CAJ | 학술논문

目的：构建及鉴定人Notch-1基因的RNA干扰（RNAi）慢病毒载体，寻找最佳RNAi慢病毒载体。方法针对人Notch-1基因序列，按照RNAi序列设计原则，设计3段RNAi靶点序列（shRNA1~3），通过限制性内切酶BamHⅠ和EcoRⅠ双酶切、T4DNA连接酶连接，将Notch-1基因序列插入慢病毒载体pLenOR-THM，构建pLenOR-THM-Notch-1重组载体。质粒转化感受态DH5α细菌，筛选阳性克隆，经KpnⅠ和EcoRⅠ双酶切及测序鉴定正确后通过脂质体将慢病毒四质粒系统共转染293T细胞，进行慢病毒包装并测定病毒滴度，观察感染效率。各组病毒载体转染ACC-M细胞后，运用定量逆转录聚合酶链反应和Westernblot检测Notch-1基因mRNA和蛋白的表达水平。结果成功构建慢病毒载体pLenOR-THM-Notch-1，四质粒共转染293T细胞后可见大量绿色荧光；浓缩后的病毒滴度为5.8×108TU·mL-1；以复感染系数为1时感染293T细胞，感染效率在90％以上。QRT-PCR和Westernblot检测结果表明，pLenOR-Notch-1-shRNA3组受抑制程度最高。结论成功构建了人Notch-1RNAi慢病毒载体。
목적：구건급감정인Notch-1기인적RNA간우（RNAi）만병독재체，심조최가RNAi만병독재체。방법침대인Notch-1기인서렬，안조RNAi서렬설계원칙，설계3단RNAi파점서렬（shRNA1~3），통과한제성내절매BamHⅠ화EcoRⅠ쌍매절、T4DNA련접매련접，장Notch-1기인서렬삽입만병독재체pLenOR-THM，구건pLenOR-THM-Notch-1중조재체。질립전화감수태DH5α세균，사선양성극륭，경KpnⅠ화EcoRⅠ쌍매절급측서감정정학후통과지질체장만병독사질립계통공전염293T세포，진행만병독포장병측정병독적도，관찰감염효솔。각조병독재체전염ACC-M세포후，운용정량역전록취합매련반응화Westernblot검측Notch-1기인mRNA화단백적표체수평。결과성공구건만병독재체pLenOR-THM-Notch-1，사질립공전염293T세포후가견대량록색형광；농축후적병독적도위5.8×108TU·mL-1；이복감염계수위1시감염293T세포，감염효솔재90％이상。QRT-PCR화Westernblot검측결과표명，pLenOR-Notch-1-shRNA3조수억제정도최고。결론성공구건료인Notch-1RNAi만병독재체。
Objective To construct and identify a lentiviral vector of RNA interference targeting human Notch-1 gene. Methods To determine the Notch-1 gene sequences, three RNAi target sequences (shRNA1-3) were designed in accordance with the RNAi sequence design principles and cloned into the lentiviral vector pLenOR-THM by endonuclease BamHⅠres-triction, EcoRⅠdouble digestion, and T4 DNA-ligase ligation. After the transformation into competent DH5αbacteria, the candidate clones were identified by KpnⅠand EcoRⅠdouble digestion and DNA sequencing. The recombinant and three packaging plasmids were co-transfected into human embryonic kidney cell line 293T cells by lipofectamine to produce the lentiviral particles. The viral titer was determined. The 293T cells were infected by the lentiviral particles obtained, and trans-fection efficiency was assessed using a fluorescent microscope. The lentiviral vector particles were also transfected into ACC-M cells. The Notch-1 expression in the transfected cells was assayed by quantitative reverse transcription polymerase chain reaction (QRT-PCR) and Western blot analysis. Results The lentiviral RNAi vector pLenOR-THM-Notch1 for Notch-1 gene was constructed successfully. Strong green fluorescence was observed in the 293T cells under fluorescent microscope after co-transfection of the cells with the four plasmids of lentiviral vector. The virus in the supernatant reached a titer of 5.8× 108 TU·mL-1. The transfection efficiency of the collected virus exceeded 90% in 293T cells with 1 as a multiplicity of in-fection. The third lentiviral vector was found to significantly inhibit the Notch-1 expression at the mRNA and protein levels. Conclusion The lentiviral RNAi vector of Notch-1 has been successfully constructed and identified.