Mammal cell expression promoter and manufacturing and use method thereof

Abstract

The invention discloses a mammal cell expression promoter and a manufacturing and use method thereof. The method comprises the following steps of 1, synthesizing a primer in a chemical synthesis mode; 2, obtaining a promoter sequence through a PCR (polymerase chain reaction); 3, through restriction enzyme cutting sites BglII and Kpn1, inserting the promoter sequence into a plasmid framework containing ori, Kana resistance genes, sv40, sv40-polyA and multi-enzyme cutting site sequences to obtain pHep / L1 plasmids; 4, transfecting E.Coli for plasmid amplification; extracting the plasmids; performing sequencing; 5, inserting the newly obtained plasmids into SEAP and IL10 target genes; extracting pure plasmids by a CsCL density gradient centrifugation method. The expression efficiency of the existing plasmids and AAV viruses is improved.

Description

technical field [0001] The present invention relates to a mammalian cell expression promoter, in particular to a mammalian cell expression promoter and its production and use method. Background technique [0002] Mammalian promoter sequences have important applications in plasmids and viral vectors, and are widely used in gene expression in life sciences, medicine and other fields. It has important applications in protein expression, plasmid construction, virus construction, target gene expression and delivery, gene therapy and other fields. The gene expression vectors commonly used at present include plasmid vectors and viral vectors, and most of the promoter sequences used are viral promoters, such as CMV. short. In response to these problems, scientists have developed some mammalian-derived promoters, such as the human serum albumin Human Serum Albumin promoter sequence, these eukaryotic animal-derived promoter sequences significantly prolong the exogenous vector in cel...

AI Technical Summary

Problems solved by technology

The gene expression vectors commonly used at present include plasmid vectors and viral vectors. Most of the promoter sequences used are viral promoters, such as CMV. short

In response to these problems, scientists have developed some mammalian-derived promoters, such as the human serum albumin Human Serum Albumin promoter sequence, these eukaryotic animal-derived promoter sequences significantly prolong the exogenous vector in cells and animals. expression time, but they also have two disadvantages. First, the expression level is significantly lower than that of viral promoter vectors. Second, this type of promoter sequence is directly copied from mammalian cells, which is generally longer and is used in some There will be limitations in viral vectors, for example, AAV vectors can only accommodate foreign DNA fragments of up to 4.7KB, and the excessively long promoter sequence limits the length and size of the target gene, reducing the application breadth of AAV vectors

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