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Method for producing human tumor necrosis factor-alpha

A tumor necrosis factor and alpha protein technology, applied in the field of efficient production of recombinant human tumor necrosis factor-alpha, can solve the problems of unsatisfactory clinical application effect, insignificant anti-tumor effect, and limited wide application.

Inactive Publication Date: 2009-04-01
SHANGHAI NEWSUMMIT BIOPHARMA
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Problems solved by technology

However, a prominent problem is that the half-life of TNF-α in the body is short, and the anti-tumor effect of low-dose injection is not significant
To achieve the level of curative effect, continuous large-dose injection is required, which far exceeds the tolerance dose of human beings, which often causes systemic symptoms such as fever, chills, fatigue, headache, shock, etc., and the clinical application effect is not ideal, thus limiting clinical application. wide application of

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  • Method for producing human tumor necrosis factor-alpha
  • Method for producing human tumor necrosis factor-alpha
  • Method for producing human tumor necrosis factor-alpha

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Experimental program
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Embodiment 1

[0032] Example 1 Construction of expression plasmids and acquisition of high-expression engineering strains

[0033]According to the natural amino acid sequence of human TNF-α, according to the codon preference, the target gene sequence of recombinant human TNF-α protein is obtained by PCR amplification under the condition of not changing the amino acid sequence, and the optimized codon sequence is introduced into the PCR primers.

[0034] The target human interferon β gene was amplified by PCR, and double-digested with XhoI+EcoRI (MBI, 2*Tango TM , 37°C) PCR product and pPIC9 plasmid (purchased from Invitrogen). The two fragments were ligated with T4 DNA ligase, and the ligated product was transformed into Escherichia coli DH5α (purchased from Promega), clones were selected on an LB plate containing ampicillin, a small amount of plasmids were prepared, and screening was performed by double enzyme digestion / PCR identification. Positive clones were verified by sequencing. The...

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Abstract

The invention provides a nucleotide sequence of a recombinant human tumor necrosis factor-Alpha, a method for efficiently producing the recombinant human tumor necrosis factor-Alpha and related techniques of construction, expression and purification of engineering cells. The optimized recombinant human tumor necrosis factor-Alpha protein gene is ideally suitable for being expressed in leaven, simultaneously, through the optimization of the ferment and purification techniques, the output thereof is increased and the advantages of high expression and high stability are realized. The invention can obtain the recombinant human tumor necrosis factor-Alpha pure products efficiently and conveniently with low cost.

Description

technical field [0001] The invention relates to the field of genetic engineering. More specifically, the present invention provides a method for efficiently producing recombinant human recombinant human tumor necrosis factor-α, as well as the nucleotide sequence of related recombinant human tumor necrosis factor-α, construction of engineered cells, recombinant human tumor necrosis factor — The expression and purification process of α. Background technique [0002] Tumor Necrosis Factor alpha (TNF-α) is a kind of cytokine with a wide range of biological effects. It is composed of 157 amino acid residues and has a molecular weight of 17,000 Daltons on SDS-PAGE. Natural TNF-α is mainly secreted by activated macrophages and some cell lines such as HL-60, U937 and other cells. After the 1980s, many laboratories have successfully cloned the gene of TNF-α, which promoted the in-depth study of the molecular structure and function of TNF-α, and provided sufficient sources for the ...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12N15/28C07K14/525C12N1/19
Inventor 黄阳滨任军朱飞云
Owner SHANGHAI NEWSUMMIT BIOPHARMA
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