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
2010年
5期
405-409
,共5页
李树颖%于德民%牛文彦%姚智
李樹穎%于德民%牛文彥%姚智
리수영%우덕민%우문언%요지
S6K1%shRNA%腺病毒载体%基因沉默
S6K1%shRNA%腺病毒載體%基因沉默
S6K1%shRNA%선병독재체%기인침묵
S6K1%shRNA%Viral vector%Gene silencing
目的 构建S6K1 shRNA基因重组腺病毒(S6K1Ax)并在细胞及小鼠肝脏验证对S6K1基因的沉默效果.方法 设计3种S6K1 shRNA序列,通过在pcPUR质粒与pcDNA3.1质粒、cosmid质粒之间的拼接将S6K1 shRNA转染进腺病毒,筛选沉默效果最佳的S6K1Ax并在293细胞扩增纯化得到高效价的S6K1Ax.感染来源于小鼠的肝脏、肌肉、脂肪细胞系,在Western blot水平评价其对S6K1蛋白表达的抑制效果.将S6K1Ax注射进C57BL/6J小鼠尾静脉,6 d后处死小鼠取肝脏,逆转录-聚合酶链反应和Western blot观察小鼠肝脏S6K1在mRNA和蛋白水平的表达.检测注射S6K1Ax前后小鼠血清丙氨酸氨基转移酶(ALT)变化.结果 Western blot证实制备的S6K1 Ax可抑制来源于小鼠的肝脏、肌肉、脂肪3种细胞系和小鼠C57BL/6J肝脏S6K1的蛋白表达.小鼠肝脏S6K1 mRNA结果显示:对照组为1.39±0.21,实验组为0.63±0.09,t=6.132,P<0.01,差异有统计学意义.S6K1Ax注射小鼠,ALT水平注射前为(15.15±4.43)U/L,注射后为(17.32±4.22)U/L,t=1.451,P>0.05,差异没有统计学意义.结论 构建的S6K1Ax可将来源于小鼠的细胞系及C57BL/6J小鼠肝脏S6K1基因沉默,为研究S6K1的基因功能提供了适合的实验工具.
目的 構建S6K1 shRNA基因重組腺病毒(S6K1Ax)併在細胞及小鼠肝髒驗證對S6K1基因的沉默效果.方法 設計3種S6K1 shRNA序列,通過在pcPUR質粒與pcDNA3.1質粒、cosmid質粒之間的拼接將S6K1 shRNA轉染進腺病毒,篩選沉默效果最佳的S6K1Ax併在293細胞擴增純化得到高效價的S6K1Ax.感染來源于小鼠的肝髒、肌肉、脂肪細胞繫,在Western blot水平評價其對S6K1蛋白錶達的抑製效果.將S6K1Ax註射進C57BL/6J小鼠尾靜脈,6 d後處死小鼠取肝髒,逆轉錄-聚閤酶鏈反應和Western blot觀察小鼠肝髒S6K1在mRNA和蛋白水平的錶達.檢測註射S6K1Ax前後小鼠血清丙氨痠氨基轉移酶(ALT)變化.結果 Western blot證實製備的S6K1 Ax可抑製來源于小鼠的肝髒、肌肉、脂肪3種細胞繫和小鼠C57BL/6J肝髒S6K1的蛋白錶達.小鼠肝髒S6K1 mRNA結果顯示:對照組為1.39±0.21,實驗組為0.63±0.09,t=6.132,P<0.01,差異有統計學意義.S6K1Ax註射小鼠,ALT水平註射前為(15.15±4.43)U/L,註射後為(17.32±4.22)U/L,t=1.451,P>0.05,差異沒有統計學意義.結論 構建的S6K1Ax可將來源于小鼠的細胞繫及C57BL/6J小鼠肝髒S6K1基因沉默,為研究S6K1的基因功能提供瞭適閤的實驗工具.
목적 구건S6K1 shRNA기인중조선병독(S6K1Ax)병재세포급소서간장험증대S6K1기인적침묵효과.방법 설계3충S6K1 shRNA서렬,통과재pcPUR질립여pcDNA3.1질립、cosmid질립지간적병접장S6K1 shRNA전염진선병독,사선침묵효과최가적S6K1Ax병재293세포확증순화득도고효개적S6K1Ax.감염래원우소서적간장、기육、지방세포계,재Western blot수평평개기대S6K1단백표체적억제효과.장S6K1Ax주사진C57BL/6J소서미정맥,6 d후처사소서취간장,역전록-취합매련반응화Western blot관찰소서간장S6K1재mRNA화단백수평적표체.검측주사S6K1Ax전후소서혈청병안산안기전이매(ALT)변화.결과 Western blot증실제비적S6K1 Ax가억제래원우소서적간장、기육、지방3충세포계화소서C57BL/6J간장S6K1적단백표체.소서간장S6K1 mRNA결과현시:대조조위1.39±0.21,실험조위0.63±0.09,t=6.132,P<0.01,차이유통계학의의.S6K1Ax주사소서,ALT수평주사전위(15.15±4.43)U/L,주사후위(17.32±4.22)U/L,t=1.451,P>0.05,차이몰유통계학의의.결론 구건적S6K1Ax가장래원우소서적세포계급C57BL/6J소서간장S6K1기인침묵,위연구S6K1적기인공능제공료괄합적실험공구.
Objective To construct S6K1 shRNA gene recombinant adenovirus(S6K1 Ax)and evaluate its gene silencing effects on mouse cell lines and C57 BL/6J mice level.Methods Three S6K1 shRNA gene sequences were designed and spliced from pcPUR plasmid,pcDNA3.1 plasmid to cosmid plasmid and transfected into adenovirus.S6K1 Ax which has best gene silencing effect was selected and proliferated in 293 cell.Silencing effect of S6K1Ax was checked on mice AML12,C2C12,3T3-L1 cell lines.C57BL/6J liver was obtained after S6K1 Ax was injected into mice tail vein six days later.S6K1 was evaluated by qRT-PCR and Western blot.Alanine transferanse(ALT)was examined before and after S6K1 Ax injected.Results S6K1Ax can silence S6K1 expression of mouse AML12,C2C12 and 3T3-L1 cell lines and liver of C57 BL/6J mice on Western blot.S6K1 mRNA expression of C57BL/6J liver were control group 1.39±0.21 vs S6K1Ax group 0.63±0.09,t=6.132.P<0.01.ALT of mice hepatic function did not change after S6K1Ax injected:before(15.15±4.43)U/L,after(17.32±4.22)U/L,t=1.451,P>0.05.Conclusion Construction of shS6K1 Ax can knockdown S6K1 gene on mice cell lines and C57BL/6J mice liver,it provides a good tool to study the function of S6K1.
