CN1651576A - Production method and process of recombination chicken beta-alexin - Google Patents
Production method and process of recombination chicken beta-alexin Download PDFInfo
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- CN1651576A CN1651576A CN 200410023566 CN200410023566A CN1651576A CN 1651576 A CN1651576 A CN 1651576A CN 200410023566 CN200410023566 CN 200410023566 CN 200410023566 A CN200410023566 A CN 200410023566A CN 1651576 A CN1651576 A CN 1651576A
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- alexin
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Abstract
A proces for preparing the recombinant chicken beta-phylaxin is disclosed. Its advantages are low cost, high curative effect and low residual in human body.
Description
Technical field:
The present invention relates to the manufacturing technology field of animal kind medicine thing, the production method and the technology of definite a kind of chicken beta-alexin of recombinating of saying so:
Background technology:
Antibiotics brings serious safety issue as antimicrobial therapy medicine and fodder additives life-time service widely, and the one, bacterial antibiotic produces serious resistance, causes many medicines more and more insensitive; The 2nd, microbiotic produces serious drug residue in animal food, has a strong impact on the healthy of the mankind, and therefore, microbiotic limiting the use of and forbidding in the animal kind medicine thing become inexorable trend.
Alexin is to have the small-molecular peptides that suppresses or kill and wound external microorganism by insect, animal, plant and a big class that the people produced.Compare with microbiotic, it has wider antimicrobial spectrum, and Gram-negative, gram positive bacterium, fungi, virus even parasite are all had lethal effect, and it participates in the immunomodulatory of body simultaneously, this be microbiotic can not compare, be ideal microbiotic substitute products.But up to the present, the large-scale production alexin is difficulty relatively.At present the alexin of report generally is to purify from biological tissue, and these method cost height, pick-up rate are low, are difficult to suitability for industrialized production.
Summary of the invention:
Purpose of the present invention is production method and the technology that a kind of chicken beta-alexin of recombinating is provided.The reorganization chicken beta-alexin cost that Using such method and technology obtain is low, pick-up rate is high, but large-scale industrial production, and have wider germ resistance.
For reaching above-mentioned technical purpose, the present invention has taked following technical scheme:
Scheme one: intestinal bacteria reorganization chicken beta-alexin fusion rotein: with the encoding gene of RT-PCR technology amplification chicken beta-alexin gaIIinacin-3 from the total RNA of chicken epithelium mucosal tissue (tongue), and at the suitable endonuclease recognition site of the two ends of gene introducing, chicken beta-alexin encoding gene is inserted into the multiple clone site of coli expression carrier PGEX-6P-1, make this gene place the downstream of glutathione S-transferase (GST) encoding gene, and form correct reading frame; Under 30-40 ° of environment, in substratum, add 0.1-2mMoI/L IPTG, shaking culture 2-10 hour, induce antigen-4 fusion protein gene to express, a kind of protein that molecular weight is about 34kDa can appear in the SDS-PAGE electrophoresis: the recombination bacillus coli after centrifugal collection is induced, and use the ultrasonic cell disintegration instrument bacteria breaking.High speed centrifugation is collected inclusion body, and uses the Triton buffer solution for cleaning; With urea-containing lysis buffer dissolving inclusion body, with renaturation buffer dilution inclusion body lysate, make renaturing inclusion bodies then.
Technical scheme two: yeast secretary express recombinant chicken beta-alexin: the encoding gene that the chicken beta-alexin gaIIinacin-3 of suitable endonuclease identification point is introduced at two ends is inserted into the multiple clone site of Yeast expression carrier pPIC9K, make this gene place the downstream of a factor, and form correct reading frame; With electrotransformation or chemical transformation, the emphasis plasmid is transformed in the yeast.Cultivated 24-96 hour containing on the YPD culture plate of Geneticin, screen positive transformed yeast bacterial strain.Choose through the PCR method and identify the recombination microzyme that carries chicken beta-alexin gene on its chromosomal DNA; Recombination microzyme in the MGY substratum, is cultivated OD for 28-38 ℃ in fermentor tank
600When value reaches 2-6, centrifugal collection yeast thalline.With the resuspended recombination microzyme of BMMY substratum, regular replenishment methyl alcohol also makes that the methyl alcohol final concentration remains on 0.1-1.0% in the nutrient solution, continues to cultivate after 24-96 hour centrifugal collection supernatant liquor; Concentrate culture supernatant, can obtain the chicken beta-alexin of recombinating to concentrated solution through SephacryaI S-200 HR chromatography column purifying.
Technical scheme three: reorganization chicken beta-alexin milk-acid bacteria structural expression: the encoding gene that the chicken beta-alexin gaIIinacin-3 of suitable endonuclease recognition site is introduced at two ends is inserted into the multiple clone site of expression vector pMG36, make this gene place the downstream of lactobacillus cremoris subspecies Mg2 protein coding gene, and form correct reading frame; With electrotransformation recombinant plasmid transformed in Lactococcus lactis, cultivate, screen positive transformed bacteria containing on the antibiotic M17 agar culture plate; Transforming bacterial strain static cultivation 12-28 hour in containing microbiotic M17-liquid of glucose substratum; Centrifugal collection recombinant lactic acid bacteria prepares recombinant plasmid from recombinant lactic acid bacteria; Identify the chicken beta-alexin gene that recombinant lactic acid bacteria is carried by plasmid with the PCR method; Positive recombinant lactic acid bacteria was cultivated 24 hours centrifugal collection reorganization thalline in M17.Ultrasonication reorganization thalline, and in centrifugal 30 minutes of 6000 rev/mins of speed, separation of supernatant; Supernatant liquor through the SephacryaIs-200 HR chromatography column purifying chicken beta-alexin fusion rotein that can obtain to recombinate.
Owing to take technique scheme: a kind of production method and technology of the chicken beta-alexin of recombinating, it is low to reach the alexin production cost, obtains the rate height, is produced on a large scale and finished product has more broad spectrum antibacterial.
Embodiment:
The present invention is further described below in conjunction with embodiment:
One, according to technical solution of the present invention one, a kind of production method and technology of the chicken beta-alexin of recombinating are: with RT-PCR technology amplification chicken beta-alexin gaIIinacin-3 encoding gene from the total RNA of chicken epithelium mucosal tissue (tongue), and at the suitable endonuclease recognition site of the two ends of gene introducing; Chicken beta-alexin encoding gene is inserted into the multiple clone site of coli expression carrier PGEX-6P-1, makes this gene place the downstream of glutathione S-transferase (GST) encoding gene, and form correct reading frame; Under producing with 37 ℃ of environment in the fermentor tank, in substratum, add 1mMoI/L IPTG, shaking culture 5 hours induces antigen-4 fusion protein gene to express, and can occur a kind of protein that molecular weight is about 34kDa in the SDS-PAGE electrophoresis; Centrifugal (2000 rev/mins, 30 minutes) collect the recombination bacillus coli after inducing, and use the ultrasonic cell disintegration instrument bacteria breaking.High speed centrifugation (20000 rev/mins, 30 minutes) is collected inclusion body, and with Triton buffer solution for cleaning 3 times; With urea-containing lysis buffer dissolving inclusion body,, make the renaturing inclusion bodies chicken beta-alexin fusion rotein that can obtain recombinating then with renaturation buffer dilution inclusion body lysate.
Two, according to technical solution of the present invention two: 2, yeast secretary express recombinant chicken beta-alexin: the encoding gene that the chicken beta-alexin gaIIinacin-3 of suitable endonuclease identification point is introduced at two ends is inserted into the multiple clone site of Yeast expression carrier pPIC9K, make this gene place the downstream of a factor, and form correct reading frame; With electrotransformation or chemical transformation, the emphasis plasmid is transformed in the yeast.Cultivated 24-96 hour containing on the YPD culture plate of Geneticin, screen positive transformed yeast bacterial strain.Choose through the PCR method and identify the recombination microzyme that carries chicken beta-alexin gene on its chromosomal DNA; Recombination microzyme in the MGY substratum, is cultivated OD for 28-38 ℃ in fermentor tank
600When value reaches 2-6, centrifugal collection yeast thalline.With the resuspended recombination microzyme of BMMY substratum, regular replenishment methyl alcohol also makes that the methyl alcohol final concentration remains on 0.20% in the nutrient solution, continues to cultivate after 96 hours centrifugal collection supernatant liquor; Concentrate culture supernatant, concentrated solution after SephacryI S24-96 hour, centrifugal collection supernatant liquor; Concentrate culture supernatant, can obtain the chicken beta-alexin of recombinating to concentrated solution through SephacryaI S-200 HR chromatography column purifying.
Three, according to technical solution of the present invention: a kind of production method and technology of the chicken beta-alexin of recombinating are: the encoding gene that the chicken beta-alexin gaIIinacin-3 of suitable endonuclease recognition site is introduced at two ends is inserted into the multiple clone site of expression vector pMG36, make this gene place the downstream of lactobacillus lactis cremoris subspecies Mg2 protein coding gene, and form correct reading frame; With electrotransformation recombinant plasmid transformed in Lactococcus lactis, containing on the antibiotic M17 agar culture plate cultivate 24 hours, screen positive transformed bacteria; Transforming bacterial strain static cultivation 24 hours in containing antibiotic M17-liquid of glucose substratum; Centrifugal collection recombinant lactic acid bacteria prepares recombinant plasmid from recombinant lactic acid bacteria; Identify the chicken beta-alexin gene that recombinant lactic acid bacteria is carried by plasmid with the PCR method; Positive recombinant lactic acid bacteria was cultivated 24 hours centrifugal collection reorganization thalline in M17.Ultrasonication reorganization thalline, and in centrifugal 30 minutes of 6000 rev/mins of speed, separation of supernatant; With supernatant liquor through the SephacryI S-200 HR chromatography column purifying chicken beta-alexin fusion rotein that can obtain to recombinate.
Claims (3)
1, a kind of production method and technology of the chicken beta-alexin of recombinating, mainly with intestinal bacteria reorganization chicken beta-alexin fusion rotein, it is characterized in that: with the encoding gene of RT-PCR technology amplification chicken beta-alexin gaIIinacin-3 from the total RNA of chicken epithelium mucosal tissue (tongue), and at the suitable endonuclease recognition site of the two ends of gene introducing, chicken beta-alexin encoding gene is inserted into the multiple clone site of coli expression carrier PGX-6P-1, make this gene place the downstream of glutathione S-transferase (GST) encoding gene, and form correct reading frame; Under 30-40 ° of environment, in substratum, add 0.1-2mMoI/L IPTG, shaking culture 2-10 hour, induce antigen-4 fusion protein gene to express, a kind of protein that molecular weight is about 34Kda can appear in the SDS-PAGE electrophoresis: the great enterobacteria after centrifugal collection is induced, bacteria breaking, high speed centrifugation is collected inclusion body, and uses the Triton buffer solution for cleaning with the ultrasonic cell disintegration instrument; With urea-containing lysis buffer dissolving inclusion body, with renaturation buffer dilution inclusion body lysate, make renaturing inclusion bodies then.
2, a kind of production method and technology of the chicken beta-alexin of recombinating, mainly with yeast secretary express recombinant chicken beta-alexin, it is characterized in that: the encoding gene that the chicken beta-alexin gaIIinacin-3 of suitable endonuclease identification point is introduced at two ends is inserted into the multiple clone site of Yeast expression carrier pPIC9K, make this gene place the downstream of a factor, and form correct reading frame; With electrotransformation or chemical transformation, the emphasis plasmid is transformed in the yeast.Cultivated 24-96 hour containing on the YPD culture plate of Geneticin, screen positive transformed yeast bacterial strain.Choose through the PCR method and identify the recombination microzyme that carries chicken beta-alexin gene on its chromosomal DNA; Recombination microzyme in the MGY substratum, is cultivated OD for 28-38 ℃ in fermentor tank
600When value reaches 2-6, centrifugal collection yeast thalline, with BMMY substratum resuspended recombination microzyme, regular replenishment methyl alcohol also makes that the methyl alcohol final concentration remains on 0.1-1.0% in the nutrient solution, continues to cultivate after 24-96 hour centrifugal collection supernatant liquor; Concentrate culture supernatant, can obtain the chicken beta-alexin of recombinating to concentrated solution through SephacryaI S-200 HR chromatography column purifying.
3, a kind of production method and technology of the chicken beta-alexin of recombinating, mainly with the structural expression of milk-acid bacteria, it is characterized in that: the encoding gene that the chicken beta-alexin gaIIinacin-3 of suitable endonuclease recognition site is introduced at two ends is inserted into the multiple clone site of expression vector pMG36, make this gene place the downstream of lactobacillus cremoris subspecies Mg2 protein coding gene, and form correct reading frame; With electrotransformation recombinant plasmid transformed in Lactococcus lactis, cultivate, screen positive transformed bacteria containing on the antibiotic M17 agar culture plate; Transforming bacterial strain static cultivation 12-28 hour in containing microbiotic M17-liquid of glucose substratum; Centrifugal collection recombinant lactic acid bacteria prepares recombinant plasmid from recombinant lactic acid bacteria; Identify the chicken beta-alexin gene that recombinant lactic acid bacteria is carried by plasmid with the PCR method; Positive recombinant lactic acid bacteria was cultivated in M17 24 hours, centrifugal collection reorganization thalline, ultrasonication reorganization thalline, and in centrifugal 30 minutes of 6000 rev/mins of speed, separation of supernatant; Supernatant liquor through the SephacryaI s-200 HR chromatography column purifying chicken beta-alexin fusion rotein that can obtain to recombinate.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100445371C (en) * | 2005-10-18 | 2008-12-24 | 甘肃亚盛盐化工业集团有限责任公司 | Production process of human-alpha phylaxin-1 protein with colibacillus |
| CN102190732A (en) * | 2011-03-22 | 2011-09-21 | 成都市金之源生物技术有限公司 | PGBD-2 fusion defensin, and preparation method and application thereof |
| CN102321712A (en) * | 2011-09-19 | 2012-01-18 | 广州和仕康生物技术有限公司 | A kind of alexinic preparation technology |
| CN104357399B (en) * | 2014-11-17 | 2018-02-13 | 武汉市畜牧兽医科学研究所 | A kind of cell line of stability and high efficiency expression chicken alexin 8 and application |
| CN111500616A (en) * | 2020-04-21 | 2020-08-07 | 北京大学第三医院（北京大学第三临床医学院） | Vector for expressing rat β -defensin 2, lactococcus lactis and application thereof |
-
2004
- 2004-02-07 CN CN 200410023566 patent/CN1651576A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100445371C (en) * | 2005-10-18 | 2008-12-24 | 甘肃亚盛盐化工业集团有限责任公司 | Production process of human-alpha phylaxin-1 protein with colibacillus |
| CN102190732A (en) * | 2011-03-22 | 2011-09-21 | 成都市金之源生物技术有限公司 | PGBD-2 fusion defensin, and preparation method and application thereof |
| CN102321712A (en) * | 2011-09-19 | 2012-01-18 | 广州和仕康生物技术有限公司 | A kind of alexinic preparation technology |
| CN102321712B (en) * | 2011-09-19 | 2014-08-20 | 广州和仕康生物技术有限公司 | Preparation process of defensins |
| CN104357399B (en) * | 2014-11-17 | 2018-02-13 | 武汉市畜牧兽医科学研究所 | A kind of cell line of stability and high efficiency expression chicken alexin 8 and application |
| CN111500616A (en) * | 2020-04-21 | 2020-08-07 | 北京大学第三医院（北京大学第三临床医学院） | Vector for expressing rat β -defensin 2, lactococcus lactis and application thereof |
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