Plasmid vector for building overexpression stable system on basis of transposase and application thereof

Abstract

The invention discloses a series of plasmid vector for building an overexpression stable system on the basis of transposase and application thereof. The plasmid vector is a double-chain annular plasmid containing an IRDR-L-IRDR-R kit; the IRDR-L-IRDR-R kit comprises an IRDR-L sequence, a promotor, a tag sequence, an attR1 sequence, a reverse selective tag gene sequence ccdB, an attR2sequence, a screening gene sequence and an IRDR-R sequence; the tag is an HA tag, an Myc tag, a Flag tag or a V5 tag. The plasmid vector has a Sleeping Beauty transposon tail end reverse repeated sequence and a Gateway Cassette sequence; the overexpression stable system can be fast and efficiently built on the basis of the transposase method; various different tag sequences Tag and screening genes GFP exist, so that the conventional requirement of expressing the target gene in eukaryotic cells can be met.

Description

Technical field [0001] The present invention belongs to the field of genetic engineering. Specifically, the present invention relates to the construction of plasmid vectors using Gateway cloning technology. The series of plasmid vectors carry different tag sequences and the screening gene GFP, and integrate the target gene into the cell through the transposon system. Genome, realizing gene transfer. In particular, it relates to a plasmid vector for establishing an overexpression stable line based on transposase and its application Background technique [0002] Overexpression stable cell line refers to the integration of foreign DNA into the host cell genome, allowing the host cell to express the target gene for a long time. Compared with the transient strain, the stable cell line can greatly facilitate experimental research and reduce experimental costs. Therefore, the construction of stable gene overexpression strains is widely used in the study of gene function and mechanism. ...

AI Technical Summary

Problems solved by technology

At present, the construction of stable cell lines is mainly achieved through virus packaging and infection of target cells. This method takes a long time, usually about two weeks

In addition, since the size of the inserted target gene fragment is limited by the size of the viral plasmid itself, the size of the target gene fragment usually cannot exceed 3Kb, and the operator has potential safety hazards of virus infection

At the same time, overexpression plasmid vectors are usually constructed by inserting into multiple cloning sites. This method is not only inefficient, but also takes a long time, which greatly affects the experimental progress of researchers.

Images