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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2019, Vol. 39 Issue (5): 88-95    DOI: 10.13523/j.cb.20190510
研究报告     
基于慢病毒系统的双荧光标记多功能自噬流监测系统建立与应用 *
马占兵1,2,党洁1,2,杨继辉3,霍正浩1,2,**(),徐广贤4()
1 宁夏医科大学基础医学院医学遗传系与细胞生物学系 银川 750004
2 宁夏回族自治区生育力保持教育部重点实验室 银川 750004
3 宁夏医科大学科技中心 银川 750004
4 宁夏医科大学临床医学院 银川 750004
Establishment and Application of Dual Fluorescent Labeling Multi-functional Autophagy Flux Monitoring System Based on Lentiviral System
Zhan-bing MA1,2,Jie DANG1,2,Ji-hui YANG3,Zheng-hao HUO1,2,**(),Guang-xian XU4()
1 Department of Medical Genetics and Cell Biology, School of Basic Medical Sciences, Ningxia Medical University, Yinchuan 750004,China
2 Key Laboratory of Fertility Conservation of Ministry of Education, Ningxia Hui Autonomous Region, Yinchuan 750004,China
3 Science and Technology Center, Ningxia Medical University, Yinchuan 750004,China
4 Clinical College, Ningxia Medical University, Yinchuan 750004,China
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摘要：

目的:构建能够用于稳定动态监测细胞自噬流变化和过表达基因的红色荧光蛋白-绿色荧光蛋白-鼠源LC3融合慢病毒多功能表达载体(PCDH-Duo-mRFP-eGFPph-LC3rat,PCDH-Duo), 并构建小鼠腹腔巨噬细胞Raw264.7稳转株观察自噬流变化。方法:应用基于PCR精确合成mRFP-eGFPph-LC3rat融合全基因,将其克隆至慢病毒表达载体PCDH-CMV-MCS-EF1a-GFP中,重组质粒经菌落PCR、酶切及测序分析正确无误后, 包装慢病毒,转染Raw264.7细胞,并利用流式分选术获取稳转株,经氯喹抑制自噬模型及Western blot鉴定eGFP蛋白表达确认其可靠性。结果:成功构建了PCDH-Duo重组慢病毒质粒,包被慢病毒并获得Raw264.7稳定细胞系(Raw264.7-PCDH-Duo),可稳定表达双荧光蛋白,经3mmol/L氯喹作用6h后,能够稳定准确指示自噬流变化。结论:成功构建了基于慢病毒系统的双荧光标记多功能自噬流监测系统,为研究细胞自噬与编码基因及非编码基因之间的关系提供了方便有力的工具。
关键词： 自噬流;  载体构建;  慢病毒;  mRFP-eGFP-LC3    
Abstract:

Objective: To construct a red fluorescent protein-green fluorescent protein-murine LC3 fusion multi-lentiviral expression vector (PCDH-Duo-mRFP-eGFPph-LC3rat, PCDH-Duo),which can be used to stably monitor the changes of autophagy flux and overexpression genes. Changes in autophagic flow were observed in the mouse peritoneal macrophage Raw264.7 stable strain.Methods:The mRFP-eGFPph-LC3rat fusion gene was synthesized by PAS and cloned into the lentiviral expression vector PCDH-CMV-MCS-EF1a-copGFP. After the recombinant plasmid was correctly analyzed by PCR, enzyme digestion and sequencing, the lentivirus was packaged. Raw264.7 cells were transfected, and stable cells were obtained by FACS. The reliability of the eGFP protein expression system was confirmed by CQ autophagy inhibition model and Western blot.Results:The recombinant plasmid of PCDH-Duo lentivirus was successfully constructed, which was coated with lentivirus and obtained stable cell line of Raw264.7. The expression of double fluorescent protein was stable. After induction by 3mmol/L CQ for 6h, it was stable and accurate. The phasing changes.Conclusion:The dual-fluorescence multi-function autophagic flux monitoring system based on lentivirus system was successfully constructed, which provides a convenient and powerful tool for studying the relationship between autophagy and coding genes and non-coding genes.
Key words: Autophagic flux    Vector construction    Lentivirus    mRFP-eGFP-LC3
收稿日期: 2018-11-08 出版日期: 2019-06-04
ZTFLH:  Q819  
基金资助: * 宁夏回族自治区自然科学基金面上项目(2018AAC03088);宁夏科技创新领军人才项目资助项目(KJT2015020)
通讯作者: 霍正浩     E-mail: huozhh@163.com;xuguangxian@nxmu.edu.cn
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马占兵
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徐广贤

引用本文:

马占兵,党洁,杨继辉,霍正浩,徐广贤. 基于慢病毒系统的双荧光标记多功能自噬流监测系统建立与应用 *[J]. 中国生物工程杂志, 2019, 39(5): 88-95.

Zhan-bing MA,Jie DANG,Ji-hui YANG,Zheng-hao HUO,Guang-xian XU. Establishment and Application of Dual Fluorescent Labeling Multi-functional Autophagy Flux Monitoring System Based on Lentiviral System. China Biotechnology, 2019, 39(5): 88-95.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20190510    或    https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I5/88

Primer
ID		 Name Sequence (5'-3')
P1 Cmv-F CGCAAATGGGCGGTAGGCGTG
P2 mRFP-EGFP-LC3-Seq1 GCCGTTACAGATCCAAGC
P3 mRFP-EGFP-LC3-Seq2 ACGCTGAGGTCAAGACCA
P4 mRFP-EGFP-LC3-V-seqR TGAAAGCCATACGGGAAG
表1  载体全长基因测序引物
图1  重组载体图谱及蛋白质同源建模
图2  全长融合基因PAS合成(a)、菌落PCR鉴定(b)、限制性内切酶分析(c)及测序验证(d)
图3  293T慢病毒包装48h荧光结果
图4  重组PCDH-Duo慢病毒(PCDH-Duo-Lv)感染Raw264.7细胞72h结果(MOI=10)
图5  流式分选双荧光标记的Raw264.7细胞
图6  EGFP 蛋白表达检测(Mock:Raw264.7)
图7  CQ抑制Raw264.7-PCDH-Duo稳转株自噬模型自噬潮检测
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