CAJ | 학술논문

目的：构建靶向犬p53基因慢病毒干扰载体，建立稳定沉默p53基因的WRD/p53-细胞系。方法设计并构建4条针对犬p53基因的特异性shRNA干扰质粒。将构建的慢病毒表达载体（pGMLV-p53）和包装质粒（packaging mix）组成的包装系统共转染293T细胞，包装病毒，收集病毒原液，超滤浓缩，并测定滴度。包装好的慢病毒感染WRD细胞，Western blot和实时荧光定量PCR方法检测不同靶点RNA干扰载体对p53基因的干扰效果，确定有效靶点。针对有效靶点慢病毒颗粒以最适感染复数（multiplicity of infection, MOI）感染WRD细胞，puromycin抗生素筛选稳定感染细胞系WRD/p53-。结果结果显示p53 RNAi慢病毒载体构建成功，成功包装四种不同靶点的p53基因RNA干扰慢病毒,病毒滴度均达到1×109 TU/ml。四种干扰载体慢病毒均具有较好的干扰效果，选用沉默效果最好的pGMLV-p53A1为最优靶点，成功建立p53 RNAi慢病毒载体稳定感染细胞系WRD/p53-。结论成功构建p53 RNAi慢病毒载体，并有效干扰WRD细胞中p53 mRNA和蛋白表达，同时成功筛选并建立p53 RNAi慢病毒稳定感染WRD/p53-细胞系。
목적：구건파향견p53기인만병독간우재체，건립은정침묵p53기인적WRD/p53-세포계。방법설계병구건4조침대견p53기인적특이성shRNA간우질립。장구건적만병독표체재체（pGMLV-p53）화포장질립（packaging mix）조성적포장계통공전염293T세포，포장병독，수집병독원액，초려농축，병측정적도。포장호적만병독감염WRD세포，Western blot화실시형광정량PCR방법검측불동파점RNA간우재체대p53기인적간우효과，학정유효파점。침대유효파점만병독과립이최괄감염복수（multiplicity of infection, MOI）감염WRD세포，puromycin항생소사선은정감염세포계WRD/p53-。결과결과현시p53 RNAi만병독재체구건성공，성공포장사충불동파점적p53기인RNA간우만병독,병독적도균체도1×109 TU/ml。사충간우재체만병독균구유교호적간우효과，선용침묵효과최호적pGMLV-p53A1위최우파점，성공건립p53 RNAi만병독재체은정감염세포계WRD/p53-。결론성공구건p53 RNAi만병독재체，병유효간우WRD세포중p53 mRNA화단백표체，동시성공사선병건립p53 RNAi만병독은정감염WRD/p53-세포계。
Objective To establish a canine cell line with p53 gene knockdown by lentivirus-mediated RNA interference (RNAi). Methods Four pairs of oligonucleotide sequences of p53 gene were synthesized and cloned into the pGMLV-SC5 RNAi vector. Following confirmation by PCR and DNA sequencing, the recombinant pGMLV-p53 plasmids and packaging mix vectors were packaged into mature lentivirus to infect 293T cells. The supernatant of the infected cells was harvested to infect WRD cells, in which p53 expression was detected using Western blotting and real-time PCR. The lentivirus with the strongest p53-silencing effect was packaged for infecting WRD cells at the optimal multiplicity of infection (MOI) to establish a stably infected cell line (WRD/p53-) screened using puromycin. Results The lentivirus carrying p53 shRNA was constructed successfully and a virus titer of 1 × 109 TU/ml. The 4 pGMLV-p53 plasmids all produced strong p53 interference affects, and pGMLV- p53A1 with the strongest effect. The stably infected cells line WRD/p53- was established successfully using pGMLV- p53A1 plasmid. Conclusion The canine cell line WRD/p53-with stable lentivirus-mediated p53 silencing has been established successfully.