Mutant type TNF-alpha gene and method of increasing the expression quantity of the antigen TNF-alpha gene

Abstract

The invention discloses a mutant type TNF-alpha gene the base composition of which is shown as the SEQ ID NO 7, and discloses a method of increasing the expression quantity of the antigen TNF-alpha gene. The method includes steps of: amplifying by adopting a human secreting type TNF-alpha gene cDNA clone as a template and by adopting the SEQ ID NO 1 and the SEQ ID NO 2 as primers, so as to obtain an amplification product T1; amplifying by adopting the SEQ ID NO 3 and the SEQ ID NO 4 as primers, so as to obtain an amplification product T2; performing overlapping PCR amplification by adopting the amplification product T1 and the amplification product T2 as templates and adopting the SEQ ID NO 1 and the SEQ ID NO 4 as primers, so as to obtain the mutant type TNF-alpha gene; subjecting the mutant type TNF-alpha gene to double digestion; inserting into a pET28a expression vector; converting escherichia coli; and performing induced culture to obtain TNF-alpha protein. Two pairs of escherichia coli rare codons close with each other in the TNF-alpha gene mutant into escherichia coli optimal codons. The expression quantity of the TNF-alpha gene after the codon optimization is increased by at least 1.5 times.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a mutant TNF-α gene and a method for increasing the expression level of the antigen TNF-α gene. Background technique [0002] Tumor necrosis factor α (Tumor necrosis factor α, TNF-α) is a class of cytokines with a wide range of biological activities, mainly produced by monocytes, macrophages, adipocytes, B cells, T cells and natural killer (NK) Cells and other immune cells are produced. TNF-α proteins in vivo include transmembrane TNF-α and secreted TNF-α. Transmembrane TNF-α has a full length of 233 amino acids, of which amino acids 1-76 are signal peptides (including transmembrane amino acids), and amino acids 77-233 are extracellular segments. The 77th-233rd amino acid sequence is also the amino acid sequence of secreted TNF-α, which has a size of 17KD and binds to the TNF receptor (TNFR) on the cell surface in the form of a trimer to mediate various biological effec...

AI Technical Summary

Problems solved by technology

The current commercialized protein TNF-α is very expensive, the price exceeds 70 yuan / μg, and the efficiency of TNF-α production in the laboratory with E. coli expression system, yeast expression system and mammalian cell expression system is not high, and innovation is urgently needed Thinking improves the expression of human TNF-α gene

Images