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Production method for human tumor necrosis factor TNF-alpha mutant

A tumor necrosis factor and mutant technology, applied in the direction of tumor necrosis factor, biochemical equipment and methods, botany equipment and methods, etc., can solve problems such as high cost, low activity, complex downstream processes, etc.

Inactive Publication Date: 2006-12-13
SHANGHAI NEWSUMMIT BIOPHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] However, genetically engineered recombinant products produced by Escherichia coli are commonly used at home and abroad. Because E. coli contains endotoxin, and recombinant proteins often form inclusion bodies, the downstream process is complicated, the cost is high, and its activity is often low.

Method used

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  • Production method for human tumor necrosis factor TNF-alpha mutant

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] The construction of embodiment 1 expression plasmid and the acquisition of high expression engineering strain

[0054] According to the amino acid sequence of the natural mature peptide of human tumor necrosis factor TNF-α, it was modified, and 7 amino acids were deleted at the N-terminal, and the Pro 8 Ser 9 Asp 10 Replaced with ArgLysArg, and replaced Leu at position 157 of the C-terminal with Phe. The amino acid sequence after mutation is shown in SEQ ID NO: 2. According to codon preference, under the condition of not changing the amino acid sequence, the whole gene is synthesized to encode human tumor necrosis factor TNF - the target gene sequence of the α mutant protein, the gene sequence is cloned into pUC19 and verified by sequencing.

[0055] The expression vector construction method is shown in Figure 1. The target human tumor necrosis factor TNF-α gene was amplified by PCR, and double-digested with BamHI+EcoRI (MBI, 2*Tango TM , 37°C) PCR product and pPIC9...

Embodiment 2

[0057] The influence of embodiment 2 different induction time on expression level

[0058] Take a single clone, inoculate it into the BMGY primary seed solution, and cultivate it for 17-20hr; secondly inoculate it in a 500ml Erlenmeyer flask with 50ml medium at a ratio of 1:10 (each condition has an auxiliary tube), and cultivate it for about 24hr, 1 % Methanol induction, samples were taken every 12 hours, and OD was measured at the same time. A total of 120 hours of induction. SDS-PAGE of samples before induction and induction at different times.

[0059] The experimental results showed that: the optimal induction time of expressing TNF-α at the shake flask level was between 24-96 hr, and the optimal induction time was 36-72 hr.

Embodiment 3

[0060] Embodiment 3 Induction stage adds different protein protectors to the influence of expression level

[0061] Take a single clone, inoculate it into the BMGY primary seed liquid, and cultivate it for 17-20hr; secondly inoculate it in a 1L Erlenmeyer flask of 250ml BMGY at a ratio of 1:10, culture it for about 4~8hr, put it into the tank for fermentation, pH 5.0, temperature 20°C, D0 > 35%, after the dissolved oxygen rises, add 50% glycerin at 10~15 rpm. After the glycerin is exhausted and the dissolved oxygen rises again, use 100% methanol to start induction at speed 1, and gradually increase the speed. In the induction phase, 300ml of CA, Peptone and Tryptone with a concentration of 10% were added to the tank (5L fermenter, 3L fermentation supernatant), and the induction was over for 48hrs. Samples were tested for SDS-PAGE and protein content to determine the best protein protectant for the induction phase.

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Abstract

The invention provides a method of producing mutant of tumour necrosis factor called TNF-alphaand the related technology about expression carrying agent and, the construction, expression and decontamination of engineering cell. The protein gene of mutant of tumour necrosis factor called TNF-alpha,which has been optimized, is very much suitable for expression in pichia, meanwhile, improves expression quantity and the rate of purification through the optimization of fermentation and decontamination, has the advantages of high expression, good stability, compact production technology. This invention can get the protein gene of mutant of tumour necrosis factor called TNF-alpha with performance, briefness and low cost.

Description

technical field [0001] The invention relates to the field of genetic engineering. More specifically, the present invention provides a human tumor necrosis factor TNF-alpha mutant sequence, its production method, as well as related expression vectors and engineering cell construction, recombinant human tumor necrosis factor TNF-alpha mutant expression and Purification process. Background technique [0002] Human tumor necrosis factor-α (human tumor necrosis factor-α, hTNF-α), also known as cachectin, is mainly produced by monocytes and macrophages, and is a cytokine with the strongest activity in killing tumor cells. [0003] The human TNF-α gene is about 2.76kb in length and consists of 4 exons and 3 introns. The human TNF-α precursor consists of 233 amino acid residues and a signal peptide of 76 amino acid residues. The post-signal peptide mature TNF-α has 157 amino acid residues and a molecular weight of 17kDa. It is a non-glycosylated protein, and two cysteines at posit...

Claims

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Application Information

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IPC IPC(8): C12N15/28C07K14/525C12N15/63C12N15/81C12P21/02C07K1/14
Inventor 孙九如任军黄阳滨丰涛蒋剑
Owner SHANGHAI NEWSUMMIT BIOPHARMA
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