MAVS-/-HeLa细胞系构建及其在干扰素信号通路中应用
Construction of MAVS-/-HeLa cell line and its application in the interferon signaling pathway
姬红 1董云霞 1那睿思 2李玉军 3程德春4
作者信息
- 1. 黑龙江省医学科学院,黑龙江哈尔滨 150081;哈尔滨医科大学寄生虫学教研室,黑龙江哈尔滨 150081
- 2. 哈尔滨医科大学寄生虫学教研室,黑龙江哈尔滨 150081
- 3. 深圳湾实验室,广东深圳 518000
- 4. 黑龙江省医学科学院,黑龙江哈尔滨 150081;哈尔滨医科大学寄生虫学教研室,黑龙江哈尔滨 150081;深圳湾实验室,广东深圳 518000
- 折叠
摘要
[目的]通过CRISPR/Cas9技术构建线粒体抗病毒信号(mitochondrial antiviral signaling,MA VS)基因敲除的HeLa细胞系,并对细胞系的生长特性、及其对干扰素信号通路的影响进行初步鉴定.[方法]针对MAVS外显子设计构建靶向MAVS基因的guide RNA,并克隆至LentiCRISPR v2载体后包装成慢病毒,感染HeLa细胞后经嘌呤霉素筛选获得MAVS基因敲除的HeLa细胞,Western Blotting验证敲除效果,MTT法检测细胞生长活力,应用聚肌胞苷酸(Poly(I∶C))刺激并检测细胞内干扰素-β(IFN-β)mRNA水平的变化.[结果]测序结果表明成功构建CRISPR-MAVS-gR质粒,包装成慢病毒并感染HeLa细胞,经筛选获得的细胞内MAVS mRNA及蛋白均不表达,生长速度及活性与野生型HeLa相同,Poly(I∶C)刺激后,细胞内IFN-β变化不明显.[结论]通过CRISPR/Cas9技术获得MAVS基因敲除的MAVS-/-HeLa细胞系,Poly(I∶C)诱导剂不能在该细胞内激活干扰素信号通路,为后续研究MAVS相关的抗病毒天然免疫信号通路奠定了基础.
Abstract
[Objective]To construct a HeLa cell line with mitochondrial antiviral signaling(MAVS)gene knockout using CRISPR/Cas9 technology,and to preliminarily identify the growth characteristics of the cell line and its impact on the interferon signaling path-way.[Method]Guide RNA targeting the exons of the MAVS gene was designed and cloned into the LentiCRISPR v2 vector,and then packaged into lentiviruses.HeLa cells were infected with the lentiviruses and selected with puromycin to obtain HeLa cells with MAVS gene knockout.The knockout efficiency was validated using Western Blotting.Cell growth vitality was measured using the MTT assay.The changes of interferon-beta(IFN-β)mRNA levels in cells stimulated with poly(I∶C)were detected by RT-qPCR.[Result]Sequencing results indicated successful construction of the CRISPR-MAVS-gR plasmid,which was packaged into lentivirus and used to infect HeLa cells.Cells that were selected did not express MAVS mRNA and protein.The growth rate and activity of these cells were similar to wild-type HeLa cells.After stimulation with Poly(I∶C),there was no significant change in IFN-β levels within the cells.[Conclusion]The MAVS gene knockout MAVS-/-HeLa cell line was successfully obtained using CRISPR/Cas9 technology.The Poly(I∶C)agonists failed to activate the interferon signaling pathway within these cells.This study lays the foundation for further research on the MAVS-related antiviral innate immune signaling pathway.
关键词
线粒体抗病毒信号蛋白/CRISPR/Cas9/HeLa/基因敲除/天然免疫/干扰素/嘌呤霉素/聚肌胞苷酸Key words
mitochondrial antiviral signaling(MAVS)/CRISPR/Cas9/HeLa/gene knockout/innate immunity/interferon/puromy-cin/poly(I∶C)引用本文复制引用
基金项目
黑龙江省自然科学基金(LH2019C086)
出版年
2024
NETL
NSTL
万方数据