(3″) specific antiserum is obtained from the immunized rabbits.This laid foundation for further investigation on the correlationship between the expressions of intI1 gene and the gene cassettes within variable regions.The expression levels of antibiotic gene cassettes that downstream of different promoters of variable regions are very different,so are the very different antibiotic resistance levels of the host bacteria.Therefore more attentions should be paid to the researches on the classification of promoters of variable regions when molecular epidemiology studies on the class 1 integrons in clinical isolates were conducted.
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