Method for preparing recombinant chicken beta-interferon and application thereof

A technology of beta interferon and production method, applied in the field of molecular immunology, can solve the problems of low expression, unfavorable industrial production, expensive cell culture costs, etc., and achieve the effect of easy degradation

Inactive Publication Date: 2011-08-17
NANJING UNIV
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Problems solved by technology

Generally speaking, exogenous proteins expressed by eukaryotic cells have the advantage of good biological activity, but also have the disadvantage of low expression, and studies have shown that the use of eukaryotic cell host systems...

Method used

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  • Method for preparing recombinant chicken beta-interferon and application thereof
  • Method for preparing recombinant chicken beta-interferon and application thereof
  • Method for preparing recombinant chicken beta-interferon and application thereof

Examples

Experimental program
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Effect test

Embodiment Construction

[0044] 1 Cloning of chicken interferon beta mature protein gene:

[0045] Extraction of chicken liver genome:

[0046] The three yellow chickens were killed by the conventional method, and about 0.05g of chicken liver tissue was quickly cut with sterile forceps, washed with sterile saline to remove the attached blood, cut into small pieces and placed in a 5ml syringe tube (remove the needle part), Push the syringe to force the liver tissue out of the needle. Put the extrudate in a glass homogenizer (pre-cooled on ice), add 800 ml tissue lysate, place it in the homogenizer, and homogenize quickly until there are no obvious tissue pieces, and then use a pipette to transfer the homogenate to into a 2ml eppendorf tube. Place the eppendorf tube containing chicken liver homogenate in a water bath at 65°C for 2 hours, then add 50 ml of proteinase K (20mg / ml), centrifuge at 14,000g for 30 minutes at 37°C, digest overnight, absorb 400ml of supernatant and add 40ml of NaAc (2.5m...

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Abstract

The invention belongs to the technical field of the molecular immunology and particularly relates to a method for efficiently preparing a chicken beta-interferon in vitro and application thereof. The technical method mainly comprises the following steps of: amplifying a chicken beta-interferon gene from a chicken liver genome by a PCR (Polymerase Chain Reaction) method; connecting the gene onto aPET28 alpha vector; constructing a gene recombination vector of a recombinant chicken beta-interferon maturation protein; transferring the gene recombination vector of the recombinant chicken beta-interferon maturation protein into a BL-21 escherichia coli expression system; efficiently expressing the chicken beta-interferon in escherichia coli; carrying out high-concentration urea denaturation on an inclusion body of the expressed chicken beta-interferon; purifying the obtained product by a NI-NTA affinity chromatography column; collecting an elution peak; and carrying out renaturation by solution with gradient urea concentration. The recombinant chicken beta-interferon in the invention has good antiviral biological activity, low production cost, high production efficiency and good biological activity and is very suitable for entrepreneurial production.

Description

technical field [0001] The invention belongs to the technical field of molecular immunology, and specifically relates to a method for efficiently preparing chicken beta interferon in vitro and an application thereof. Background technique [0002] Interferon beta is an important type I interferon, which is mainly secreted by fibroblasts and leukocytes under the stimulation of interferon-inducing substances such as viral nucleic acid and polyinosinic acid polycytidylic acid (Poly IC) A glycoprotein. Interferon-beta not only plays an important role in the process of anti-viral innate immunity, but also has anti-tumor, anti-proliferation and immune regulation functions. In some cases where alpha interferon fails (multiple sclerosis) or ineffective (severe acute respiratory syndrome, SARS), beta interferon can play a very good role. [0003] Judging from the scientific research value and clinical application value of beta interferon, chicken beta interferon will definitely play...

Claims

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Application Information

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IPC IPC(8): C12N15/22C12N15/70C07K14/565C07K1/22A61K38/21A61P31/12
Inventor 沈萍萍蔡梅红
Owner NANJING UNIV
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