Establishment and Application of Dual Fluorescent Labeling Multi-functional Autophagy Flux Monitoring System Based on Lentiviral System

doi:10.13523/j.cb.20190510

China Biotechnology

2019, Vol. 39

Issue (5): 88-95 DOI: 10.13523/j.cb.20190510

Establishment and Application of Dual Fluorescent Labeling Multi-functional Autophagy Flux Monitoring System Based on Lentiviral System

Zhan-bing MA^1,²,Jie DANG^1,²,Ji-hui YANG³,Zheng-hao HUO^1,^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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Abstract

Objective: To construct a red fluorescent protein-green fluorescent protein-murine LC3 fusion multi-lentiviral expression vector (PCDH-Duo-mRFP-eGFP_ph-LC3_rat, 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-eGFP_ph-LC3_rat 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

Received: 08 November 2018 Published: 04 June 2019

ZTFLH:

Q819

Corresponding Authors: Zheng-hao HUO E-mail: huozhh@163.com;xuguangxian@nxmu.edu.cn

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	Zhan-bing MA
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Cite this article:

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.

URL:

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

Table 1 Sequencing primer for full-length gene

Fig.1 Recombinant vector map and protein homology modeling

Fig.2 Full-length fusion gene synthesis via PAS (a),colony PCR identification (b),restriction endonuclease analysis (c) and sequencing verification (d)

Fig.3 The fluorescence results of lentivirus packaging in 293T after 48h

Fig.4 The results of Raw264.7 infected with PCDH-Duo-Lv at 72h

Fig.5 Flow cytometry of dual fluorescently labeled Raw264.7 cells

Fig.6 Detection EGFP through Western blot

Fig.7 Autophagy flux detection in CQ inhibition of Raw264.7-PCDH-Duo stable cell model Mock: Raw264.7-PCDH-Duo (a) P<0.01 (b) P<0.05


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