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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p GL4-WT和p GL4-MUT,得到野生型和无义突变荧光素酶编码c DNA,分别插入到慢病毒载体p LVX-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m RNA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c DNA,与p LVX-IRES-Neo连接获得重组慢病毒载体p LVX-WT、p LVX-MUT,EcoRⅠ单酶切、NheⅠ与BamHⅠ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m RNA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 establish a cell line stably expressing luciferase containing nonsense mutation site to screen for a new nonsense mutation. Methods: Plasmids pGL4-WT and pGL4-MUT were digested to obtain wild-type and non-sense mutant luciferase-encoding cDNA and inserted into the lentiviral vector p LVX-IRES-Neo multi-cloning site respectively. After digestion with restriction endonucleases and PCR, the recombinant plasmids were packaged into lentivirus particles and infected with HEK 293 cells. The stable cell lines were screened by monoclonal cell resistance and the total RNA was extracted. The expression of luciferase mRNA was verified by reverse transcription PCR. Finally, the cells were treated with G 418, a known positive nonsense mutation, and luciferase expression was detected by Western blot before and after treatment. The luciferase activity was also analyzed. Results: The 2.7 kb wild-type and nonsense mutant luciferase-encoding c DNA was obtained by restriction enzyme digestion and ligated with p LVX-IRES-Neo to obtain recombinant lentiviral vector p LVX-WT, p LVX-MUT and EcoR I digestion, Nhe Ⅰ and BamH Ⅰ double digestion results showed that the correct insertion of the sequence, PCR also amplified the target fragment; stably infected with lentivirus HEK293WT and HEK293MUT cells by reverse transcription PCR method to detect luciferase m RNA expression; positive reading G 418 treatment After HEK293WT cells were treated with HEK293WT cells, luciferase protein was expressed both before and after treatment, and luciferase activity was also basically the same. However, HEK293MUT cells did not express luciferase protein and did not have luciferase activity before treatment, and partially fluorescein Enzyme protein expression, whose luciferase activity is equivalent to 20% of HEK293WT cells. Conclusion: The cell line stably expressing nonsense mutant luciferase was successfully established and could be used to screen novel nonsense mutants.