CN102816226A - Preparation method of porcine small intestine antibacterial peptides PR39 - Google Patents
Preparation method of porcine small intestine antibacterial peptides PR39 Download PDFInfo
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- CN102816226A CN102816226A CN2012103059722A CN201210305972A CN102816226A CN 102816226 A CN102816226 A CN 102816226A CN 2012103059722 A CN2012103059722 A CN 2012103059722A CN 201210305972 A CN201210305972 A CN 201210305972A CN 102816226 A CN102816226 A CN 102816226A
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- chitterlings
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- 101100244894 Sus scrofa PR39 gene Proteins 0.000 title claims abstract description 19
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 210000000813 small intestine Anatomy 0.000 title abstract description 7
- 108090000765 processed proteins & peptides Proteins 0.000 title abstract description 4
- 230000000844 anti-bacterial effect Effects 0.000 title abstract 5
- 102000004196 processed proteins & peptides Human genes 0.000 title abstract 3
- 239000006228 supernatant Substances 0.000 claims abstract description 36
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 21
- 238000003756 stirring Methods 0.000 claims abstract description 12
- 229920005654 Sephadex Polymers 0.000 claims abstract description 9
- 239000012507 Sephadex™ Substances 0.000 claims abstract description 9
- 239000007788 liquid Substances 0.000 claims abstract description 7
- 229920000936 Agarose Polymers 0.000 claims abstract description 6
- 238000009792 diffusion process Methods 0.000 claims abstract description 6
- 239000000203 mixture Substances 0.000 claims abstract description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 6
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 3
- 210000004347 intestinal mucosa Anatomy 0.000 claims abstract description 3
- 239000003910 polypeptide antibiotic agent Substances 0.000 claims description 29
- 101100400378 Mus musculus Marveld2 gene Proteins 0.000 claims description 10
- 238000002386 leaching Methods 0.000 claims description 10
- 238000001641 gel filtration chromatography Methods 0.000 claims description 6
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims description 5
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 5
- 230000003385 bacteriostatic effect Effects 0.000 claims description 5
- 238000004108 freeze drying Methods 0.000 claims description 5
- 239000001632 sodium acetate Substances 0.000 claims description 5
- 235000017281 sodium acetate Nutrition 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 238000009210 therapy by ultrasound Methods 0.000 claims description 5
- 238000000034 method Methods 0.000 abstract description 5
- 230000008569 process Effects 0.000 abstract description 4
- 238000000605 extraction Methods 0.000 abstract description 3
- 108090000623 proteins and genes Proteins 0.000 abstract description 3
- 102000004169 proteins and genes Human genes 0.000 abstract description 3
- 238000009835 boiling Methods 0.000 abstract 1
- 238000001816 cooling Methods 0.000 abstract 1
- 238000005336 cracking Methods 0.000 abstract 1
- 238000001035 drying Methods 0.000 abstract 1
- 239000003480 eluent Substances 0.000 abstract 1
- 238000011010 flushing procedure Methods 0.000 abstract 1
- 238000007710 freezing Methods 0.000 abstract 1
- 230000008014 freezing Effects 0.000 abstract 1
- 239000005457 ice water Substances 0.000 abstract 1
- 238000002156 mixing Methods 0.000 abstract 1
- 239000002244 precipitate Substances 0.000 abstract 1
- 238000005070 sampling Methods 0.000 abstract 1
- 241000282898 Sus scrofa Species 0.000 description 9
- 201000010099 disease Diseases 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 230000000968 intestinal effect Effects 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 206010012735 Diarrhoea Diseases 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 238000005360 mashing Methods 0.000 description 3
- 210000004400 mucous membrane Anatomy 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 2
- 206010037660 Pyrexia Diseases 0.000 description 2
- 230000007123 defense Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 208000012153 swine disease Diseases 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- 108700042778 Antimicrobial Peptides Proteins 0.000 description 1
- 102000044503 Antimicrobial Peptides Human genes 0.000 description 1
- 208000034657 Convalescence Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 201000000297 Erysipelas Diseases 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 206010028470 Mycoplasma infections Diseases 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 230000012173 estrus Effects 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000010822 slaughterhouse waste Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
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- Peptides Or Proteins (AREA)
Abstract
The invention discloses a preparation method of porcine small intestine antibacterial peptides PR39, which comprises the following specific steps of: 1, taking porcine small intestinal mucosa and flushing; 2, adding a mixture of acetic acid and ice water with the volume fraction of 4-5%, and smashing to be made into a homogenate; 3, carrying out a boiling water bath for 10min, and cooling to 0-10 DEG C; 4, centrifuging, cracking a supernatant by ultrasonic waves for 30s, stirring and extracting, centrifuging, and taking the supernatant; 5, mixing a precipitate with acetic acid with the volume fraction of 4-5%, stirring and extracting, centrifuging, and taking the supernatant; 6, combining the supernatants, centrifuging and taking the supernatant; 7, sampling the supernatant to a Sephadex G2100 gel column for chromatography, and collecting an eluate; and 8, detecting the antibacterial property of liquid in each collection tube by an agarose diffusion method, collecting the eluent with an antibacterial effect to obtain the porcine small intestine antibacterial peptides PR39, and storing by freezing and drying in a vacuum. The process is simple, the cost is low, most of impure proteins are removed, and the extraction rate is high.
Description
Technical field
The present invention relates to the preparation method of a kind of chitterlings antibacterial peptide PR39, belong to biological pharmacy technical field.
Background technology
In recent years; Swine disease is increasing; Especially the abuse of medicine makes a lot of germs produce resistance; The appearance of some variation strains has increased difficulty for especially complicated swine disease prevention, to the development of pig industry produce very big obstruction grice diarrhoea and various virus disease such as swine fever breathe with breeding syndrome etc. be the thorny disease of aquaculture always.At present, sow and piglet health care more and more are familiar with by people, but this also is the difficult problem of pendulum in face of people.The application of antibacterial peptide can address this problem well.
Antibacterial peptide is one type of little peptide of defense that organism produces when resisting pathogenic microbes, is the important component part of biological immune system of defense.Be present in widely in insect, plant, animal and the human body.Antibacterial peptide has non-specific anti-gram positive organism, gram-negative bacteria, fungi, the effect of virus and the effect of killing tumor cell, but to normal eukaryotic cell nontoxic or low toxicity.At present, antibacterial peptide is widely used in a plurality of fields such as animal genetic engineering, drug development, industry, agricultural, livestock industry.Use antibacterial peptide and can impel oestrus of sow, increase sow milk, improve the piglet reguarity, reduce weak pig and cad pig, prevention baby pig diarrhoea, diarrhoea can also promote to search for food and improve the speed of growth simultaneously.When disease appears in swinery, in feed, add antibacterial peptide or Perorally administrable antimicrobial peptide liquid, control disease spreads and shortens the period of convalescence very soon; Explain that it has the assisting therapy effect to a lot of diseases, also begin to use the antibacterial peptide prevention clinically now and treat a lot of diseases, such as swine fever; Pig erysipelas, pig streptococcicosis, eperythrozoonosis; Blue otopathy etc. have all obtained fabulous effect.
Because the intestinal tissue complex structure, foreign protein is of a great variety, the technology of the extraction antibacterial peptide that adopts at present, and complicated operation often will pass through the antibacterial peptide that the separating for several times purifying just can obtain purifying, cost of idleness, and also yield is not high yet.
Summary of the invention
The preparation method who the purpose of this invention is to provide a kind of chitterlings antibacterial peptide PR39 produces resistance to solve virus to medicine, reduces cost, and improves yield.
In order to realize above purpose, the technical scheme that the present invention adopted is to provide the preparation method of a kind of chitterlings antibacterial peptide PR39, and concrete steps are following:
1) getting pig intestinal mucosa washes with sterile saline;
2) add the Acetic Acid Glacil water mixture of volume(tric)fraction 4-5% according to the ratio of 20ml/g, smash system homogenate to pieces;
3) 90-100 ℃ of water-bath 10min, after be cooled to 0-10 ℃ rapidly;
4) centrifugal, get supernatant and put ultrasonic treatment 30s on ice, 4 ℃ of stirring and leaching, centrifugal again, get supernatant;
5) acetate of centrifugal throw out with isopyknic volume(tric)fraction 4-5% is mixed, stirring and leaching, centrifugal, get supernatant;
6) combining step 4) and the supernatant that obtains of step 5), regulate pH=5-6.5, centrifugal, get supernatant;
7) supernatant is splined on Sephadex G2100 gel filtration chromatography, collects elutriant;
8) adopt the agarose diffusion method to detect the bacterinertness of each collection tube liquid, collect the elutriant that bacteriostatic action is arranged, obtain piglet small intestine antibacterial peptide PR39, vacuum lyophilization is preserved.
Described Sephadex G2100 chromatography elutriant is the 0.2mol/L sodium acetate solution, and the speed of constant flow pump is 30r/min.
Described centrifuging temperature is 4 ℃, and rotating speed is 8000r/min, and the time is 30min.
The present invention adopts Sephadex G2100 gel filtration chromatography, compared with prior art, disposablely can obtain chitterlings antibacterial peptide PR39, has removed macromolecular substance, and method is simple, has practiced thrift time and cost, and the chitterlings antibacterial peptide purity that obtains is higher.
The present invention adopts in the intestines mucosa of slaughterhouse waste and extracts antibacterial peptide, has effectively utilized waste, has not only reduced the pollution of environment, and has turned waste into wealth, and has good economic benefit.Technology of the present invention is simple, and is with low cost, and ultrasonic degradation is to let albumen wherein discharge, and adopts the separator column chromatography to remove most foreign protein, can once obtain the antibacterial peptide of purifying, and extraction yield is high.The antibiotic Toplink of the present invention preparation overcomes the shortcoming of the spinoff that resistance that microbiotic produces and vaccine produced, and body is not had toxic action.
Embodiment
Embodiment 1
The preparation method of the chitterlings antibacterial peptide PR39 that present embodiment provides, concrete steps are following:
1) gets chitterlings, remove fat and content, take by weighing the 20g mucous membrane of small intestine, wash with sterile saline;
2) add the Acetic Acid Glacil water mixture of volume(tric)fraction 5% according to the ratio of 20ml/g, process homogenate through tissue mashing machine;
3) 100 ℃ of water-bath 10min, after be cooled to 0 ℃ rapidly;
4) 4 ℃, the centrifugal 30min of 8000rpm gets supernatant and puts ultrasonic treatment 30s on ice, 4 ℃ of stirring and leaching, and the centrifugal 30min of 8000rpm gets supernatant;
5) the centrifugal throw out is mixed with the acetate of isopyknic volume(tric)fraction 5%, stirring and leaching once more, the centrifugal 30min of 8000rpm gets supernatant;
6) combining step 4) and the supernatant that obtains of step 5), regulating pH=6, the centrifugal 30min of 8000rpm gets supernatant;
7) supernatant is splined on Sephadex G2100 gel filtration chromatography, with 0.2mol/L sodium acetate wash-out, the speed of constant flow pump is 30r/min, after the nucleic acid-protein detector detects, collects every pipe 1.5mL with automatic collector;
8) adopt the agarose diffusion method to detect each collection tube liquid to the intestinal bacteria bacterinertness, collect the elutriant that bacteriostatic action is arranged, obtain chitterlings antibacterial peptide PR39, vacuum lyophilization is preserved.
Embodiment 2
The preparation method of the chitterlings antibacterial peptide PR39 that present embodiment provides, concrete steps are following:
1) gets chitterlings, remove fat and content, take by weighing the 20g mucous membrane of small intestine, wash with sterile saline;
2) add the Acetic Acid Glacil water mixture of volume(tric)fraction 4.5% according to the ratio of 20ml/g, process homogenate through tissue mashing machine;
3) 90 ℃ of water-bath 10min, after be cooled to 5 ℃ rapidly;
4) 4 ℃, the centrifugal 30min of 8000rpm gets supernatant and puts ultrasonic treatment 30s on ice, 4 ℃ of stirring and leaching, and the centrifugal 30min of 8000rpm gets supernatant;
5) the centrifugal throw out is mixed with the acetate of isopyknic volume(tric)fraction 4.5%, stirring and leaching once more, the centrifugal 30min of 8000rpm gets supernatant;
6) combining step 4) and the supernatant that obtains of step 5), regulating pH=6.5, the centrifugal 30min of 8000rpm gets supernatant;
7) supernatant is splined on Sephadex G2100 gel filtration chromatography, with 0.2mol/L sodium acetate wash-out, the speed of constant flow pump is 30r/min, after the nucleic acid-protein detector detects, collects every pipe 1.5mL with automatic collector;
8) adopt the agarose diffusion method to detect each collection tube liquid to the intestinal bacteria bacterinertness, collect the elutriant that bacteriostatic action is arranged, obtain chitterlings antibacterial peptide PR39, vacuum lyophilization is preserved.
Embodiment 3
The preparation method of the chitterlings antibacterial peptide PR39 that present embodiment provides, concrete steps are following:
1) gets chitterlings, remove fat and content, take by weighing the 20g mucous membrane of small intestine, wash with sterile saline;
2) add the Acetic Acid Glacil water mixture of volume(tric)fraction 4% according to the ratio of 20ml/g, process homogenate through tissue mashing machine;
3) 95 ℃ of water-bath 10min, after be cooled to 10 ℃ rapidly;
4) 4 ℃, the centrifugal 30min of 8000rpm gets supernatant and puts ultrasonic treatment 30s on ice, 4 ℃ of stirring and leaching, and the centrifugal 30min of 8000rpm gets supernatant;
5) the centrifugal throw out is mixed with the acetate of isopyknic volume(tric)fraction 4%, stirring and leaching once more, the centrifugal 30min of 8000rpm gets supernatant;
6) combining step 4) and the supernatant that obtains of step 5), regulating pH=5, the centrifugal 30min of 8000rpm gets supernatant;
7) supernatant is splined on Sephadex G2100 gel filtration chromatography, with 0.2mol/L sodium acetate wash-out, the speed of constant flow pump is 30r/min, after the nucleic acid-protein detector detects, collects every pipe 1.5mL with automatic collector;
8) adopt the agarose diffusion method to detect each collection tube liquid to the intestinal bacteria bacterinertness, collect the elutriant that bacteriostatic action is arranged, obtain chitterlings antibacterial peptide PR39, vacuum lyophilization is preserved.
Claims (3)
1. the preparation method of a chitterlings antibacterial peptide PR39 is characterized in that concrete steps are following:
1) getting pig intestinal mucosa washes with sterile saline;
2) add the Acetic Acid Glacil water mixture of volume(tric)fraction 4-5% according to the ratio of 20ml/g, smash system homogenate to pieces;
3) 90-100 ℃ of water-bath 10min, after be cooled to 0-10 ℃ rapidly;
4) centrifugal, get supernatant and put ultrasonic treatment 30s on ice, 4 ℃ of stirring and leaching, centrifugal again, get supernatant;
5) acetate of centrifugal throw out with isopyknic volume(tric)fraction 4-5% is mixed, stirring and leaching, centrifugal, get supernatant;
6) combining step 4) and the supernatant that obtains of step 5), regulate pH=5-6.5, centrifugal, get supernatant;
7) supernatant is splined on Sephadex G2100 gel filtration chromatography, collects elutriant;
8) adopt the agarose diffusion method to detect the bacterinertness of each collection tube liquid, collect the elutriant that bacteriostatic action is arranged, obtain chitterlings antibacterial peptide PR39, vacuum lyophilization is preserved.
2. the preparation method of a kind of chitterlings antibacterial peptide PR39 according to claim 1 is characterized in that, described Sephadex G2100 chromatography elutriant is the 0.2mol/L sodium acetate solution, and the speed of constant flow pump is 30r/min.
3. the preparation method of a kind of chitterlings antibacterial peptide PR39 according to claim 1 is characterized in that, described centrifuging temperature is 4 ℃, and rotating speed is 8000rpm/min, and the time is 30min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012103059722A CN102816226A (en) | 2012-08-23 | 2012-08-23 | Preparation method of porcine small intestine antibacterial peptides PR39 |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012103059722A CN102816226A (en) | 2012-08-23 | 2012-08-23 | Preparation method of porcine small intestine antibacterial peptides PR39 |
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| Publication Number | Publication Date |
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| CN102816226A true CN102816226A (en) | 2012-12-12 |
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| CN2012103059722A Pending CN102816226A (en) | 2012-08-23 | 2012-08-23 | Preparation method of porcine small intestine antibacterial peptides PR39 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103417574A (en) * | 2013-08-08 | 2013-12-04 | 福州派生特生物科技有限公司 | Preparation method of recombinant antimicrobial peptide extracted from small intestine of pig and application thereof |
| CN104448006A (en) * | 2014-12-13 | 2015-03-25 | 青岛宏昊生物科技有限公司 | Hybrid antibacterial peptide CE-PR and application thereof |
| CN107345207A (en) * | 2017-06-29 | 2017-11-14 | 王德亮 | With animal intestinal mucosa production enteric microorganism powder co-production small-molecular peptides, liquaemin, the method for alkaline phosphatase and application |
-
2012
- 2012-08-23 CN CN2012103059722A patent/CN102816226A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| 张继杰: "《中药化学》", 31 October 1994, 人民卫生出版社 * |
| 马卫明 等: "猪小肠抗菌肽的提取及部分生物学活性研究", 《科学技术与工程》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103417574A (en) * | 2013-08-08 | 2013-12-04 | 福州派生特生物科技有限公司 | Preparation method of recombinant antimicrobial peptide extracted from small intestine of pig and application thereof |
| CN103417574B (en) * | 2013-08-08 | 2015-10-28 | 福州派生特生物科技有限公司 | The Preparation method and use of pig small intestine recombinant antimicrobial peptide |
| CN104448006A (en) * | 2014-12-13 | 2015-03-25 | 青岛宏昊生物科技有限公司 | Hybrid antibacterial peptide CE-PR and application thereof |
| CN107345207A (en) * | 2017-06-29 | 2017-11-14 | 王德亮 | With animal intestinal mucosa production enteric microorganism powder co-production small-molecular peptides, liquaemin, the method for alkaline phosphatase and application |
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Application publication date: 20121212 |