Method for producing porcine alpha-interferon

A production method and interferon technology, which are applied in the field of treatment of swine infectious diseases, can solve the problems of difficult separation, complicated operation, drug safety risks, etc., and achieve the effects of good stability, high activity and high purity

Inactive Publication Date: 2012-07-11
GUANGDONG ZIJIN ZHENGTIAN PHARMA
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AI Technical Summary

Problems solved by technology

There are following deficiencies in cell crushing: first, the cost is high and the operation is complicated; second, the cell crushing process has a certain effect on interferon, which affects the activity and stability of interferon; Miscellaneous proteins and other impurities are mixed in interferon and are difficult to separate, which reduces the purity and biological activity of interferon products
If Escherichia coli is used as the expression system, the destruction of the bacteria will cause the endotoxin to be mixed in the interferon, which will pose a cert

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0022] 1. Production of interferon

[0023] Take a single clone, inoculate it into the primary seed liquid, cultivate it for 17-20 hours, inoculate it in the Erlenmeyer flask for the secondary stage according to the ratio of 1:10, cultivate it for 4-8 hours, put it into the tank for fermentation, pH5.0, temperature 20℃, DO>35% , when the dissolved oxygen rises to 10-15, add 50% glycerin to the speed. After the glycerol was exhausted and the dissolved oxygen rose again, the induction was started with a methanol-containing mixture at a speed of 1, and the product concentration was detected after 48 hours of induction.

[0024] 2. Purification of interferon

[0025] Take 50L of fermentation broth and filter it through a two-stage series filter. The filter membranes have a pore size of 0.45 μm and 0.22 μm respectively. The filtrate is filtered through a 22KD ultrafiltration system, and the obtained filtrate is then concentrated through an 18KD ultrafiltration system. The volume o...

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Abstract

The invention relates to a method for producing porcine alpha-interferon, and belongs to the field of the treatment of porcine infection diseases. The invention mainly provides a method for secreting, separating and purifying interferon simply, and aims to simplify a production process, reduce production cost and obtain a recombinant porcine alpha-interferon product with high activity and high stability. The production method comprises the following steps of: producing interferon by using a mixture of methanol; purifying the interferon by multiple-effect ultrafiltration; and making ultrafiltration membranes with different molecular weight cutoff according to the molecular weight of the interferon into a filter, and filtering, so that impure protein and impurities of which the molecular weight is greater than that of the interferon and foreign protein, inorganic salt and water of which the molecular weight is smaller than that of the interferon in a fermentation liquor are removed, and only the interferon and little water are reserved; and freezing and drying to obtain an interferon finished product which is high in purity, activity and stability.

Description

technical field [0001] The invention belongs to the field of treating porcine infectious diseases, and mainly provides an efficient and simple interferon secretion, separation and purification method. Background technique [0002] Interferon was first divided into type Ⅰ and type Ⅱ. Type Ⅰ was once called leukocyte interferon, and type Ⅱ was once called fibroblast interferon. Later, it was found that there are two components in type I interferon. Although they share receptor components, the structure of the two is different and the immunogenicity is completely different, so type I is divided into two types: α and β. Among them, IFN-α is a family of 20 structurally related polypeptides with a molecular weight of about 18KDa, each encoded by an independent gene; IFN-β is a single gene product, a glycoprotein with a molecular weight of 20KDa. Later, type II was designated as γ type. IFN-γ is a glycoprotein composed of subunits of about 21-24KDa in the form of homologous double...

Claims

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Application Information

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IPC IPC(8): C12P21/02C07K1/34C12R1/84
Inventor 王庆福刘易通李振天
Owner GUANGDONG ZIJIN ZHENGTIAN PHARMA
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