CAJ | 학술논문

目的构建用于RNA干扰(RNAi)的小发夹RNAshRNA表达载体并检测其对ezrin基因的沉默效果.方法以ezrin为靶基因,以pGenesil-1质粒为载体,设计和构建重组体,根据GeneBank数据库提供的ezrin核苷酸序列,按照Tuschl设计原则,设计2条小发夹结构的DNA序列,经退火形成互补双链,克隆到空载体pGenesil-1中,转化DH5a菌株,提取质粒,予以酶切和测序鉴定；重组质粒转染786-0肾癌细胞株,运用实时荧光定量PCR和蛋白质印迹法进行筛选鉴定.结果酶切及测序鉴定表明成功地构建重组质粒shRNA-ezrinl、shRNA-ezrin2；荧光实时定量PCR和蛋白质印迹法检测结果显示,肾癌细胞中shRNA-ezrinl、shRNA-ezrin2 mRNA相对表达量分别为(0.3376±0.0166)及(0.4661±0.0266),shRNA-ezrinl与shRNA-ezrin2相对表达量差异有统计学意义(P＜0.01),根据基相对表达量算出shRNA-ezrinl,shRNA-ezrin2 ezrin-mRNA的表达量抑制率分别为66.33％及53.29％.转染重组质粒后显著抑制786-0细胞中ezrin mRNA和蛋白表达,其中shRNA-ezrinl的抑制效率最高.结论成功构建ezrin基因的shRNA表达载体,并筛选出抑制率较高的重组质粒载体shRNA-ezrinl,为进一步研究ezrin基因沉默对肾癌786-0细胞株生物学行为的影响奠定基础.
목적 구건용우RNA간우(RNAi)적소발협RNAshRNA표체재체병검측기대ezrin기인적침묵효과.방법 이ezrin위파기인,이pGenesil-1질립위재체,설계화구건중조체,근거GeneBank수거고제공적ezrin핵감산서렬,안조Tuschl설계원칙,설계2조소발협결구적DNA서렬,경퇴화형성호보쌍련,극륭도공재체pGenesil-1중,전화DH5a균주,제취질립,여이매절화측서감정；중조질립전염786-0신암세포주,운용실시형광정량PCR화단백질인적법진행사선감정.결과 매절급측서감정표명성공지구건중조질립shRNA-ezrinl、shRNA-ezrin2；형광실시정량PCR화단백질인적법검측결과현시,신암세포중shRNA-ezrinl、shRNA-ezrin2 mRNA상대표체량분별위(0.3376±0.0166)급(0.4661±0.0266),shRNA-ezrinl여shRNA-ezrin2상대표체량차이유통계학의의(P＜0.01),근거기상대표체량산출shRNA-ezrinl,shRNA-ezrin2 ezrin-mRNA적표체량억제솔분별위66.33％급53.29％.전염중조질립후현저억제786-0세포중ezrin mRNA화단백표체,기중shRNA-ezrinl적억제효솔최고.결론 성공구건ezrin기인적shRNA표체재체,병사선출억제솔교고적중조질립재체shRNA-ezrinl,위진일보연구ezrin기인침묵대신암786-0세포주생물학행위적영향전정기출.
Objective To construct the expression vector of small hairpin RNA(shRNA) and to test its efficiency in silencing ezrin gene.Methods According to the ezrin cDNA sequence in GenBank,2 pair oligo nucleotides were designed and synthesized.After primer annealing,they were inserted into plasmid pGenSil-l to construct the shRNA eukaryotic expression vector.The recombinant plasmid was transformed into DH5a and the positive strain was identified by enzyme digestion and sequence analysis.The recombinant plasmid were transfected into 786-0 cells with Lipofectamine 2000.Then the expression level of ezrin gene was analyzed by the real-time fluorescent quantitative polymerase chain reaction (qRT-PCR)and Western bloting.Results The restriction enzyme analyses demonstrated that shRNA was inserted into vectors.Sequencing analysis demonstrated that shRNA was inserted into vectors and sequencing analyses demonstrated that the sequences were as same as the designed.The results of qRT-PCR and Western bloting showed that the expression of ezrin gene was reduced dramatically in mRNA and at protein level in shRNA-ezrinl.Conclusions We have successfully constructed shRNA eukaryotic expression vectors targeting at ezrin gene.shRNA-ezrinl can efficiently suppress ezrin expression in 786-0 cells,which is important for further research on ezrin gene function in 786-0 cell and the future RNAi experimental research in vivo and in vitro.