PERK干扰慢病毒载体的构建及其在人牙髓细胞中的表达

doi:10.19439/j.sjos.2017.04.003

上海口腔医学 ›› 2017, Vol. 26 ›› Issue (4): 363-367.doi: 10.19439/j.sjos.2017.04.003

PERK干扰慢病毒载体的构建及其在人牙髓细胞中的表达

文扬, 朱亚琴

上海交通大学医学院附属第九人民医院·口腔医学院口腔综合科,上海市口腔医学重点实验室, 上海市口腔医学研究所,国家口腔疾病临床研究中心,上海 200011

收稿日期:2016-12-22 修回日期:2017-04-10 出版日期:2017-08-25 发布日期:2017-09-01
通讯作者: 朱亚琴, E-mail: zyq1590@163.com
作者简介:文扬（1992-）,女,硕士,E-mail：wenyang_480@163.com
基金资助:
国家自然科学基金（81271134,81300867）; 高等学校博士学位点专项科研基金（博导类,20130073110013）; 上海高校高峰高原学科建设项目

Construction of a lentiviral vector of RNA interference of PERK gene and identification in human dental pulp cells

WEN Yang, ZHU Ya-qin

Department of General Dentistry, Shanghai Ninth People's Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine; Shanghai Key Laboratory of Stomatology and Shanghai Research Institute of Stomatology; National Clinical Research Center of Stomatology. Shanghai 200011, China

Received:2016-12-22 Revised:2017-04-10 Online:2017-08-25 Published:2017-09-01

摘要/Abstract

摘要： 目的构建人PERK基因的shRNA慢病毒载体,检测其对人牙髓细胞（dental pulp cells,DPCs）PERK基因表达的抑制作用。方法针对人PERK基因的cDNA序列,设计并合成针对人PERK基因的shRNA表达序列,将其连接到载体hU6-MCS-CMV-EGFP中。测序正确后,将构建的目的载体和包装质粒共转染293T细胞,72 h后收获并浓缩得到重组慢病毒颗粒。筛选最佳感染复数（multiplicity of infection, MOI）,将病毒感染人牙髓细胞,通过实时荧光定量PCR（quantitative real-time PCR,RT-PCR）和蛋白质免疫印迹（Western blot）技术检测PERK基因mRNA和蛋白的表达水平,验证干扰效果。采用SPSS24.0软件包对数据进行统计学分析。结果成功构建LV-PERK-RNAi慢病毒载体,病毒滴度为3×108 TU/mL,最适MOI=30;RT-PCR和Western印迹法检测显示,与空白对照组相比,PERK基因的mRNA和蛋白质表达水平显著下降,在mRNA水平的表达抑制率约为63%。结论成功构建PERK干扰慢病毒表达载体,为后续的研究创造了条件。

关键词: PERK, RNAi, 慢病毒载体

Abstract: To construct an expression vector of a small hairpin RNA (shRNA) targeting human PERK gene and to observe gene-silencing effects of PERK in human dental pulp cells (DPCs). METHODS: According to PERK gene cDNA sequence, shRNA was designed and synthesized, which was then annealed into hU6-MCS-CMV-EGFP vector. After identified by sequencing, hU6-MCS-CMV-EGFP vector and packaging vector were co-transfected into 293 T cells. 72 hours later, the recombinant lentiviruses were obtained after harvesting and concentrating. Then LV-PERK-RNAi vectors were transfected into DPCs at an appropriate multiplicity of infection. To verify the interference effect, real- time PCR and Western blot were used to detect expression levels of PERK mRNA and protein in the transfected DPCs. The data were analyzed with SPSS 24.0 software package. RESULTS: LV-PERK-RNAi vectors were successfully constructed with a high titer of 3×108 TU/mL. The results of RT-PCR and Western blot demonstrated that after infection with LV-PERK-RNAi vector at a multiplicity of infection of 30, the expression level of PERK gene in DPCs was significantly down-regulated compared with control group. At mRNA level, the interference rate was about 63%. CONCLUSIONS: An effective lentiviral shRNA expression vector targeting the PERK gene is successfully constructed and can be used for further study on the function of PERK gene.

Key words: PERK, RNA interference, Lentiviral vector

中图分类号:

R781.31

文扬, 朱亚琴. PERK干扰慢病毒载体的构建及其在人牙髓细胞中的表达[J]. 上海口腔医学, 2017, 26(4): 363-367.

WEN Yang, ZHU Ya-qin. Construction of a lentiviral vector of RNA interference of PERK gene and identification in human dental pulp cells[J]. Shanghai Journal of Stomatology, 2017, 26(4): 363-367.

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PERK干扰慢病毒载体的构建及其在人牙髓细胞中的表达

Construction of a lentiviral vector of RNA interference of PERK gene and identification in human dental pulp cells

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