CN102559819A - Method for producing porcine alpha-interferon - Google Patents
Method for producing porcine alpha-interferon Download PDFInfo
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- CN102559819A CN102559819A CN2012100612460A CN201210061246A CN102559819A CN 102559819 A CN102559819 A CN 102559819A CN 2012100612460 A CN2012100612460 A CN 2012100612460A CN 201210061246 A CN201210061246 A CN 201210061246A CN 102559819 A CN102559819 A CN 102559819A
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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
The invention belongs to the treatment pig and infect the disease field, mainly provided an efficient simple Interferon, rabbit secretion, separation purification method.
Background technology
Interferon, rabbit is divided into I type and II type the earliest, and the I type once was called as LeIF, the once called fiblaferon of II type.Found in the interferon type to also have two kinds of compositions afterwards, though their shared receptor components, the structure of the two is different, and immunogenicity is also different fully, so the I type is divided into two types of α and β again.Wherein IFN-α is the peptide family that the molecular weight of 20 structurally associateds is about 18KDa, and each is by genes encoding independently; IFN-β is a single-gene product, is the gp of molecular weight 20KDa.Then be decided to be the γ class to the II type afterwards, the gp that exists with the homodimer form that IFN-γ is made up of the subunit of about 21-24KDa, responsive to 56 ℃, pH2 and 0.1%SDS.Therefore, Interferon, rabbit just is divided into α, β, γ three major types.In recent years, also found the Interferon, rabbit of types such as ω, τ.
Interferon type is claimed antiviral Interferon, rabbit again, its BA with antiviral be main; Interferon type is claimed type II interferon again, and its BA is adjusted to the master with immunity.The method of conventional fermentation expression Interferon, rabbit will be carried out bacterial cell disruption when reclaiming Interferon, rabbit.There is following weak point in bacterial cell disruption: the one, and cost is high, complicated operation; The 2nd, bacterial cell disruption technology have certain broken can acting on to interference, and interferon activity and stability are affected; The 3rd, bacterial cell disruption can produce a lot of foreign proteins and other impurity, is blended in the Interferon, rabbit, is difficult to separate, and the Interferon, rabbit product purity is descended, and biological activity descends.If with intestinal bacteria as expression system, bacterial cell disruption can make intracellular toxin be blended in the Interferon, rabbit, causes certain risk to drug safety.If when from fermented liquid, reclaiming Interferon, rabbit, adopt salting-out process usually, there is following weak point in salting-out process: the one, and salting-out process does not have selectivity, and foreign protein also can be blended in the Interferon, rabbit by deposition, and degree of disturbance purity is descended; The 2nd, there is certain influence in salting-out process to interferon activity, the part Interferon, rabbit in precipitation process because destructurized and lost activity.Porcine alpha-interferon is mainly used in infection such as blue otopathy poison, CSFV, PCV-II, Pseudorabies virus, influenza virus, epidemic diarrhea virus, TGE, rotavirus, also can adjust the animal body immunologic function simultaneously.
Summary of the invention
The object of the invention is intended to simplify production technique, reduces production costs, and obtains high reactivity, high stability recombinant IFN-alpha product.
Working method provided by the present invention is: the one, produce Interferon, rabbit with methanol mixture; The 2nd, adopt multiple-effect ultrafiltration purification Interferon, rabbit.Select the ultra-filtration membrane of PSPP to make strainer according to the interferon molecule amount; Make in the fermented liquid greater than the foreign protein of interferon molecule amount and impurity, foreign protein, inorganic salt and water through filtration and to be gone out less than the interferon molecule amount; Only keep Interferon, rabbit and a spot of water, high, active high, the good stability of the Interferon, rabbit finished product purity that lyophilize obtains.
A kind of working method of porcine alpha-interferon, its step comprises:
One, produces Interferon, rabbit with methanol mixture
The first step, get the raw materials ready: mono-clonal, glycerine, methanol mixture;
Second step, mono-clonal are inoculated into primary seed solution, cultivate 17～20h;
The 3rd the step, in XX:YY=1:10 ratio two-stage inoculation in Erlenmeyer flask, the cultivation 4～8h;
The 4th step, fermentation
PH5.0,20 ℃ of temperature, DO＞35% is after dissolved oxygen rises to 10～15; Rotating speed adds 50% glycerine; Treat that glycerine exhausts, after dissolved oxygen rises once more;
The 5th step, methanol mixture are induced and are obtained alpha-interferon
Begin to induce with rotating speed 1 with containing methanol mixture, induce 48h after, obtain alpha-interferon.
Two, purifying alpha-interferon
The 6th step, alpha-interferon liquid filter through the two-stage tandem strainer, one-level in-line filter serial connection two-stage tandem strainer, and one-level in-line filter filter membrane aperture is that 0.45 μ m, two-stage tandem strainer filter membrane aperture are 0.22 μ m;
The 7th step, filtrating are filtered through the 22KD ultrafiltration system;
The 8th step, gained filtrating concentrate through the 18KD ultrafiltration system again;
The purer alpha-interferon that obtains after the 9th step, the lyophilize.
The present invention adopts improved Pichia anomala expression system secreting, expressing porcine alpha-interferon, through optimization for fermentation technology, simplifies purifying process, obtains high expression level, high reactivity, high stability, and reaches the porcine alpha-interferon of tool industrialization value.
Embodiment
Embodiment:
1, the generation of Interferon, rabbit
Get mono-clonal, be inoculated in the primary seed solution, cultivate 17～20h, in Erlenmeyer flask, cultivate 4～8h in 1:10 ratio two-stage inoculation, last jar of fermentation, pH5.0,20 ℃ of temperature, DO＞35%, after dissolved oxygen rose to 10～15, rotating speed added 50% glycerine.Treat that glycerine exhausts, dissolved oxygen rise once more the back begin to induce with rotating speed 1 with containing methanol mixture, induce 48h after, the detection production concentration.
2, the purifying of Interferon, rabbit
Get fermented liquid 50L; Filter through the two-stage tandem strainer, the filter membrane aperture is respectively 0.45 μ m and 0.22 μ m, will filtrate and filter through the 22KD ultrafiltration system; Gained filtrating concentrates through the 18KD ultrafiltration system again; The solution to be concentrated volume stops to concentrate during for 0.5L, and the qualified back of samples contg in the liquid concentrator is aseptic subpackaged to cillin bottle, lyophilize.
Above embodiment has been merely the present invention has been further described, and scope of the present invention does not receive the limitation of the embodiment that lifts.
Claims (1)
1. the working method of a porcine alpha-interferon, its step comprises:
One, produces Interferon, rabbit with methanol mixture
The first step, get the raw materials ready: mono-clonal, glycerine, methanol mixture;
Second step, mono-clonal are inoculated into primary seed solution, cultivate 17～20h;
The 3rd the step, in XX:YY=1:10 ratio two-stage inoculation in Erlenmeyer flask, the cultivation 4～8h;
The 4th step, fermentation
PH5.0,20 ℃ of temperature, DO＞35% is after dissolved oxygen rises to 10～15; Rotating speed adds 50% glycerine, treats that glycerine exhausts, after dissolved oxygen rises once more;
The 5th step, methanol mixture are induced and are obtained alpha-interferon
Begin to induce with rotating speed 1 with containing methanol mixture, induce 48h after, obtain alpha-interferon;
Two, purifying alpha-interferon
The 6th step, alpha-interferon liquid filter through the two-stage tandem strainer, one-level in-line filter serial connection two-stage tandem strainer, and one-level in-line filter filter membrane aperture is that 0.45 μ m, two-stage tandem strainer filter membrane aperture are 0.22 μ m;
The 7th step, filtrating are filtered through the 22KD ultrafiltration system;
The 8th step, gained filtrating concentrate through the 18KD ultrafiltration system again;
The purer alpha-interferon that obtains after the 9th step, the lyophilize.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012100612460A CN102559819A (en) | 2012-03-09 | 2012-03-09 | Method for producing porcine alpha-interferon |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012100612460A CN102559819A (en) | 2012-03-09 | 2012-03-09 | Method for producing porcine alpha-interferon |
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| CN102559819A true CN102559819A (en) | 2012-07-11 |
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| CN2012100612460A Pending CN102559819A (en) | 2012-03-09 | 2012-03-09 | Method for producing porcine alpha-interferon |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110606884A (en) * | 2019-11-01 | 2019-12-24 | 山东仙普爱瑞科技股份有限公司 | Post-extraction process of porcine alpha-interferon |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1435486A (en) * | 2002-01-28 | 2003-08-13 | 上海贸基生物工程科技有限公司 | Human composite alpha-interferon engineered gene, its expression and production thereof |
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2012
- 2012-03-09 CN CN2012100612460A patent/CN102559819A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1435486A (en) * | 2002-01-28 | 2003-08-13 | 上海贸基生物工程科技有限公司 | Human composite alpha-interferon engineered gene, its expression and production thereof |
Non-Patent Citations (2)
| Title |
|---|
| 刘国强等: "降低诱导温度对改善猪α-干扰素发酵生产性能和ATP再生效率的作用", 《化工学报》 * |
| 张玉忠等: "《液体分离膜技术与应用》", 31 January 2004 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110606884A (en) * | 2019-11-01 | 2019-12-24 | 山东仙普爱瑞科技股份有限公司 | Post-extraction process of porcine alpha-interferon |
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Application publication date: 20120711 |
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