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Post-extraction technology of chicken alpha-disruptor

An extraction process and interferon technology, applied in the field of post-extraction process of chicken alpha-interferon, can solve problems such as high cost, low yield, complicated production process, etc., and achieve mild elution conditions, environmental protection, cost, and cost saving Effect

Inactive Publication Date: 2019-12-31
山东仙普爱瑞科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, recombinant interferon has been successfully expressed by Escherichia coli and Pichia pastoris, but so far no recombinant chicken interferon has obtained a new veterinary drug certificate in my country, and no imported recombinant chicken interferon has obtained an import registration certificate
One of the biggest obstacles is the complex production process, especially the post-extraction process
[0003] The purpose of the post-extraction process is to obtain interferon with higher purity. The current commonly used method is divided into two steps. One is preliminary purification, such as salting out, isoelectric point, organic solvent precipitation, etc.; Phase chromatography, metal affinity chromatography, ion exchange, hydrophobic chromatography and gel filtration chromatography, and reversed-phase interaction chromatography, in order to obtain ideal results, it is usually necessary to combine and optimize several methods, and finally lead to extraction, separation and purification The process is complicated, the technology is various, the yield is low, and the cost is high
At present, there is no ideal post-extraction process for obtaining high-purity recombinant chicken α-interferon

Method used

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  • Post-extraction technology of chicken alpha-disruptor
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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Embodiment 1. The present invention produces the construction of the engineered bacterium of recombinant chicken α-interferon

[0079] Step 1. Obtaining the target gene

[0080] We found the original gene sequence of α-interferon of red jungle fowl (SEQ ID NO.3) on NCBI, deleted the original signal peptide of α-interferon of red jungle fowl, and counted the amino acids with degenerate codons Analysis and selection of codons with high usage frequency to obtain a new recombinant chicken α-interferon gene sequence (SEQ ID NO.1), entrust Sangon Bioengineering (Shanghai) Co., Ltd. to perform whole gene synthesis to obtain the target gene.

[0081] Design primers, add EcoR I restriction site to the 5' end of the target gene, and add Not I restriction site to the 3' end by PCR reaction. After the PCR product was subjected to 1.5% agarose gel electrophoresis, a 513bp fragment was recovered with a DNA purification and recovery kit. The DNA sequence of the fragment is shown in S...

Embodiment 2

[0107] Embodiment 2. The fermentation process of recombinant chicken α-interferon

[0108] 1. Screening of multi-copy strains

[0109] Streak-inoculate the single colonies of positive clones obtained in Example 1 onto YPD solid medium containing 300 μg / ml, 600 μg / ml, and 900 μg / ml bleomycin in sequence, and screen out stable multi-copy high-yielding strains. Specific steps are as follows:

[0110] Inoculate the single colonies of positive clones on the YPD plate containing 100 μg / ml bleomycin to the YPD selective plate medium containing 300 μg / ml bleomycin using the “three-step streaking method” and culture at 30°C for 3-5 days. Colonies were observed after days.

[0111] Inoculate a single colony with good growth on the YPD plate containing 300 μg / ml bleomycin to the YPD selective plate medium containing 600 μg / ml bleomycin using the “three-step streaking method” and culture at 30°C for 3- Colonies were observed after 5 days.

[0112] Inoculate a single colony with good g...

Embodiment 3

[0129] Embodiment 3. Post-extraction process of chicken alpha-interferon

[0130] Step 1: Put the jar

[0131] First, the preserved recombinant Pichia strain (CCTCC M 2019748) is carried out according to the method of Example 2 for the screening of multi-copy strains, primary seed culture, secondary seed cultivation, and then the secondary seed liquid is pressed by 15% (v / v ) is transferred to 15T (tons) BMMY liquid medium for fermentation and cultivation (method is the same as in Example 2), and the Pichia fermented liquid is obtained. The chicken α-interferon quantitative detection kit was used to measure the concentration of the recombinant chicken α-interferon in the fermentation liquid of Pichia pastoris, and the concentration of the chicken α-interferon in the fermentation liquid was measured to be 20-22 g / L.

[0132] Step 2: Centrifuge

[0133] Centrifuge the fermented liquid in the storage tank through a centrifuge, the centrifuge speed is 12000rpm, and the centrifug...

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Abstract

The invention relates to a post-extraction technology of a chicken alpha-disruptor, and belongs to the technical field of production of medicines for livestock. The technology comprises the steps of S1, performing centrifugation on fermentation liquid containing the chicken alpha-disruptor, and reserving supernatant; S2, filtering the supernatant with a 50KDa ceramic membrane, filtering filtrate through a 10KDa ceramic membrane, and collecting trapped liquid; S3, adding a Cu<2+>-IDA-glucan carrier and a NaCl solution, with the same volume to the trapped liquid in the step S2, and performing anadsorption reaction to obtain reaction liquid; S4, adding a phosphate buffer solution with the same volume to the reaction liquid in the step S3, performing uniform mixing, performing filtering withthe 50KDa ceramic membrane, and collecting trapped liquid; S5, adding eluent to the trapped liquid in the step S4, then performing filtering through the 30KDa ceramic membrane, and collecting filtrate; and S6, filtering the filtrate in the step S5 through the 10KDa ceramic membrane for desalinating, and collecting trapped liquid to obtain the chicken alpha-disruptor. The yield is 61% or above, thepurity is 98% or above, and the activity yield is 96.73% or above.

Description

technical field [0001] The invention relates to a post-extraction process of chicken alpha-interferon, which belongs to the technical field of poultry drug production. Background technique [0002] my country is a big chicken raising country in the world. In 2018, more than 3.9 billion chickens were slaughtered. The annual losses due to virus infection are immeasurable. There is an urgent need to find a new product for the prevention and treatment of chicken viral diseases that is efficient, safe and non-toxic. At present, recombinant interferon has been successfully expressed by Escherichia coli and Pichia pastoris, but so far no recombinant chicken interferon has obtained a new veterinary drug certificate in my country, and no imported recombinant chicken interferon has obtained an import registration certificate. One of the biggest obstacles is the complex production process, especially the post-extraction process. [0003] The purpose of the post-extraction process is t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/56C07K1/36C07K1/34C07K1/22C12P21/02C12R1/84
CPCC07K14/56
Inventor 郭海岩刘刚王兴业韩国英苗玉和韩法杰王红军孙秀丽
Owner 山东仙普爱瑞科技股份有限公司
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