發(fā)布時(shí)間：2018-01-18 11:18

  本文關(guān)鍵詞：無(wú)義突變型熒光素酶穩(wěn)定表達(dá)細(xì)胞株的建立　出處：《藥物分析雜志》2017年04期 　論文類型：期刊論文

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【摘要】：目的:建立穩(wěn)定表達(dá)含無(wú)義突變位點(diǎn)熒光素酶的細(xì)胞株,用以篩選新的無(wú)義突變通讀劑。方法:酶切質(zhì)粒p GL4-WT和p GL4-MUT,得到野生型和無(wú)義突變熒光素酶編碼c DNA,分別插入到慢病毒載體p LVX-IRES-Neo多克隆位點(diǎn)。酶切及PCR鑒定后,將重組載體包裝成慢病毒顆粒,感染HEK 293細(xì)胞,單克隆細(xì)胞抗性篩選獲得穩(wěn)定細(xì)胞株,提取總RNA,經(jīng)逆轉(zhuǎn)錄PCR方法驗(yàn)證熒光素酶m RNA表達(dá)。最后用已知陽(yáng)性無(wú)義突變通讀劑G 418處理細(xì)胞,Western blot方法檢測(cè)處理前后熒光素酶蛋白表達(dá)水平,同時(shí)分析熒光素酶活性。結(jié)果:經(jīng)酶切獲得2.7 kb長(zhǎng)野生型和無(wú)義突變熒光素酶編碼c DNA,與p LVX-IRES-Neo連接獲得重組慢病毒載體p LVX-WT、p LVX-MUT,EcoRⅠ單酶切、NheⅠ與BamHⅠ雙酶切結(jié)果證明序列正確插入,PCR也擴(kuò)增出目的片段;穩(wěn)定感染慢病毒的HEK293WT和HEK293MUT細(xì)胞經(jīng)逆轉(zhuǎn)錄PCR方法成功檢測(cè)到熒光素酶m RNA表達(dá);陽(yáng)性通讀劑G 418處理細(xì)胞后發(fā)現(xiàn),HEK293WT細(xì)胞在處理前后均表達(dá)熒光素酶蛋白,熒光素酶活性也基本相同,而HEK293MUT細(xì)胞在處理前不表達(dá)熒光素酶蛋白,無(wú)熒光素酶活性,處理后恢復(fù)了部分熒光素酶蛋白表達(dá),其熒光素酶活性相當(dāng)于HEK293WT細(xì)胞的20%。結(jié)論:成功建立了穩(wěn)定表達(dá)無(wú)義突變熒光素酶的細(xì)胞株,可用于篩選新的無(wú)義突變通讀劑。
[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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