Post-extraction process for swine alpha-interferon

An extraction process and interferon technology, which is applied in the direction of interferon, cytokines/lymphokines/interferons, peptide preparation methods, etc., can solve the problems of complex extraction process, high cost, poor yield, etc., and achieve high purity, Cost savings and mild elution conditions

Inactive Publication Date: 2019-12-24
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

The existing post-extraction process of porcine α-interferon is complex, with low purity, poor yield and high cost
At present, there is no ideal post-extraction process to obtain high-purity porcine α-interferon

Method used

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  • Post-extraction process for swine alpha-interferon
  • Post-extraction process for swine alpha-interferon
  • Post-extraction process for swine alpha-interferon

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1. Construction of Recombinant Porcine α-Interferon Engineering Bacteria

[0078] Step 1. Obtaining the target gene

[0079] We found the original gene sequence of boar α-interferon (SEQ ID NO.3) on the NCBI website, deleted the original signal peptide of boar α-interferon, and performed statistical analysis on the amino acids with degenerate codons , selected codons with high usage frequency, obtained a new recombinant porcine α-interferon gene sequence (SEQ ID NO.1), entrusted Sangon Bioengineering (Shanghai) Co., Ltd. to carry out whole gene synthesis, and obtained the target gene.

[0080] 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 525bp fragment was recovered with a DNA purification and recovery kit. The DNA sequence of the fragment is shown in SEQ ID NO.2.

[0081] The nucleotide seque...

Embodiment 2

[0110] Embodiment 2. The fermentation process of recombinant porcine alpha-interferon

[0111] 1. Screening of multi-copy strains

[0112] 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:

[0113] 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.

[0114] 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.

[0115] Inoculate a single colony with go...

Embodiment 3

[0132] Embodiment 3. Post-extraction process of porcine alpha-interferon

[0133] Step 1: Put the jar

[0134] First, the recombinant Pichia strain (CCTCC M 2019749) is carried out according to the method of Example 2 for the screening of multi-copy bacterial strains, primary seed culture, and secondary seed cultivation, and then the secondary seed liquid is pressed at 15% (v / v) The inoculum size was transferred to 15T (ton) induction medium for fermentation culture (the method is the same as in Example 2) to obtain Pichia pastoris fermentation broth. The porcine alpha interferon quantitative detection kit was used to measure the concentration of porcine recombinant alpha interferon in the Pichia pastoris fermentation broth, and the concentration of porcine alpha interferon in the fermentation broth was measured to be 20-22g / L.

[0135] Step 2: Centrifuge

[0136] Centrifuge the fermented liquid in the storage tank through a centrifuge, the centrifuge speed is 12000rpm, and ...

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Abstract

The invention relates to a post-extraction process for swine alpha-interferon and belongs to the technical field of production of drugs for livestock and poultry. The process comprises the steps: S1.subjecting swine alpha-interferon containing fermentation broth to centrifugation, and reserving supernatant; S2. filtering the supernatant through a 50KDa ceramic membrane, then, filtering filter liquor through a 10KDa ceramic membrane, and collecting trapped fluid; S3. adding an equal volume of Ni<2+>-IDA-glucan vector and a NaCl solution into the trapped fluid obtained in the step S2, and carrying out an adsorption reaction, so as to obtain a reaction solution; S4. adding an equal volume of phosphate buffer solution into the reaction solution obtained in the step S3, carrying out uniform mixing, then, carrying out filtering through a 50KDa ceramic membrane, and collecting trapped fluid; S5. adding an eluent into the trapped fluid obtained in the step S4, then, carrying out filtering through a 30KDa ceramic membrane, and collecting filter liquor; and S6. filtering the filter liquor obtained in the step S5 through a 10KDa ceramic membrane to remove salts, and collecting trapped fluid,thereby obtaining the swine alpha-interferon. According to the process, the yield is 62% or more, the purity is 98% or more, and the active yield is 100%.

Description

technical field [0001] The invention relates to a post-extraction process of porcine α-interferon, belonging to the technical field of poultry drug production. Background technique [0002] my country is a big pig raising country in the world. In 2018, more than 693.82 million pigs were slaughtered. Every year, the outbreak of viral diseases causes tens of billions of losses to the pig breeding industry. There is an urgent need to seek a new product for the prevention and treatment of porcine viral diseases with high efficiency, safety and no side effects. Porcine leukocyte interferon is currently the most used in the market, produced by Sichuan Shihong Biotechnology Co., Ltd. through the induction method, and obtained the new veterinary drug certificate of the Ministry of Agriculture in 1997 [(97) New Veterinary Drug Certificate No. 01]. [0003] At present, recombinant interferon can be successfully expressed by Escherichia coli and Pichia pastoris, but so far no recombin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/56C07K1/22C07K1/34C12N15/81
CPCC07K14/56C12N15/815
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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