Method for preparing mutant code cDNA of apoptosis induction ligand related to human tumor necrosis factor, and application

Abstract

This invention provides the coding cDNA of recombinant human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mutant peptides. TRAIL mutant peptides are manufactured by: constructing the expression vector containing the coding cDNA, and transferring into host cells (especially prokaryotic cells) to express TRAIL mutant peptides. The method has higher expression efficiency, increased soluble expression proportion, and higher activity of TRAIL mutant peptides than the present technique. This invention overcomes the disadvantages of low yield, high cost and low product activity faced by the present technique, and can largely reduce the utilization cost of TRAIL mutant peptides. This invention realizes the industrial production of TRAIL mutant peptides, and accelerates the application of TRAIL mutant peptides in basic research and medicine manufacture.

Description

technical field [0001] The present invention mainly relates to the field of genetic engineering, in particular to a new cDNA encoding human tumor necrosis factor-related apoptosis-inducing ligand mutant and its preparation method and application. Background technique [0002] Human tumor necrosis factor-related apoptosis-inducing ligand (Tumor necrosis factor-relatedapoptosis-inducing Ligand, TRAIL) is a cytokine that belongs to the tumor necrosis factor family member, and its cDNA was first cloned by Wiley et al. in 1995 [1] . [0003] TRAIL cDNA encodes a precursor protein with a full length of 281 amino acids, of which amino acids 114-281 are soluble fragments. TRAIL protein has the structural characteristics of a type II transmembrane protein. The N-terminal of the molecule is located inside the cell membrane without a signal peptide sequence. The outside of the membrane is divided into a C segment with a hydrophilic end. [2] . [0004] Studies have shown that the sol...

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Problems solved by technology

[0006] However, the coding cDNA of the human TRAIL deletion mutant disclosed in the above invention still has major deficiencies, such as: (1) the coding gene of the human TRAIL mutant is a mammalian cell preference codon, and the expression efficiency in prokaryotic cells is low, and the expression The protein accounts for about 21.15% of the total bacterial protein; (2) Most of the expression products of the recombinant expression vector exist in the form of inclusion bodies, and the proportion of soluble expression products is low, and the yield is small; (3) TRAIL mutant proteins need to be denatured by inclusion bodies , Refolding method to purify the target protein, the inclusion body purification method is not only cumbersome to operate, the recovery rate of the target protein is low, but also cannot directly obtain the target protein with the correct conformation, the target protein is difficult to refold, and the biological activity is low [8]

These deficiencies make the yield of the TRAIL mutant low, the production process complicated, and the cost too high, which greatly affects its further application

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