无义突变型荧光素酶稳定表达细胞株的建立

发布时间：2018-01-18 11:18

  本文关键词：无义突变型荧光素酶稳定表达细胞株的建立　出处：《药物分析杂志》2017年04期 　论文类型：期刊论文

  更多相关文章： 基因治疗药物筛选 无义突变通读剂 提前终止密码子 荧光素酶 蛋白质截短试验 双荧光素酶报告基因检测方法

【摘要】：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。
[Abstract]:Objective: to establish a cell line expressing luciferase with nonsense mutation site and to screen a new reading agent. Methods: plasmids p GL4-WT and p GL4-MUT were digested by enzyme. Wild type and nonsense mutant luciferase encoding c DNAs were obtained and inserted into the polyclonal site of lentivirus vector p LVX-IRES-Neo respectively. After restriction endonuclease digestion and PCR identification. The recombinant vector was packaged into lentivirus particles and infected with HEK 293 cells. Stable cell lines were obtained by monoclonal cell resistance screening and total RNA was extracted. The expression of luciferase m RNA was confirmed by reverse transcription PCR. Finally, the cells were treated with G418. The expression of luciferase protein was detected by Western blot before and after treatment. Results: 2.7 kb long wild-type and nonsense mutant luciferase encoding c DNA was obtained by enzyme digestion. The recombinant lentivirus vector pLVX-WTP LVX-MUTEcoR 鈪，

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