CN102108096A - Escherichia coli recombinant chicken alpha-gamma divalent interferon and preparation method thereof - Google Patents
Escherichia coli recombinant chicken alpha-gamma divalent interferon and preparation method thereof Download PDFInfo
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- CN102108096A CN102108096A CN 200910256418 CN200910256418A CN102108096A CN 102108096 A CN102108096 A CN 102108096A CN 200910256418 CN200910256418 CN 200910256418 CN 200910256418 A CN200910256418 A CN 200910256418A CN 102108096 A CN102108096 A CN 102108096A
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Abstract
The invention relates to an escherichia coli recombinant chicken alpha-gamma divalent interferon and a preparation method thereof. The invention aims at providing the interferon which has good anti-tumor effect and can improve immunity of livestock and poultry and realize control over diseases within a short time. The interferon comprises the components such as an escherichia coli recombinant chicken alpha-interferon and an escherichia coli recombinant chicken gamma-interferon; and the preparation method comprises the steps of gene design, construction of an expression vector, induction of expression, purification of a target protein and activity detection. The medicine is an alternative medicine for treating viral diseases of the livestock and the poultry, and escherichia coli is utilized for expressing the chicken alpha-interferon and the chicken gamma-interferon, thereby having a broad-spectrum antivirus role. The divalent interferon can have immunoregulation activity and a certain antivirus activity after purifying the protein, and the two interferons have obvious synergy when being combined and applied clinically. The divalent interferon prepared by the invention is better than any one interferon which is applied separately in the effects of treating and preventing newcastle disease and bursal disease.
Description
Technical field
The present invention relates to a kind of intestinal bacteria recombination chicken alpha-γ divalence Interferon, rabbit and preparation method thereof.
Background technology
Virus diseases such as the chicken Marek's disease that current poultry neoplastic disease and virus disease infection cause, newcastle disease, infectious bursal disease, infectious bronchitis, infectious laryngotracheitis, encephalomyelitis, influenza, infectious anemia, viral hepatitis are brought enormous economic loss to aviculture, have become one of the most serious disease of harm chicken industry this year.Effective treatment to poultry neoplastic disease and virus disease is one of great difficult problem of puzzlement poultry disease prevention and control always.Immunity system of chicken and the immune response that is produced when pathogen infection or vaccinate are similar to the PURU animal, come control disease so can consider the application cell factor.Comparatively speaking, the raising of bird is simple, cheap, and the time of coming out of steamer is short, and quantity is bigger, and this just needs to consider the economic worth of used vaccine or medicine.The startup of Green Foodstuff Project, people are to the intensification of the attention of food safety, and the serious drug residue that antiviral commonly used is brought jeopardizes human beings'health, and these problems obtain the common concern of society.So aviculture presses for the natural methods of treatment of a new generation.In pathogen infection and vaccine immunity, cytokines such as Interferon, rabbit are adjustable immunoreactive type and response intensity, be a kind of efficiently, natural methods of treatment.And treat according to traditional method, be difficult in the short period of time inner control state of an illness, use genetic engineering interferon then can make the antiviral rapidly propagation of body, antitumor hyperplasia, and can improve immunizing power.Use clinically, not only effect is obvious, and drug residue free, can in early days it is effectively treated in viral infection.Utilizing the biotechnology means to develop livestock and poultry is at chemicals forbidding back opposing livestock and poultry pestilence powerful mean with Interferon, rabbit (IFN), IFN has the antivirus action of wide spectrum, show that malicious proliferative amount reduces, the cell injury degree reduces, and has the dual function of enhance immunity power simultaneously.Can be used for preventing and treating newcastle disease, bird flu, fabricius bursa.The present invention selects the gene engineering expression product of Interferon, rabbit to develop green high-efficient anti-infection bio preparation, realizes the industrialization of gene engineering expression Interferon, rabbit product.
A kind of recombination chicken alpha that the present invention relates to-γ divalence Interferon, rabbit has antiviral and immunoregulatory double effects, does not see the report of clinical combined utilization.
Summary of the invention
1, right according to chicken IFN-α gene design primer among the GenBank, utilize pcr clone, design a pair of primer in mature peptide gene both sides, add proper restriction site respectively, with its subclone on expression vector PET, transform BL21 (DE3) back IPTG abduction delivering, with SDS-PAGE electrophoretic analysis expression-form, the result is that albumen is with the inclusion body formal representation, after broken thalline extracts inclusion body purification, utilize aminothiopropionic acid-aminothiopropionic acid to reoxidize the inclusion body sex change liquid of method after and carry out segmentation dilution method renaturation purifying.Antiviral activity by cytopathic-effect inhibition assay mensuration renaturation liquid obtains chicken alpha-interferon.
2, according to chicken IFN-gamma gene sequences among the GenBank, remove the signal peptide district, its mature peptide district is carried out the transformation of e. coli codon preference and mRNAde 5 ' end Headphone structure to be analyzed, set up BgI II and Hand III restriction enzyme site at the sequence two ends simultaneously, synthesize and the checking of TA cloning and sequencing.Pass through structure, the conversion of recombinant expression vector, the abduction delivering of expression vector again,, detect, obtained chicken interferon-gamma at last by ni-sepharose purification target protein and antiviral activity through determining expression-form behind the electrophoresis.
3, by gene design with synthetic, the structure of expressor carrier, abduction delivering, target protein purifying and obtain active substance and make.With the ripe gene clone of chicken alpha-interferon and chicken interferon-gamma to pet vector, be converted among the host bacterium BL21 (DE3), through the IPTG abduction delivering, obtained target protein, have very high antiviral activity through vesicular stomatitis virus-chick embryo fibroblast systems measurement.
The invention has the advantages that and utilize the escherichia coli expression chicken alpha-interferon, with the inclusion body formal representation, by having the antivirus action of wide spectrum after the sex change renaturation, and escherichia coli expression chicken interferon-gamma secreting, expressing, by having immunoregulatory activity and certain antiviral activity behind the purifying protein, the two combined utilization has the obvious synergistic effect, and gained divalence Interferon, rabbit of the present invention can be better than using separately treatment and the prevention of any Interferon, rabbit to the newcastle disease and the fabricius bursa.
Embodiment
Right according to chicken IFN-α gene design primer among the GenBank, utilize pcr clone IFN-α gene, design a pair of primer in mature peptide gene both sides, add proper restriction site respectively, with its subclone on expression vector PET, transform BL21 (DE3) back IPTG abduction delivering, with SDS-PAGE electrophoretic analysis expression-form, the result is that albumen is with the inclusion body formal representation, after broken thalline extracts inclusion body purification, utilize aminothiopropionic acid-aminothiopropionic acid to reoxidize the inclusion body sex change liquid of method after and carry out segmentation dilution method renaturation purifying.Antiviral activity by cytopathic-effect inhibition assay mensuration renaturation liquid obtains chicken alpha-interferon.Again according to chicken IFN-gamma gene sequences among the GenBank, remove the signal peptide district, its mature peptide district is carried out the transformation of e. coli codon preference and mRNAde 5 ' end Headphone structure to be analyzed, set up BgI II and Hand III restriction enzyme site at the sequence two ends simultaneously, synthesize and the checking of TA cloning and sequencing.Pass through structure, the conversion of recombinant expression vector, the abduction delivering of expression vector again,, detect, obtained chicken α-IFN-at last by ni-sepharose purification target protein and antiviral activity through determining expression-form behind the electrophoresis.
Claims (3)
1. intestinal bacteria recombination chicken alpha-γ divalence Interferon, rabbit is characterized in that its main component is intestinal bacteria recombination chicken alpha-γ divalence Interferon, rabbit, is colourless clear liquid to little Huang, the visible insolubles of no naked eyes.
2. a kind of intestinal bacteria recombination chicken alpha according to claim 1-γ divalence Interferon, rabbit, its feature is made the divalence Interferon, rabbit at chicken alpha-interferon and chicken interferon-gamma with escherichia coli expression.
3. the preparation method of a kind of intestinal bacteria recombination chicken alpha according to claim 2-γ divalence Interferon, rabbit is characterized in that: by gene design with synthetic, the structure of expressor carrier, abduction delivering, target protein purifying and obtain active substance and make.With the ripe gene clone of chicken alpha-interferon and chicken interferon-gamma to pet vector, be converted among the host bacterium BL21 (DE3), through the IPTG abduction delivering, obtained target protein, have very high antiviral activity through vesicular stomatitis virus-chick embryo fibroblast systems measurement.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN 200910256418 CN102108096A (en) | 2009-12-25 | 2009-12-25 | Escherichia coli recombinant chicken alpha-gamma divalent interferon and preparation method thereof |
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| CN 200910256418 CN102108096A (en) | 2009-12-25 | 2009-12-25 | Escherichia coli recombinant chicken alpha-gamma divalent interferon and preparation method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103585628A (en) * | 2013-11-21 | 2014-02-19 | 青岛润达生物科技有限公司 | Immunoglobulin preparation for treating newcastle disease and preparation method thereof |
| CN108576399A (en) * | 2018-03-22 | 2018-09-28 | 中国农业科学院生物技术研究所 | Composite interference promotor composition and its preparation and application |
-
2009
- 2009-12-25 CN CN 200910256418 patent/CN102108096A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103585628A (en) * | 2013-11-21 | 2014-02-19 | 青岛润达生物科技有限公司 | Immunoglobulin preparation for treating newcastle disease and preparation method thereof |
| CN103585628B (en) * | 2013-11-21 | 2016-08-17 | 青岛润达生物科技有限公司 | A kind of immunoglobulin preparation preventing Newcastle disease and preparation method thereof |
| CN108576399A (en) * | 2018-03-22 | 2018-09-28 | 中国农业科学院生物技术研究所 | Composite interference promotor composition and its preparation and application |
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| Date | Code | Title | Description |
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| C06 | Publication | ||
| PB01 | Publication | ||
| C53 | Correction of patent for invention or patent application | ||
| CB02 | Change of applicant information |
Address after: 266000 Shandong city of Qingdao province Chengyang Qingda Industrial Park, the first northbound dual Applicant after: Qingdao KDN Pharmaceutical Co., Ltd. Applicant after: Qingdao Conlinent Animal Pharmaceutical Co., Ltd. Address before: 266061 Shandong city of Qingdao province high tech park of Laoshan district by the No. 29 Shandong Road, building 605 high speed Applicant before: Qingdao Continent Biotech Co., Ltd. Applicant before: Qingdao Liuhe Medicine Co., Ltd. |
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| CB03 | Change of inventor or designer information |
Inventor after: Wang Lei Inventor after: Wang Honghua Inventor after: Ling Hongli Inventor after: Jia Deqiang Inventor before: Hao Zhihui Inventor before: Song Li Inventor before: Jia Deqiang Inventor before: Zhang Ruili Inventor before: Qiu Mei Inventor before: Wang Chunyuan |
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| COR | Change of bibliographic data |
Free format text: CORRECT: INVENTOR; FROM: HAO ZHIHUI SONG LI JIA DEQIANG ZHANG RUILI QIU MEI WANG CHUNYUAN TO: WANG LEI WANG HONGHUA LING HONGLI JIA DEQIANG |
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| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20110629 |