无义突变型荧光素酶稳定表达细胞株的建立

目的：建立稳定表达含无义突变位点荧光素酶的细胞株，用以筛选新的无义突变通读剂。方法：酶切质粒pGL4-WT和pGL4-MUT，得到野生型和无义突变荧光素酶编码cDNA，分别插入到慢病毒载体pLVX-IRES-Neo多克隆位点。酶切及PCR鉴定后，将重组载体包装成慢病毒颗粒，感染HEK 293细胞，单克隆细胞抗性筛选获得稳定细胞株，提取总RNA，经逆转录PCR方法验证荧光素酶mRNA表达。最后用已知阳性无义突变通读剂G 418处理细胞，Western blot方法检测处理前后荧光素酶蛋白表达水平，同时分析荧光素酶活性。结果：经酶切获得2.7 kb长野生型和无义突变荧光素酶编码cDNA，与pLVX-IRES-Neo连接获得重组慢病毒载体pLVX-WT、pLVX-MUT，EcoR Ⅰ单酶切、Nhe Ⅰ与BamH Ⅰ双酶切结果证明序列正确插入，PCR也扩增出目的片段；稳定感染慢病毒的HEK293WT和HEK293MUT细胞经逆转录PCR方法成功检测到荧光素酶mRNA表达；阳性通读剂G 418处理细胞后发现，HEK293WT细胞在处理前后均表达荧光素酶蛋白，荧光素酶活性也基本相同，而HEK293MUT细胞在处理前不表达荧光素酶蛋白，无荧光素酶活性，处理后恢复了部分荧光素酶蛋白表达，其荧光素酶活性相当于HEK293WT细胞的20%。结论：成功建立了稳定表达无义突变荧光素酶的细胞株，可用于筛选新的无义突变通读剂。

Objective: To construct cells stably expressing luciferase containing nonsense mutation for rapid screen of compounds which can induce readthrough of nonsense mutations. Methods: Plasmids pGL4-WT and pGL4-MUT were digested by endonuclease to give cDNA encoding wild type luciferase and nonsense mutant, which were then inserted into the multiple cloning site of lentiviral vector pLVX-IRES-Neo respectively. After endonuclease digestion and identification by PCR, the recombinant lentiviral vectors were packaged into lentiviral particles for the infection of HEK293 cells. Stably infected HEK293 cells were obtained by resistance screening monoclonal cells, total DNA extraction, and validation for expression of luciferase mRNA by reverse transcription PCR. Finally, stably infected HEK 293 cells were treated with G 418, a positive nonsense mutation readthrough agent, and the protein level and the activity of luciferase were detected using Western blot and chemiluminescence assay, respectively. Results: After digestion, 2.7 kb wild type luciferase cDNA and nonsense mutant were both obtained. The recombinant lentiviral vectors pLVX-WT and pLVX-MUT were successfully constructed, confirmed by the results of EcoR Ⅰ digestion, Nhe Ⅰ and BamH Ⅰ digestion, as well as PCR. The result of reverse transcription PCR of luciferase demonstrated that HEK 293 cells stably infected with pLVX-WT and pLVXMUT were successfully selected, which were named as HEK293WT and HEK293MUT respectively. It was found that HEK293WT cells stably expressed luciferase and showed the same luciferase activities treated with G 418 or not. HEK293MUT cells did not express luciferase or show any luciferase activity before G 418 treatment. After G418 treatment, HEK293MUT cells expressed luciferase whose activity is equal to 20% of that in HEK293WT. Conclusion: HEK293 cells stably expressing luciferase containing nonsense mutations were successfully established and may be helpful for screening new compounds which can induce readthrough of nonsense mutations.

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