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Production method of recombination human interferon gamma

A technology of interferon and gamma protein, applied in the field of genetic engineering, can solve the problems of low interferon activity, difficult protein renaturation, complex process, etc.

Inactive Publication Date: 2006-11-29
SHANGHAI NEWSUMMIT BIOPHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0011] At present, Escherichia coli expression system is mostly used at home and abroad to express human interferon gamma (such as Chinese patent 941120910). Interferon gamma is expressed in inclusion bodies, which involves problems such as difficult protein renaturation and complicated processes. Yeast intracellular expression is also used (see China Patent 021368538), there are also problems such as ultrasonic crushing of bacteria, complex purification process, and low activity of interferon obtained

Method used

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  • Production method of recombination human interferon gamma
  • Production method of recombination human interferon gamma

Examples

Experimental program
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Effect test

Embodiment 1

[0062] Example 1 Construction of expression plasmids and acquisition of high-expression engineering strains

[0063] According to the natural amino acid sequence of human interferon gamma, according to the codon preference, under the condition of not changing the amino acid sequence, the target gene sequence encoding the mature peptide of human interferon gamma protein was synthesized, and the gene sequence was cloned into pUC19 and sequenced verify.

[0064] For expression vector construction methods, see figure 1 . The target human interferon gamma 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 do...

Embodiment 2

[0066] Example 2 Effect of different pH on expression level

[0067] 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, the pH of the induction stage needs to be adjusted (in the BMMY medium, the pH is directly adjusted with a phosphate buffer system). Sampling was taken at 24 hours after induction, OD and pH were measured at the same time, and 1% methanol was added to continue induction. A total of 48hr induction. SDS-PAGE of samples before induction and induction at different times.

[0068] The experimental results showed that the optimum pH for expressing IFN-γ at the shake flask level was between 5 and 9, and the optimum pH was 5-7.

Embodiment 3

[0069] Example 3 The effect of adding different protein protectants on the expression level during the induction phase

[0070] 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 relates to a reconstruction interferon gamma nucleotide sequence and the manufacture method to produce reconstruction interferon gamma, and the relative expression carrier and engineer cell construction, expression and purification technology. The method improves the expression quantity and the purification yield. It has the advantage of high expression, good stability, and simple technology. It could gain reconstruction human interferon gamma high efficiently, conveniently and in 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 gamma, as well as the construction of related expression vectors and engineered cells, the expression and purification process of recombinant human interferon gamma. 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 main...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/23C07K14/57C12N15/63C12N15/81C12P21/02
Inventor 孙九如任军黄阳滨丰涛蒋剑
Owner SHANGHAI NEWSUMMIT BIOPHARMA
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