国际检验医学杂志
國際檢驗醫學雜誌
국제검험의학잡지
INTERNATIONAL JOURNAL OF LABORATORY MEDICINE
2015年
8期
1009-1011,1014
,共4页
付瑞%刘华%段勇%王玉明%单斌
付瑞%劉華%段勇%王玉明%單斌
부서%류화%단용%왕옥명%단빈
产肠毒素大肠杆菌%不耐热肠毒素%小干扰RNA技术%RNA干扰
產腸毒素大腸桿菌%不耐熱腸毒素%小榦擾RNA技術%RNA榦擾
산장독소대장간균%불내열장독소%소간우RNA기술%RNA간우
Enterotoxigenic Escherichia coli%heat-labile enterotoxin%small interfering RNA technology%RNA interfer-ence
目的:应用小干扰RNA(siRNA)技术抑制产肠毒素大肠杆菌(ETEC)LT 基因的表达。方法针对 LT基因设计siRNAs序列,在ETEC培养过程中,将针对LT的siRNA、非特异性对照siRNA、阴性对照siRNA和LB培养基分别加入siRNA组(siRNA‐LT1组、siRNA‐LT2组)、siRNA‐coa3组、siRNA‐NC组和空白对照组,分3个时间点加入,每次1 nmol。在首次加入siRNA后45 min(A时间点)、90 min(B时间点)、135 min(C时间点)留取菌液,应用实时荧光定量PCR检测上述3个时间点5组LT mRNA的表达水平。应用蛋白免疫印迹法(Western blot)检测siRNA‐LT1组、siRNA‐LT2组和空白对照组在3个时间点的LT蛋白表达水平。结果实时荧光定量PCR结果显示,siRNA‐LT1在A、B、C 3个时间点对LT mRNA表达的抑制率分别为70.9%、70.1%、72.5%,siRNA‐LT2在A、B、C 3个时间点对LT mRNA表达的抑制率分别为70.1%、69.2%、70.5%,siRNA‐LT1组、siRNA‐LT2组对LT mRNA表达的抑制率与相应处理时间的siRNA‐NC组、siRNA‐coa3组、空白对照组比较差异均有统计学意义(P<0.05)。Western Blot结果显示,siRNA‐LT1和siRNA‐LT2在A、B、C 3个时间点对LT蛋白表达的抑制率分别为43.1%、18.4%、5.0%和38.2%、15.4%、30.1%。结论针对L T基因设计合成的siRN A在体外能有效地抑制L T 基因的表达。
目的:應用小榦擾RNA(siRNA)技術抑製產腸毒素大腸桿菌(ETEC)LT 基因的錶達。方法針對 LT基因設計siRNAs序列,在ETEC培養過程中,將針對LT的siRNA、非特異性對照siRNA、陰性對照siRNA和LB培養基分彆加入siRNA組(siRNA‐LT1組、siRNA‐LT2組)、siRNA‐coa3組、siRNA‐NC組和空白對照組,分3箇時間點加入,每次1 nmol。在首次加入siRNA後45 min(A時間點)、90 min(B時間點)、135 min(C時間點)留取菌液,應用實時熒光定量PCR檢測上述3箇時間點5組LT mRNA的錶達水平。應用蛋白免疫印跡法(Western blot)檢測siRNA‐LT1組、siRNA‐LT2組和空白對照組在3箇時間點的LT蛋白錶達水平。結果實時熒光定量PCR結果顯示,siRNA‐LT1在A、B、C 3箇時間點對LT mRNA錶達的抑製率分彆為70.9%、70.1%、72.5%,siRNA‐LT2在A、B、C 3箇時間點對LT mRNA錶達的抑製率分彆為70.1%、69.2%、70.5%,siRNA‐LT1組、siRNA‐LT2組對LT mRNA錶達的抑製率與相應處理時間的siRNA‐NC組、siRNA‐coa3組、空白對照組比較差異均有統計學意義(P<0.05)。Western Blot結果顯示,siRNA‐LT1和siRNA‐LT2在A、B、C 3箇時間點對LT蛋白錶達的抑製率分彆為43.1%、18.4%、5.0%和38.2%、15.4%、30.1%。結論針對L T基因設計閤成的siRN A在體外能有效地抑製L T 基因的錶達。
목적:응용소간우RNA(siRNA)기술억제산장독소대장간균(ETEC)LT 기인적표체。방법침대 LT기인설계siRNAs서렬,재ETEC배양과정중,장침대LT적siRNA、비특이성대조siRNA、음성대조siRNA화LB배양기분별가입siRNA조(siRNA‐LT1조、siRNA‐LT2조)、siRNA‐coa3조、siRNA‐NC조화공백대조조,분3개시간점가입,매차1 nmol。재수차가입siRNA후45 min(A시간점)、90 min(B시간점)、135 min(C시간점)류취균액,응용실시형광정량PCR검측상술3개시간점5조LT mRNA적표체수평。응용단백면역인적법(Western blot)검측siRNA‐LT1조、siRNA‐LT2조화공백대조조재3개시간점적LT단백표체수평。결과실시형광정량PCR결과현시,siRNA‐LT1재A、B、C 3개시간점대LT mRNA표체적억제솔분별위70.9%、70.1%、72.5%,siRNA‐LT2재A、B、C 3개시간점대LT mRNA표체적억제솔분별위70.1%、69.2%、70.5%,siRNA‐LT1조、siRNA‐LT2조대LT mRNA표체적억제솔여상응처리시간적siRNA‐NC조、siRNA‐coa3조、공백대조조비교차이균유통계학의의(P<0.05)。Western Blot결과현시,siRNA‐LT1화siRNA‐LT2재A、B、C 3개시간점대LT단백표체적억제솔분별위43.1%、18.4%、5.0%화38.2%、15.4%、30.1%。결론침대L T기인설계합성적siRN A재체외능유효지억제L T 기인적표체。
The distinctive LT siRNAs were designed according to the LT sequence .During the process of cultivation ,siRNA targeting the LT gene ,non‐specific control siRNA ,negative control siRNA and culture medium were added into siRNA group (siRNA‐LT1 group , siRNA‐LT2 group) ,siRNA‐coa3 group ,siRNA‐NC group and blank control group ,respectively ,and three times in each group (1 nmol each time) .After siRNA added at the first time ,bacteria was collected in 45 min (A) ,90 min (B) and 135 min (C) time points .The expression of mRNA in three time points (A ,B and C) were detected by real‐time fluorescence quantitative PCR .The protein level of LT in siRNA‐LT1 group ,siRNA‐LT2 group and blank control group were detected by Western blot in three time points .Results The results of real‐time fluorescence quantitative PCR showed that inhibition of siRNA‐LT1 on the expression of LT mRNA at the three time points(A ,B and C)were 70 .9% ,70 .1% ,72 .5% respectively ,and inhibition of siRNA‐LT2 on the ex‐pression of LT mRNA at the three time points(A ,B and C)were 70 .1% ,69 .2% and 70 .5% respectively .In the three time points (A ,B and C)the inhibition rate of the expression of LT mRNA in siRNA‐LT1 group and siRNA‐LT2 group were statistically lower than that in the siRNA‐NC group ,siRNA‐coa3 group and blank control group (P<0 .05) .The results of Western blot showed that in siRNA‐LT1 group the inhibitory rate of expression of LT protein in the three time points were 43 .1% ,18 .4% and 5 .0% ,re‐spectively ;in the siRNA‐LT2 group were 38 .2% ,15 .4% and 30 .1% ,respectively .Conclusion The specific siRNA could inhibit the expression of LT gene in vitro .