Slow virus green fluorescence autophagy report vector and building method thereof

Abstract

The invention belongs to the field of cell research, and relates to a lentiviral green fluorescent autophagy reporter carrier based on a lentiviral carrier skeleton, which is used for screening stable strains and observing changes in autophagosomes in cells and a construction method. The invention discloses a lentiviral green fluorescent autophagy reporter carrier, which comprises ampicillin resistance, puromycin selection marker and green fluorescent autophagy marker protein LC3 gene sequence. The green fluorescent autophagy marker protein LC3 plasmid of the present invention uses lentivirus as the backbone, which is conducive to the establishment of stably expressing green fluorescent cell lines, and due to the wide applicability of lentivirus to mammalian cells, the green fluorescent plasmid of the present invention can be used for various cells. Compared with the expensive price and specificity of using common vectors, the cost is very low.

Description

technical field [0001] The invention belongs to the field of cell research, and relates to a lentiviral green fluorescent autophagy reporter carrier based on a lentiviral carrier skeleton, which is used for screening stable strains and observing changes in autophagosomes in cells and a construction method. Background technique [0002] Autophagy is a phenomenon of "self-eating" in cells, and apoptosis is a phenomenon of "suicide". The two share the same stimulating factors and regulatory proteins, but the induction thresholds and thresholds are different. How to switch and coordinate is still unclear . Autophagy refers to the membrane wrapping part of the cytoplasm and the organelles and proteins that need to be degraded in the cell to form autophagosomes, and finally fuse with lysosomes to form autophagy lysosomes, which degrade the contents wrapped by them, so as to achieve cell homeostasis and Cellular renewal. [0003] Since the autophagy process is very fast, dynamic ...

AI Technical Summary

Problems solved by technology

[0004] At present, there are some commercial green fluorescent vectors for autophagy detection, but there are some limitations: 1. The vector backbone is a common plasmid vector, and it is difficult to obtain stable expression cell lines; 2. The price is expensive and the use is limited.

The price ranges from 5,000 yuan to tens of thousands of yuan; 3. The carrier plasmid containing green fluorescent protein cannot specifically observe the changes of autophagosomes

4. Some vectors contain G418 screening gene, the screening cycle is long and the stability is not high

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