| 易国辉,姚孟霞,张菁芸,陈锦萍,何小稳,彭江龙.食品级乳酸乳球菌pNZ8149-luc质粒的构建及表达[J].,2019,19(9):1649-1652 |
| 食品级乳酸乳球菌pNZ8149-luc质粒的构建及表达 |
| Construction and Expression of a Plasmid pNZ8149-luc in Food-Grade Lactococcus Lactis |
| 投稿时间：2018-11-05 修订日期：2018-11-26 |
| DOI：10.13241/j.cnki.pmb.2019.09.010 |
| 中文关键词: 乳酸乳球菌 食品级 荧光素酶 基因表达 |
| 英文关键词: Lactococcus lactis Food-grade Luciferase Gene expression |
| 基金项目:国家自然科学基金项目（81360456） |
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| 中文摘要: |
| 摘要 目的：构建能够稳定表达萤火虫荧光素酶报告基因（luc）的乳酸乳球菌(Lactococcus lactis, L.lactis)食品级表达系统，以便后续研究对目的基因进行示踪。方法：从pGL4.10质粒中PCR扩增萤火虫荧光素酶报告基因，测序，克隆至载体pNZ8149，构建pNZ8149-luc表达质粒；电击转化宿主乳酸乳球菌NZ3900，采用乳糖筛选法获得重组的乳酸乳球菌，Nisin诱导，采用微孔板发光检测仪检测荧光素酶的存在，Western Blot检测目标蛋白luc的表达。结果：PCR扩增的荧光素酶报告基因成功克隆至pNZ8149质粒，并电击转化宿主乳酸乳球菌NZ3900，得到乳酸乳球菌表达系统NZ3900/pNZ8149-luc。Nisin诱导后，检测到荧光素酶随诱导时间的延长活性逐渐增强，时间超过24 h之后荧光素酶活性逐渐下降。Western Blot检测到目标蛋白luc在胞内表达。结论：成功构建了pNZ8149-luc表达载体，并能够在乳酸乳球菌体内稳定表达。 |
| 英文摘要: |
| ABSTRACT Objective: A food-grade expression system for Lactococcus lactis was constructed to stably express the firefly lu- ciferase reporter gene for subsequent study of the target gene. Methods: The luciferase reporter gene was amplified by PCR from pGL4.10 plasmid, sequenced and cloned into vector pNZ8149. The expression plasmid pNZ8149-luc was constructed, which was transformed into the host L.lactis NZ3900 by electric shock. Recombinant L.lactis was obtained by lactose screening and induced through using Nisin. The presence of luciferase was detected by microplate fluorescence detector and the expression of Luc was detected by Western Blot. Results: The luciferase reporter gene was successfully cloned into the pNZ8149 plasmid that was transformed into the host L.lactis NZ3900 to obtain the L.lactis expression system NZ3900/pNZ8149-luc. After induction by Nisin, the activity of luciferase increased gradually with the prolongation of induction time, and decreased gradually after 24 hours. Western Blot detected intracellular expression of the tar- get protein luc. Conclusion: The pNZ8149-luc expression vector has been successfully constructed and can be expressed stably in L. lactis. |
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