Production of recombinant human interferon beta

A technology of interferon β and nucleotide sequence, which is applied in the field of efficient production of recombinant human interferon β, and can solve problems such as complex processes

Inactive Publication Date: 2006-11-01
SHANGHAI NEWSUMMIT BIOPHARMA
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, it is expressed in CHO cells, and the process is more complicated

Method used

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  • Production of recombinant human interferon beta
  • Production of recombinant human interferon beta

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Experimental program
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Embodiment 1

[0064] The construction of embodiment 1 expression plasmid and the acquisition of high expression engineering strain

[0065] According to the natural amino acid sequence of human interferon β, according to the codon preference, under the condition of not changing the amino acid sequence, the target gene sequence of the recombinant human interferon β protein was synthesized, and the gene was cloned into pUC19 and verified by sequencing

[0066] The expression vector construction method is shown in Figure 1. 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 Company), clones were selected on an LB plate containing ampicillin, a small amount of plasmid was prepared, and screening was performed by double enzyme dige...

Embodiment 2

[0068] Embodiment 2 Induction phase adds different protein protectors to the influence of expression level

[0069] 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, DO > 35%, after the dissolved oxygen rises, add 50% glycerol 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.

Embodiment 3

[0070] The influence of different Peptone amounts in the induction stage of embodiment 3 on the expression level

[0071] 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, DO > 35%, after the dissolved oxygen rises, add 50% glycerol 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 stage, different volumes of 10% Peptone flow were added to a 5L fermenter (fermentation supernatant 3L), and the induction ended for 48 hours. Samples were tested for SDS-PAGE and protein content to determine the optimal amount of Peptone for the induction phase.

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Abstract

The invention provides a nucleotide sequence of recombinant human interferon beta, a production method for efficiently producing recombinant human interferon beta, and related engineering cell construction, expression and purification techniques. The optimized recombinant human interferon beta protein gene is very suitable for expression in Pichia pastoris. At the same time, through optimization of fermentation and purification process, the expression level has been increased, and it has the advantages of high expression and high stability. The invention can obtain the pure product of recombinant human interferon beta efficiently, conveniently and at 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 interferon beta, and related engineering cell construction, recombinant human interferon beta expression and purification processes. Background technique [0002] Interferon is a soluble protein produced by a variety of cells with a wide range of antiviral, antitumor and immunomodulatory effects. Interferon is not a uniform molecule as a whole, and can be divided into three types according to the producing cells: α-type produced by leukocytes; β-type produced by fibroblasts; γ-type produced by T cells. According to comprehensive factors such as interferon-producing cells, receptors and activity, it can be divided into two types: type I and type II. [0003] Type I interferon is also known as antiviral interferon, and its biological activity is mainly antiviral. Type I interferons come in 3 ...

Claims

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Application Information

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IPC IPC(8): C12N15/22C07K14/435C12N1/19C12N15/62C12N15/63C12N15/79C12P21/02
Inventor 孙九如任军黄阳滨黄智华丰涛蒋剑
Owner SHANGHAI NEWSUMMIT BIOPHARMA
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