Construction and application of mammal cell high-efficiency expression vector

Abstract

The invention relates to a mammal cell high-efficiency expression vector pSNEO. The vector is constructed on the basis of neomycin resistance screening gene NEO by combined strategy of weakening screening gene expression and reinforcing target gene expression. The screening gene weakening comprises the following two sides: introducing deletion mutant into the promoter to implement transcription weakening of the screening gene, and introducing a hairpin structure sequence before the translation initial site of the screening gene to implement the translation weakening of the screening gene. The expression reinforcement of the target gene is implemented by adding a transcription control element WPRE sequence between polyclone site and polyA tail of the target gene expression frame. The pSNEO vector can conveniently and quickly complete the construction of the stable high-expression cell strain of the target gene, and provides a new tool for preparing the high-polymer recombinant protein.

Description

technical field [0001] The invention relates to biopharmaceutical gene engineering expression technology, in particular to the construction and application of a mammalian cell high-efficiency expression vector. Background technique [0002] The acquisition of high-efficiency mammalian cell expression system and high-expression engineered cell line is the bottleneck in the research and production of genetically engineered drugs, and expression vector and host cell are the two basic components of mammalian cell expression system. The expression systems currently used to prepare genetically engineered drugs include prokaryotic expression systems with Escherichia coli as hosts and eukaryotic expression systems with yeast, insect cells, plant cells and mammalian cells as hosts. [0003] The CHO expression system is one of the most widely used eukaryotic expression systems and one of the few animal cells that can be cultured in high-density suspension. The exogenous gene product ...

AI Technical Summary

Problems solved by technology

Studies have shown that: in CHO cells, the transcriptional activity of the CMV promoter is about 10 times and 30 times that of the SV40 promoter and the LTR (Rous sarcoma virus gene) promoter, respectively. In previous studies, we passed a relatively strong promoter A promoter such as CMV promoter to drive the expression of the target gene, and a weaker promoter such as SV40 promoter to express the screening gene, but there are still many clones formed in the end, and high-expression cell lines are selected from thousands of transfectants is still difficult and often takes a long time

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