RHBDF2-shRNA慢病毒载体的构建和Neuro-2a-RHBDF2-shRNA稳转细胞系的建立

doi:10.13481/j.1671-587x.20190606

吉林大学学报(医学版) ›› 2019, Vol. 45 ›› Issue (06): 1224-1230.doi: 10.13481/j.1671-587x.20190606

RHBDF2-shRNA慢病毒载体的构建和Neuro-2a-RHBDF2-shRNA稳转细胞系的建立

李胜男¹, 陈吴海², 陈少凤³, 邓福³, 朱佩仪³, 李友¹

1. 广东省衰老相关心脑疾病重点实验室, 广东湛江 524001;
2. 中国热带农业科学院农产品加工研究所, 广东湛江 524001;
3. 广东医科大学附属医院神经病学研究所, 广东湛江 524001

收稿日期:2019-03-29 出版日期:2019-12-05 发布日期:2019-12-05
通讯作者: 李友,副研究员,硕士研究生导师(Tel:0759-2386772,E-mail:youli805@163.com) E-mail:youli805@163.com
作者简介:李胜男(1992-),女,安徽省淮北市人,研究实习员,理学硕士,主要从事神经退行性疾病发病机制方面的研究。
基金资助:
国家自然科学基金资助课题（81571157）

Construction of RHBDF2-shRNA lentiviral vector and establishment of stably transfected Neuro-2a-RHBDF2-shRNAcell line

LI Shengnan¹, CHEN Wuhai², CHEN Shaofeng³, DENG Fu³, ZHU Peiyi³, LI You¹

1. Key Laboratory of Age-Related Cardiac and Cerebral Diseases, Guangdong Province, Zhanjiang 524001, China;
2. Agricultural Products Processing Research Institute, Chinese Academy of Tropical Agricultural Sciences, Zhanjiang 524001, China;
3. Institute of Neurology, Affiliated Hospital, Guangdong Medical University, Zhanjiang 524001, China

Received:2019-03-29 Online:2019-12-05 Published:2019-12-05

摘要/Abstract

摘要： 目的：构建RHBDF2-shRNA慢病毒载体，建立Neuro-2a-RHBDF2-shRNA稳转细胞系，并检测稳转细胞株中RHBDF2 mRNA和蛋白表达水平。方法：根据NCBI提供的RHBDF2基因序列，设计并合成针对RHBDF2基因干扰靶点的shRNA-oligo，退火后将其连接到经EcoRⅠ和AgeⅠ酶切的慢病毒载体FV067-RNAi-EGFP-Puro中，构建FV067-RHBDF2-shRNA慢病毒质粒，通过PCR筛选阳性克隆并测序鉴定。使用HEK293T细胞和Neuro-2a细胞作为实验材料，FV067 Vector作为对照组，FV067-RHBDF2-shRNA作为实验组。通过Lipofectamine 2000将FV067 Vector和FV067-RHBDF2-shRNA慢病毒质粒分别与慢病毒辅助质粒psPAX2和pMD2G共转染至HEK293T细胞中，转染48 h收集并纯化慢病毒，分别将对照组FV067 Vector和实验组FV067-RHBDF2-shRNA慢病毒在感染复数（MOI）为50时感染Neuro-2a细胞，荧光显微镜下观察细胞中绿色荧光蛋白的表达，采用2 mg·L^-1嘌呤霉素筛选出RHBDF2基因稳定沉默的细胞系。实时荧光定量PCR法检测2组细胞中RHBDF2 mRNA表达水平，免疫印迹法检测2组细胞中RHBDF2蛋白表达水平。结果：PCR和测序，RHBDF2-shRNA慢病毒载体中的干扰序列与设计合成的RHBDF2-shRNA-oligo序列完全一致。FV067 Vector慢病毒的滴度为5×10⁸ TU·mL^-1，FV067-RHBDF2-shRNA慢病毒的滴度为3×10⁸ TU·mL^-1。实时荧光定量PCR检测，实验组稳转细胞株中RHBDF2 mRNA表达水平比对照组降低了52.26%（t=11.44，P=0.0076）。免疫印迹法检测，与对照组比较，实验组RHBDF2-shRNA稳转细胞系中RHBDF2蛋白表达水平降低了47%（t=6.374，P=0.0078）。结论：成功构建了FV067-RHBDF2-shRNA慢病毒载体并建立了Neuro-2a-RHBDF2-shRNA稳转细胞系，且Neuro-2a-RHBDF2-shRNA稳转细胞系中RHBDF2mRNA和蛋白表达水平明显降低。

关键词: RHBDF2, RNA干扰, 慢病毒, HEK 293T细胞Neuro-2a细胞, 稳转细胞系

Abstract: Objective: To construct the RHBDF2-shRNA lentiviral vector and to establish a stably transfected Neuro-2a-RHBDF2-shRNA cell line,and to detect the expression levels of RHBDF2 mRNA and protein in the stably transfected cell line. Methods: According to the RHBDF2 sequence in NCBI,a shRNA-oligo targeting RHBDF2 gene was designed and synthesized.It was cloned into the lentiviral vector FV067-RNAi-EGFP-Puro digested by EcoRⅠand AgeⅠrestriction endonuclease to construct the FV067-RHBDF2-shRNA lentiviral plasmid.The positive clones were screen by PCR and verified by sequencing.The HEK293T cells and Neuro-2a cells were used as the experimental materials.The Neuro-2a-FV067 Vector was used as control group,and the Neuro-2a-FV067-RHBDF2-shRNA was used as experiment group.The FV067 Vector and FV067-RHBDF2-shRNA plasmids were co-transfected into the HEK293T cells with lentiviral-assisted plasmids psPAX2 and pMD2G using Lipofectamine 2000,respectively.The lentivirus was harvested and purified 48 h after transfection.The FV067 Vector lentivirus in control group and the FV067-RHBDF2-shRNA lentivirus in experiment group were used to infect the Neuro-2a cells when the multiplicity of infection(MOI) was 50,respectively,and the expression of green fluorescence was detected under fluorescence microscope.Then 2 mg·L^-1 puromycin was used to screen the cell line stably silencing RHBDF2.Real-time fluorescence quantitative PCR was used to detect the expression levels of RHBDF2 mRNA in the cells in two groups and Western blotting method was used to detect the expression levels of RHBDF2 protein in the cells in two groups. Results: The PCR and the sequencing analysis results indicated that the interference sequence of RHBDF2-shRNA lentiviral vector was identical with the sequence of designed and synthesized RHBDF2-shRNA-oligo.The titer of FV067 Vector lentivirus was 5×10⁸ TU·mL^-1 and the titer of FV067-RHBDF2-shRNA lentivirus was 3×10⁸ TU·mL^-1.The real-time fluorescence quantitative PCR results revealed that the expression level of RHBDF2 mRNA in the stably transfencted cell line in experiment group was significantly decreased by 52.26% compared with control group(t=11.44,P=0.007 6).The Western blotting results indicated that the expression level of RHBDF2 protein in the stably transfected cell line was significantly decreased by 47% compared with control group(t=6.374,P=0.007 8). Conclusion: The FV067-RHBDF2-shRNA lentiviral vector is constructed successfully and the Neuro-2a-RHBDF2-shRNA cell line is established,and the expression levels of RHBDF2 mRNA and protein in the Neuro-2a-RHBDF2-shRNA stably transfected cell line is decreased significantly.

Key words: RHBDF2, RNA interference, lentivirus, Neuro-2a cells, stable cell line

中图分类号:

Q343.37

李胜男, 陈吴海, 陈少凤, 邓福, 朱佩仪, 李友. RHBDF2-shRNA慢病毒载体的构建和Neuro-2a-RHBDF2-shRNA稳转细胞系的建立[J]. 吉林大学学报(医学版), 2019, 45(06): 1224-1230.

LI Shengnan, CHEN Wuhai, CHEN Shaofeng, DENG Fu, ZHU Peiyi, LI You. Construction of RHBDF2-shRNA lentiviral vector and establishment of stably transfected Neuro-2a-RHBDF2-shRNAcell line[J]. Journal of Jilin University(Medicine Edition), 2019, 45(06): 1224-1230.

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RHBDF2-shRNA慢病毒载体的构建和Neuro-2a-RHBDF2-shRNA稳转细胞系的建立

Construction of RHBDF2-shRNA lentiviral vector and establishment of stably transfected Neuro-2a-RHBDF2-shRNAcell line

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