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
2011年
11期
961-966
,共6页
宋莹%邢铭友%许东%龚凤云%夏超%王丽丽%谢旭华%申爱霞%占伟丽%宋建新
宋瑩%邢銘友%許東%龔鳳雲%夏超%王麗麗%謝旭華%申愛霞%佔偉麗%宋建新
송형%형명우%허동%공봉운%하초%왕려려%사욱화%신애하%점위려%송건신
siRNA%mexB基因%Western blot%铜绿假单胞菌
siRNA%mexB基因%Western blot%銅綠假單胞菌
siRNA%mexB기인%Western blot%동록가단포균
siRNA%mexB gene%Western blot%Pseudomonas aeruginosa
目的 观察siRNA干扰质粒转入铜绿假单胞菌后,MexB蛋白表达量的变化.方法 针对mexB基因设计合成特异性siRNA分子,与pGPU6/GFP/Neo载体连接,构建pGPU6/GFP/NeosiRNA重组质粒.构建重组表达质粒pET22b+/mexB,并转化大肠杆菌BL21( DE3) plysS,诱导表达MexB蛋白,蛋白纯化后免疫家兔制备多克隆抗体.siRNA质粒分别电转化铜绿假单胞菌野生株、临床耐药株及mexB基因高表达株,运用Western blot观察转化8、12、24h后MexB蛋白表达量的变化.结果 成功构建pGPU6/GFP/Neo-siRNA重组质粒.成功表达铜绿假单胞菌外排泵蛋白MexB,并制备多克隆兔抗.siRNA质粒对铜绿假单胞菌野生株、临床耐药株及mexB基因高表达株的mexB基因均具有良好的沉默效果,而且沉默效果存在时间差异性.结论 siRNA质粒转化3株铜绿假单胞菌后,在8h及12 h时均可观察到MexB蛋白表达量明显减少,而在24 h时MexB蛋白表达量无改变.
目的 觀察siRNA榦擾質粒轉入銅綠假單胞菌後,MexB蛋白錶達量的變化.方法 針對mexB基因設計閤成特異性siRNA分子,與pGPU6/GFP/Neo載體連接,構建pGPU6/GFP/NeosiRNA重組質粒.構建重組錶達質粒pET22b+/mexB,併轉化大腸桿菌BL21( DE3) plysS,誘導錶達MexB蛋白,蛋白純化後免疫傢兔製備多剋隆抗體.siRNA質粒分彆電轉化銅綠假單胞菌野生株、臨床耐藥株及mexB基因高錶達株,運用Western blot觀察轉化8、12、24h後MexB蛋白錶達量的變化.結果 成功構建pGPU6/GFP/Neo-siRNA重組質粒.成功錶達銅綠假單胞菌外排泵蛋白MexB,併製備多剋隆兔抗.siRNA質粒對銅綠假單胞菌野生株、臨床耐藥株及mexB基因高錶達株的mexB基因均具有良好的沉默效果,而且沉默效果存在時間差異性.結論 siRNA質粒轉化3株銅綠假單胞菌後,在8h及12 h時均可觀察到MexB蛋白錶達量明顯減少,而在24 h時MexB蛋白錶達量無改變.
목적 관찰siRNA간우질립전입동록가단포균후,MexB단백표체량적변화.방법 침대mexB기인설계합성특이성siRNA분자,여pGPU6/GFP/Neo재체련접,구건pGPU6/GFP/NeosiRNA중조질립.구건중조표체질립pET22b+/mexB,병전화대장간균BL21( DE3) plysS,유도표체MexB단백,단백순화후면역가토제비다극륭항체.siRNA질립분별전전화동록가단포균야생주、림상내약주급mexB기인고표체주,운용Western blot관찰전화8、12、24h후MexB단백표체량적변화.결과 성공구건pGPU6/GFP/Neo-siRNA중조질립.성공표체동록가단포균외배빙단백MexB,병제비다극륭토항.siRNA질립대동록가단포균야생주、림상내약주급mexB기인고표체주적mexB기인균구유량호적침묵효과,이차침묵효과존재시간차이성.결론 siRNA질립전화3주동록가단포균후,재8h급12 h시균가관찰도MexB단백표체량명현감소,이재24 h시MexB단백표체량무개변.
Objective To investigate the alteration of MexB protein expression by plasmid containing siRNA template strand was transformed into Pseudomonas aeruginosa.Methods siRNA molecules specifically against mexB were designed and ligated into pGPU6/GFP/Neo vector to construct the recombinant plasmid pGPU6/GFP/Neo-siRNA.MexB gene was cloned into expression vector pET22b+ to construct plasmid pET22b+/mexB,the recombinant expression plasmid was transformed into E.coli BL21 (DE3)plysS and protein MexB was induced to express,then purified protein MexB was used to prepare specific antibodies in rabbits.The plasmids with siRNA molecules specifically against mexB were transformed into wild type strain,clinical multiresistant strain and mexB overexpression strain of Pseudomonas aeruginosa by electroporation respectively,and the changes of the expression of MexB were detected in 8,12,24 h respectively by Western blot.Results pGPU6/GFP/Neo-siRNA was constructed successfully.Protein MexB was expressed successfully and the rabbit polyclonal antibodies against MexB was prepared well.The gene silence of mexB by siRNA molecules was effective in the three strains of Pseudomonas aeruginosa,but it depended on the silencing time.Conclusion The expression of MexB was reduced in 8 h and 12 h,but in 24 h,the expression was unchanged.
