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Preparation method of recombinant horse interferon-alpha-1

A GS115, induced expression technology, applied in the field of genetic engineering, can solve the problems of not being able to obtain a large number of natural horses and the limited source of horse white blood cells, and achieve the effect of ingenious design, enhanced immune response, and improved ability

Active Publication Date: 2017-06-20
SHANGHAI SERUM BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In view of the fact that the preparation of horse Interferon-alpha-1 requires a large amount of horse leukocytes, and the source of horse leukocytes is extremely limited, it is impossible to obtain a large amount of natural horse Interferon-alpha-1. Therefore, it is urgent to develop a method for the preparation of recombinant horse Interferon-alpha-1 , which can obtain biologically active recombinant equine Interferon-alpha-1 with high expression and high purity, which is of great significance for the guarantee of raw materials for the production of antiserum products

Method used

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  • Preparation method of recombinant horse interferon-alpha-1
  • Preparation method of recombinant horse interferon-alpha-1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment I

[0036] Embodiment 1: expression sequence acquisition and optimization

[0037] In order to achieve the purpose of high expression of horse Interferon-alpha-1 in yeast cells, the amino acid sequence of the complete gene (ie, the amino acid sequence shown in SEQ ID NO: 4) was obtained by searching the literature. According to this amino acid sequence, the nucleotide sequence encoding horse Interferon-alpha-1 (i.e. the sequence shown in SEQ ID NO: 1) was determined, and the nucleotide sequence (i.e. the sequence shown in SEQ ID NO: 2) was designed through codon optimization sequence), insert the nucleotide sequence into an expression vector, and then transform yeast cells. The corresponding inducers are then used to induce expression. A Pichia pastoris inducible expression system can be used.

Embodiment 2

[0038] Example 2: Construction of expression cell lines and screening of expression clones

[0039]In a preferred embodiment, the nucleotide sequence containing codon-optimized horse Interferon-alpha-1 (i.e. the sequence shown in SEQ ID NO: 1) adds an XhoI site and an AAA AGA nucleotide sequence at the 5' end, An EcoRI site was added at the 3' end. The pPIC9 plasmid (Invitrogen) was selected and linearized with XhoI and EcoRI double digestion. Equine Interferon-alpha-1 DNA obtained by digestion with XhoI and EcoRI was ligated with pPIC9 plasmid (Invitrogen) linearized with XhoI and EcoRI (see figure 1 ), and then transformed into Escherichia coli strain DH5ɑ (Invitrogen), which was named pPIC9-Interferon-alpha-1. It can be understood that the present invention is not limited to equine Interferon-alpha-1 factor, but also relates to Interferon-alpha-1 from other animal sources (dog, sheep, rabbit, etc.).

[0040] pPIC9-Interferon-alpha-1 was digested with SalI to make it line...

Embodiment 3

[0043] Embodiment 3: interferon purification

[0044] Take 50 ml of SP-Sepharose FF and load it into a 2.6x10cm chromatography column. The gel is washed successively with 2 volumes of 1M NaCl and 2 volumes of 0.5 N NaOH, and then equilibrated with pH 4.0 and 50 mM acetate buffer.

[0045] Four 2000ml Erlenmeyer flasks, each containing 500ml of YPD medium, were inoculated with the best selected

[0046] Interferon-alpha-1 expression clone, grow at 28-30°C shaker 200-250rpm for 48 hours, centrifuge to discard the supernatant, add 500 ml of YP medium containing 0.5% (v / v) methanol under sterile conditions, 28 Grow on a shaker at -30°C at 200-250 rpm for 72 hours, and add 2.5 ml of methanol every 24 hours. After 72 hours, the supernatant was collected by centrifugation, and the pH of the supernatant was adjusted to 4.0 with 20% acetic acid, and loaded onto a balanced SP-Sepharose FF ion-exchange chromatography column. After sample loading, the column was washed with pH 4.0, 50 m...

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Abstract

The invention relates to a preparation method of recombinant horse interferon-alpha-1. According to the preparation method, a nucleotide sequence which is optimized by codon, and is used for coding horse interferon-alpha-1 is introduced into a yeast cell induced expression vector, and then is introduced into yeast cells for culturing and induced expression, and purification is carried out so as to obtain recombinant horse interferon-alpha-1, wherein preferably, the yeast cell induced expression vector is a secretory expression vector, after induced expression, a cell fermentation supernatant is obtained via culturing, and liquid phase column chromatography is adopted for purification of the cell fermentation supernatant. Design of the preparation method is skillful; recombinant horse interferon-alpha-1 with biological activity can be obtained via high expression at high purity, the recombination recombinant horse interferon-alpha-1, can be used to preventing houses from virus infection, and improving virus resistance of animals.

Description

technical field [0001] The present invention relates to the technical field of genetic engineering, in particular to the technical field of Interferon-alpha-1 (Interferon α1) eukaryotic cell expression and preparation, specifically refers to the recombination and secretion of a recombinant horse Interferon-alpha-1 in yeast cells Expression and preparation method; In addition, the present invention also relates to a codon-optimized nucleotide sequence encoding equine Interferon-alpha-1. Background technique [0002] Equine antibodies play an irreplaceable role in passive immunotherapy and prevention. Equine antivenom antibodies are the fastest and most effective medication for snakebite. [0003] Shanghai Sailun Biotechnology Co., Ltd. uses horses to produce anti-biological toxin antibodies, such as anti-viper serum, anti-cobra serum, anti-bungare snake serum, and anti-slap snake serum. These sera specifically treat patients with corresponding venomous snake bites. In my...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N15/21
CPCC07K14/56C12N15/815C12N2800/22
Inventor 孙九如史小月柏伟范志和
Owner SHANGHAI SERUM BIOTECH
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