CN103417574B - The Preparation method and use of pig small intestine recombinant antimicrobial peptide - Google Patents
The Preparation method and use of pig small intestine recombinant antimicrobial peptide Download PDFInfo
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Abstract
The invention discloses a kind of Preparation method and use of pig small intestine recombinant antimicrobial peptide, the present invention first adopts meat grinder, colloid mill by the homogenate of pig small intestine tissue mashing, multigelation, preliminary smudge cells.Carry out water proof subsequently and boil heat treatment, then the antibacterial peptide of more amount can be obtained with acetic acid solution lixiviate.Centrifugal afterwards, the amount of foreign protein is reduced greatly, and centrifugal gained supernatant is successively through slipstream micro-filtration membrane, cross-flow ultrafiltration membrane filtration, after adjust ph, and dialysis desalination concentrated through slipstream nanofiltration membrane, degerming after regulating osmotic pressure again, obtains pig small intestine recombinant antimicrobial peptide.The purposes of the pig small intestine recombinant antimicrobial peptide that the present invention proposes, use the effect of antibacterial peptide in mucosal immunity, immunostimulant, new antibiotic etc., antibacterial peptide is used for mucosal immunity and plays the transmissible gastroenteritis of swine-epidemic diarrhea of critical function and the prevention and control of chicken virus mycoplasma disease, and alternative conventional antibiotic, Be very effective.
Description
Technical field
The invention belongs to animal and veterinary, biological product, field of biological pharmacy, be specifically related to a kind of Preparation method and use of pig small intestine recombinant antimicrobial peptide.
Background technology
Antibacterial peptide is the small peptide of the class antimicrobial that produces in the defense reaction process resisting pathogenic microorganism of living organism and some malignant cells." antimicrobial peptide " (Antimicrobial peptides, AMPs) or " peptide antibiotic " (peptide antibiotics) is used in some international literatures.Antibacterial peptide has antibacterial action, and some also has protozoacide, antifungal, antiviral and suppression or the effect such as killing tumor cell, parasite.It has the features such as molecular weight little (3-10 KD), good water solubility, heat-resisting, non-immunogenicity, can become a kind of efficient, low toxicity and antibacterial, the antiviral of noresidue harm and PTS.
A lot of microorganism at present creates extensive drug resistance to conventional antibiotic, and antibacterial peptide still remains its powerful antibacterial efficacy in the evolution course that have passed through millions of years, and microorganism is not easily developed immunity to drugs to it.Due to the antibacterial activity of antibacterial peptide broad-spectrum high efficacy and the antibacterial mechanisms of uniqueness thereof, make antibacterial peptide especially noticeable.
To 2003, Italian Trieste university antibacterial peptide data base collects 28 boar derived antimicrobial peptides or antibacterial peptide precursor molecule, and the antibacterial peptide or the antibacterial peptide precursor molecule that wherein derive from pig small intestine have 8 kinds:
| Molecular weight | Antibacterial peptide kind | Concrete molecular weight |
| 3000-8000D | 5 kinds | 3339D、3910.5D、4720D、5972D、6150D |
| 8000-10000D | 3 kinds | 8178D、8925D、9341D |
| More than 10000D | 1 kind | 19495D |
New Antibacterial Peptide Extracted from Pig Small Intestine is had again to be found, as 5972Da Antibacterial Peptide Extracted from Pig Small Intestine is found by Ma Weiming afterwards successively.
Due to intestinal tissue complex structure, foreign protein is of a great variety, and antibacterial peptide is usually present in cytoplasmic granule, and its molecular weight is lower, and content is in the tissue lower, and thus abstraction and purification is more difficult.The method in the past extracting micromolecule polypeptide mainly contains: method of organic solvent extraction, water extract method and organic acid extraction process etc.Then the centrifugal crude extract obtained contains protein, salt and other micromolecule.Next step separation and purification, method comprises: filtration chromatography (medium has Sephadex, Biogel, Sephacryl), ion-exchange chromatography, hydrophobic exchange chromatography, gel electrophoresis and reverse-phase HPLC etc.As: someone adopts water proof to boil rear acid extraction, centrifugally obtains crude extract, and successively through Sephadex G-100 gel column, Sephadex G-25 or BiogelP10 gel filtration column chromatography, the protein do not waited by molecular size respectively and salt are separately.Preliminary purification gained Antibacterial Peptide Extracted from Pig Small Intestine semifinished product, through 10kD ultra-filtration centrifuge tube except thermal source, then is further purified through 3-5KD ultra-filtration centrifuge tube.These method complicated operations, just will can obtain antibacterial peptide through separation and purification repeatedly, cost of idleness, yield is not high, and what often obtain is the Antibacterial Peptide Extracted from Pig Small Intestine of unimodal molecular weight, is difficult to obtain recombinant antimicrobial peptide.
In membrane separation technique, usually there is the filtration of two types: conventional vertical filters (Normal FlowFiltration, NFF) and tangential flow filtration (Tangential Flow Filtration, TFF).In conventional vertical filters, all fluids are directly by filter membrane, and the material buildup film be trapped is surperficial.Due to the accumulation of these materials, decline rapidly until stop completely, as Fig. 1 by the flow of filter membrane; And in tangential flow filtration, fluid flows through filter membrane as shown in Figure 2 and Figure 3 cross.
Tangential flow filtration filters different from conventional vertical, and liquid tangentially flows through film surface, and part solution is pressed through filter membrane by the transmembrane pressure that fluid produces, and retains part then circulating reflux in systems in which.In whole process, liquid continues to flow through filter membrane surface with certain speed, also washes away filter membrane surface while filtration, makes film surface not form gel layer, thus makes the granule in feed liquid can not block filter membrane very soon, maintains the stable rate of filtration.
Microorganism requires isotonic to residing osmotic pressure, and the osmotic pressure value of isosmotic solution is 280-320mosm/kg.Osmotic pressure value claims hypisotonic solution, as distilled water etc. lower than the solution of this value; Osmotic pressure value claims hyperosmotic solution higher than the solution of this value, as the Glucose Liquid of 10% or the Glucose Liquid etc. of 50%.Microorganism is in hyperosmotic solution, plasmolysis shrinkage easily occurs dead; If be in hypisotonic solution on the contrary, easy imbibition is broken death.
Application number is the preparation method that patent discloses a kind of Antibacterial Peptide Extracted from Pig Small Intestine PR39 of 201210305972.2, it adopts the albumen in ultrasonic degradation release intestinal mucosa, detached dowel chromatography is adopted to remove most foreign protein, obtain antibacterial peptide, the method is extracted monistic PR39 antibacterial peptide from intestinal mucosa, complex process, yield be not high, be not suitable for large-scale production, and gained Antibacterial Peptide Extracted from Pig Small Intestine PR39 concentration is not high.
Summary of the invention
First technical problem that the present invention will solve is to provide a kind of preparation method of pig small intestine recombinant antimicrobial peptide, and the method comprises the steps:
(1) fresh pig small intestinal is clean with deionized water rinsing, remove content, remove serous coat and degrease after use water for injection cleaning down, then freezingly shred, meat grinder just twists 1-3 time, then uses colloid mill homogenate, makes homogenate;
Pig small intestine of the present invention refer to arbitrary section or several sections of pig duodenum, jejunum or ileum;
(2) by after described homogenate multigelation 3-6 time, 100 DEG C of water proofs boil 15-25min, then the rear cold preservation of cooling rapidly, and refrigerated storage temperature is 1-4 DEG C, and cold preservation time is 0-5 hour;
(3) in homogenate, the acetic acid solution that concentration is 5.00% is added, stirring and leaching, it is centrifugal that 4 DEG C of conditions carry out first time, by the supernatant freezen protective centrifugal first time, described concentration be 5.00% acetic acid solution water for injection prepare, the volume ratio of described acetic acid solution and homogenate is 1:1;
(4) in precipitate centrifugal for the first time, the acetic acid solution that concentration is 6.67%-5.00% is added, stirring and leaching, it is centrifugal that 4 DEG C of conditions carry out second time, and by supernatant freezen protective centrifugal for second time, described concentration is that the acetic acid solution water for injection of 6.67%-5.00% is prepared;
(5) in the precipitate that second time is centrifugal, the acetic acid solution that concentration is 8.75%-5.63% is added again, stirring and leaching, it is centrifugal that 4 DEG C of conditions carry out third time, and obtain third time centrifugal supernatant, described concentration is that the acetic acid solution water for injection of 8.75%-5.63% is prepared;
(6) merge above-mentioned three supernatant, by the supernatant that merges through 0.1,0.22 or 0.45um slipstream micro-filtrate membrane filtration, collect MF permeate thing, obtain clear liquor; Clear liquor, through 8 or 10KD cross-flow ultrafiltration membrane filtration, is collected ultrafiltration through thing, is obtained ultrafiltrate; Regulate ultrafiltrate pH value to 6.5-7.5, obtain pig small intestine recombinant antimicrobial peptide semifinished product;
(7) pig small intestine recombinant antimicrobial peptide semifinished product is concentrated and dialysis desalination through 1 or 3KD slipstream nanofiltration membrane, collect nanofiltration retentate; Regulate osmotic pressure to 280-320mosm/kg, more degerming through the sterilizing filter of 0.1um, obtain pig small intestine recombinant antimicrobial peptide.
Further, step (3), (4) described cryogenic temperature are less than-20 DEG C, and cooling time is no more than 30 days; Step (3), (4), (5) described stirring and leaching are for stirring 8-14 hour, and temperature is 1-6 DEG C; Step (3), (4), (5) described centrifugation time are 15-25min, and centrifuge speed is 6000-8000rpm.
Further, described first time centrifugal sediment and the weight/volume (W:V) of acetic acid solution be 1Kg:0.6-1L.
Further, the weight/volume (W:V) of described second time centrifugal sediment and acetic acid solution is 1Kg:0.4-0.8L.
The present invention also will protect the pig small intestine recombinant antimicrobial peptide adopting this preparation method to obtain.
Second technical problem that the present invention will solve is to provide a kind of purposes of pig small intestine recombinant antimicrobial peptide, and described pig small intestine recombinant antimicrobial peptide uses as poultry new antibiotic, immunostimulant and immunological adjuvant.
Further, described pig small intestine recombinant antimicrobial peptide separately or with vaccine combined immunization.
Further, described pig small intestine recombinant antimicrobial peptide 1-6mL separately or with transmissible gastroenteritis of swine-epidemic diarrhea vaccine 1-3 head part combined immunization every pig.
Further, described pig small intestine recombinant antimicrobial peptide 0.01-0.2mL separately or with the every plumage chicken of chicken virus mycoplasma vaccine 1-3 plumage part combined immunization.
The present invention has following beneficial effect:
The present invention first adopts meat grinder, colloid mill by the homogenate of pig small intestine tissue mashing, multigelation, preliminary smudge cells.Carry out water proof subsequently and boil heat treatment, the further cell lysis of one side heat treatment mode, the antibacterial peptide in endochylema is discharged as much as possible, make foreign protein degeneration on the other hand, then the antibacterial peptide of more amount can be obtained with acetic acid solution lixiviate.Centrifugal afterwards, the amount of foreign protein is reduced greatly.Centrifugal gained supernatant successively through 0.1,0.22 or 0.45um slipstream micro-filtration membrane, 8 or 10KD cross-flow ultrafiltration membrane filtration, that is: the mode adopting slipstream to filter step by step, technique is simple, production efficiency and antibacterial peptide yield high.After regulating pH value, concentrated and desalination process adopts 1 or 3KD slipstream nanofiltration membrane, and than adopting Rotary Evaporators deacidification desalination or adopting Sephadex G-25, Biogel P10 isogel post filtration chromatography, technique is more efficient, practical, is applicable to large-scale production.Carry out osmotic pressure to be subsequently adjusted to isotonic (280-320mosm/kg), isosmotic solution had both been conducive to reducing immunological stress, was conducive to again the combined immunization of gained pig small intestine recombinant antimicrobial peptide and vaccine.Sterilizing filter finally by 0.1um is degerming, than the sterilizing filter adopting 0.22um, is more conducive to removing the microorganisms such as mycoplasma.
Present invention process is simply efficient, and gained pig small intestine composite antibacterial peptide concentration is high, yield is high.
The purposes of the pig small intestine recombinant antimicrobial peptide that the present invention proposes, use the effect of antibacterial peptide in mucosal immunity, immunostimulant, new antibiotic etc., antibacterial peptide is used for mucosal immunity and plays the transmissible gastroenteritis of swine-epidemic diarrhea of critical function and the prevention and control of chicken virus mycoplasma disease, Be very effective.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail
Fig. 1 is that conventional vertical filters schematic diagram;
Fig. 2 is tangential flow filtration cleaning effect schematic diagram;
Fig. 3 is tangential flow filtration schematic diagram.
Detailed description of the invention
Embodiment 1
1, the preparation of pig small intestine recombinant antimicrobial peptide
(1) by 7.5Kg fresh pig small intestinal (comprising duodenum, jejunum), clean with deionized water rinsing, remove content, remove serous coat and degrease, clean with water for injection cleaning down, freezingly to shred, meat grinder just twists 2 times and obtain pig small intestine colloid;
(2) by above-mentioned pig small intestine colloid through colloid mill homogenate, obtained tissue homogenate 3.8L, is then sub-packed in-40 DEG C, apyrogenic bottle freezing by homogenate, 37 DEG C of flowing water thaw, homogenate 100 DEG C of water proofs are boiled 15min, are cooled to rapidly 4 DEG C by multigelation 3 times;
Under (3) 4 DEG C of conditions, it is the acetic acid solution 3.8L stirring and leaching 12 hours of 5.00% by the 3.8L homogenate after freeze thawing and concentration, it is centrifugal that mixture after stirring and leaching carries out first time in 4 DEG C of condition 8000rpm, centrifugal 15min, obtain first time supernatant 4.4L,-20 DEG C of preservations, described concentration be 5.00% acetic acid solution water for injection prepare;
Under (4) 4 DEG C of conditions, the acetic acid solution 2.24L(W:V being 6.07% by first time centrifugal gained precipitate 3.2Kg and concentration is 1:0.7) stirring and leaching 12 hours, repeat above-mentioned centrifugal process, obtain second time supernatant 1.54L,-20 DEG C of preservations, described concentration be 6.07% acetic acid solution water for injection prepare;
Under (5) 4 DEG C of conditions, the acetic acid solution 1.95L(W:V being 7.5% by the centrifugal gained precipitate 3.9Kg of second time and concentration is 1:0.5) stirring and leaching 12 hours, repeat above-mentioned centrifugal process again, obtain third time supernatant 1.32L, described concentration be 7.5% acetic acid solution water for injection prepare;
(6) merge above-mentioned three centrifugal gained 7.26L supernatant, by supernatant through 0.45um micro-filtrate membrane filtration, 8KD ultrafiltration membrance filter, collect ultrafiltration through thing, obtain ultrafiltrate; Regulate ultrafiltrate pH value to 7.0, obtain pig small intestine recombinant antimicrobial peptide semifinished product;
Wherein, a.0.45um microfiltration is by 7.26L supernatant, through 0.45um boxlike film tangential flow systems (Millipore company Pellicon2Durapore0.45um1.0m
2film bag) microfiltration clarification (5 times concentrate), obtain microfiltration clear liquor 5.81L, MF retentate 1.45L does 3 times of equal-volumes dialysis again and to get back clear liquor 4.35L, obtains 10.16L clear liquor altogether.
0.45um boxlike film tangential flow systems mean parameter is as follows:
B.8KD ultrafiltration is by microfiltration clear liquor 10.16L, through 8KD boxlike film tangential flow systems (Millipore company Pellicon2Biomax8KD0.5m
2film bag) ultrafiltration (15 times concentrate), ultrafiltration obtains ultrafiltrate 9.48L, and ultrafiltration retentate 0.68L does 3 times of equal-volumes dialysis again and to get back ultrafiltrate 2.04L, obtains 11.52L ultrafiltrate altogether, regulates ultrafiltrate pH value to 7.0, be pig small intestine recombinant antimicrobial peptide semifinished product.
8KD boxlike film tangential flow systems mean parameter is as follows:
(7) pig small intestine recombinant antimicrobial peptide semifinished product is concentrated and dialysis desalination through 1KD nanofiltration membrane, collect nanofiltration retentate; After the osmotic pressure of the nanofiltration retentate of collection is adjusted to 280mosm/kg, the sterilizing filter through 0.1um is degerming, obtains pig small intestine recombinant antimicrobial peptide 8.1L.
Wherein, a.1KD concentrated the and dialysis desalination of nanofiltration is by pig small intestine recombinant antimicrobial peptide semifinished product, through 1KD boxlike film tangential flow systems (Millipore company Pellicon2PLAC1KD2.0m
2film bag) nanofiltration concentrated (1.3 times concentrate), collect nanofiltration retentate 8.86L, do 2 times of equal-volume dialysis with 17.72L water for injection for dialysis solution again, obtain 8.86L nanofiltration concentrate dialysate altogether, the osmotic pressure of the nanofiltration concentrate dialysate of collection is adjusted to 280mosm/kg;
1KD boxlike film tangential flow systems mean parameter is as follows:
B.0.1um sterilizing filter is degerming is by 8.86L nanofiltration concentrate dialysate, and degerming through 0.1um sterilizing filter, obtain pig small intestine recombinant antimicrobial peptide 8.1L, quality inspection is qualified.
Pig small intestine recombinant antimicrobial peptide embodiment 1 prepared is as poultry new antibiotic, immunostimulant and immunological adjuvant, and pig small intestine recombinant antimicrobial peptide 0.02mL is the every plumage chicken of immunity separately.
2, the quality inspection method of pig small intestine recombinant antimicrobial peptide
(1) character this product is micro-yellow or light yellow transparent liquid;
(2) steriling test this product is tested by existing " Chinese veterinary pharmacopoeia " annex, asepsis growth;
(3) pH value and osmometry this product are tested by existing " Chinese veterinary pharmacopoeia " annex, and pH value should be 6.5-7.5, and osmotic pressure value should be 280-320mosm/kg;
(4) 20% sulfosalicylic acid 2ml and sample 2ml mixes by protein qualitative determination, without turbidity and precipitation after reaction, reacts for feminine gender;
(5) determining content of peptides this product measures by Forint phenol method, and every milliliter of content of peptides should be not less than 2.0mg;
Wherein Forint phenol method
Reagent
Alkaline copper Solutions Solution I carries out 5 times and dilutes rear and solution II, and mix with the ratio of 50:1, be alkaline copper solution after mixing, this reagent can only with 1 day, expire.
Phenol solution
Maneuver
1., the preparation of reference substance solution gets bovine serum albumin reference substance, adds water in making every milliliter containing the solution of 250ug.
2., that this product is got in the preparation of need testing solution is appropriate, carries out suitably dilution as need testing solution with water.
3., 7 test tubes are got in the preparation of standard curve, precision measure reference substance solution 0.0,0.1,0.2,0.4,0.6,0.8,1.0ml, put respectively in band plug scale test tube, respectively add water to 1.0ml, then add alkaline copper solution 5.0ml respectively, shake up, put 10min in ambient temperatare, respectively add phenol solution 0.5ml, mix immediately, put room temperature and place 30min, then measure trap in the wavelength place of 500nm; Simultaneously not add solution in standard protein test tube (0 pipe) as blank, with reference substance solution concentration for abscissa, trap is vertical coordinate, and drawing standard curve line linearity of going forward side by side returns, and correlation coefficient is not less than 0.99.
4., algoscopy precision measures need testing solution 1.0ml, the method under the preparation of sighting target directrix curve, from " adding alkaline copper solution respectively again ", with 0 pipe in standard curve for blank, measures trap in accordance with the law.From regression equation, try to achieve the concentration of need testing solution according to trap, and be multiplied by extension rate, obtain test sample content of peptides.
(6) Ribose concentration measures this product and measures by Ribose concentration algoscopy, and every milliliter of Ribose concentration should lower than 85 μ g;
Ribose concentration algoscopy
Reagent
1., 5.00% trichloroacetic acid takes trichloroacetic acid 5g, and adding distil water is dissolved into 100ml.
2., 0.1% ferric chloride-hydrochloric acid solution takes ferric chloride (FeCl
36H
2o) 0.5g, enriching dissolving with hydrochloric acid becomes 500ml, can life-time service.
3., 1%3,5-orcins (orcin) take 3,5-orcin 1g, being dissolved in 100ml0.1% ferric chloride-hydrochloric acid solution, facing with newly joining.
Operational approach
The preparation precision of reference substance solution takes D ribose reference substance in right amount, makes every milliliter containing the solution of ribose 20ug, shake up with 5.00% trichloroacetic acid.
The preparation of need testing solution gets this product in right amount, carries out suitably dilution as need testing solution with 5.00% trichloroacetic acid.
The preparation precision of standard curve measures reference substance solution 0.0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0ml, put in band plug scale test tube respectively, respectively add 5.00% trichloroacetic acid solution to 2.0ml, add 1%3 respectively again, 5-orcin solution 2.0ml, shake up, put in boiling water bath and accurately heat 30min, be cooled to room temperature rapidly, with No. 0 pipe for blank, according to spectrophotography (see existing " Chinese Pharmacopoeia " annex), trap is measured at the wavelength place of 650nm, take concentration as abscissa, trap is vertical coordinate, drawing standard curve go forward side by side line linearity return, correlation coefficient is not less than 0.995.
Algoscopy precision measures need testing solution 2.0ml, the preparation method of sighting target directrix curve, rises, measure trap in accordance with the law from " adding 1%3,5-orcin solution 2.0ml more respectively ".From regression equation, try to achieve the concentration of need testing solution according to trap, and be multiplied by extension rate, obtain test sample Ribose concentration.
(7) bacterial endotoxin measures this product and measures by detection of bacterial endotoxin method, and every milliliter of endotoxin content should be no more than 10EU;
Detection of bacterial endotoxin method
Detection of bacterial endotoxin test kit is adopted to detect.
Preheating utensil used in 37 DEG C of incubators.
Test sample sodium chloride injection (endotoxin < 0.125EU/ml) is done 10 times of dilutions, to be measured.
Get the test sample 0.5ml after dilution and carefully add sample detection pipe (SPL), and shake up gently.
Add Sample Positive detector tube (PPC) with the test sample that 0.25ml drawn carefully by pyrogen-free suction pipe from sample detection pipe and mix 60 seconds gently.
By completely mixing after sample detection pipe and Sample Positive detector tube be positioned over 37 DEG C cultivate 30min.
Be inverted sample detection pipe and Sample Positive detector tube, observe with or without coagulation.
Sentence test when the complete coagulation of positive control detector tube (PPC) to set up.
If sample detection pipe has agglutination, illustrate that in this dilution test sample, endotoxic content is not less than 1.0EU/ml; If sample detection Guan Zhongwu agglutination, illustrate that in this dilution test sample, endotoxin content is lower than 1.0EU/ml.Endotoxin content in former test sample is calculated according to dilution factor.
(8) safety verification
Pyrogen inspection adopts body weight 1.7-3.0Kg healthy rabbits (doe should without pregnant) 3, raises 2 separately, without exception.Survey body temperature once every 30min, survey twice, twice temperature difference is no more than 0.2 DEG C, using the meansigma methods of this body temperature of twice as the normal body temperature of this rabbit.The rabbit that the same day uses, normal body temperature is in the scope of 38.0-39.6 DEG C, and between each rabbit, regular using warming therapy difference is no more than 1 DEG C.The syringe tested, syringe needle and the vessel that all contact with this product, pyrogen of should going out.Rabbit is in its normal body temperature 15min of mensuration, this product that prescribed dose (injection volume is per kilogram of body weight 0.5ml) is warmed to about 38 DEG C is slowly injected from ear vein, then every 30min thermometric once, survey 6 times altogether, normal body temperature is once deducted with the highest in 6 body temperature, be the body temperature (when rabbit is heated up as negative value, all in 0 DEG C) that this rabbit raises.The temperature that 3 rabbit body temperatures raise all should lower than 0.6 DEG C, and 3 rabbit body temperatures raise summations should lower than 1.4 DEG C.
Check with 1 age in days SPF chickling 10 with chicken, after this product physiological saline solution is diluted 10 times, every oral 1ml of chicken, 24 hours repeat once oral, observe 5.Chickling all should be good for and be lived, and spirit, search for food, drink water, feather, feces should be all without exception.
Abnormal toxicity test body weight 18-22g healthy guinea pig 5, every mouse peritoneal injects this product 0.5ml, observes 7.Mice all should be good for and be lived, and spirit, search for food, drink water, feather, feces should be all without exception.
Animal allergy inspection body weight 250-350g healthy guinea pig 6, the next day every only each lumbar injection this product 1.0ml, totally three times, carry out sensitization.Then be divided into 2 groups, often organized 3, after injecting first the 14th day and the 21st day respectively, attacked by intramuscular injection this product 1.0ml.Observe behavior and the sign of every animal every day.Observe after attacking in 30min, animal is without symptoms of allergic such as perpendicular hair, dyspnea, tics.In intramuscular injection this product 2 hours, anaphylaxis must not be there is.If any 2 kinds in perpendicular hair, sneeze, retch, continuously phenomenon such as cough 3 sound and dyspnea etc. or two or more, or have a convulsion, suffer a shock, the one of the phenomena of mortality, sentence this product against regulation.
Embodiment 2
(1) by 150Kg fresh pig small intestinal (comprising duodenum, jejunum, ileum), clean with deionized water rinsing, remove content, remove serous coat and degrease, clean with water for injection cleaning down, freezingly to shred, meat grinder just twists 3 times and obtain pig small intestine colloid;
(2) by above-mentioned pig small intestine colloid through colloid mill homogenate, obtained tissue homogenate 80L, then homogenate is sub-packed in-40 DEG C, apyrogenic bottle freezing, 37 DEG C of flowing water thaw, multigelation 5 times, homogenate 100 DEG C of water proofs are boiled 20min, is cooled to rapidly 4 DEG C;
Under (3) 4 DEG C of conditions, it is the acetic acid solution 80L stirring and leaching 10 hours of 5.00% by the homogenate 80L after freeze thawing and concentration, it is centrifugal that mixture after stirring and leaching carries out first time in 4 DEG C of condition 8000rpm, centrifugal 25min, obtain first time supernatant 93L,-20 DEG C of preservations, described concentration be 5.00% acetic acid solution water for injection prepare;
Under (4) 4 DEG C of conditions, the acetic acid solution 53.6L(W:V being 5.63% by first time centrifugal gained precipitate 67Kg and concentration is 1:0.8) stirring and leaching 9 hours, repeat above-mentioned centrifugal process, obtain second time supernatant 34L,-20 DEG C of preservations, described concentration be 5.63% acetic acid solution water for injection prepare;
Under (5) 4 DEG C of conditions, the acetic acid solution 52L(W:V being 6.67% by the centrifugal gained precipitate 86Kg of second time and concentration is 1:0.6) stirring and leaching 12 hours, repeat above-mentioned centrifugal process, obtain third time supernatant 32L, described concentration be 6.67% acetic acid solution water for injection prepare;
(6) above-mentioned three centrifugal gained 159L supernatant are merged; By supernatant through 0.22um micro-filtrate membrane filtration, 10KD ultrafiltration membrance filter, collect ultrafiltration through thing, obtain ultrafiltrate; Regulate ultrafiltrate pH value to 7.0, obtained pig small intestine recombinant antimicrobial peptide semifinished product;
Wherein a.0.22um microfiltration is by 159L supernatant, through 0.22um boxlike film tangential flow systems (Millipore company CUF-50Durapore0.22um5.0m
2film bag) microfiltration clarification (6 times concentrate), obtain microfiltration clear liquor 133L, MF retentate 26L does 2 times of dialysis again and to get back clear liquor 52L, obtains 185L clear liquor altogether.
0.22um boxlike film tangential flow systems mean parameter is as follows:
B.10KD ultrafiltration is by microfiltration clear liquor 185L, through 10KD boxlike film tangential flow systems (Millipore company Pellicon2Biomax10KD2.5m
2film bag) ultrafiltration (15 times concentrate), ultrafiltration obtains ultrafiltrate 173L, and ultrafiltration retentate 12L does 2.5 times of equal-volumes dialysis again and to get back ultrafiltrate 30L, obtains 203L ultrafiltrate altogether, regulates ultrafiltrate pH value to 7.0, be pig small intestine recombinant antimicrobial peptide semifinished product.
10KD boxlike film tangential flow systems mean parameter is as follows:
(7) pig small intestine recombinant antimicrobial peptide semifinished product is concentrated and dialysis desalination through 3KD nanofiltration membrane, collect nanofiltration retentate; After the osmotic pressure of the nanofiltration retentate of collection is adjusted to 280mosm/kg, the sterilizing filter through 0.1um is degerming, obtains pig small intestine recombinant antimicrobial peptide 100.3L.
Wherein, a.3KD concentrated the and dialysis desalination of nanofiltration is by pig small intestine recombinant antimicrobial peptide semifinished product, through 3KD boxlike film tangential flow systems (Millipore company Pellicon2PLBC3KD2.5m
2film bag) nanofiltration concentrated (2 times concentrate), collect nanofiltration retentate 101.5L, do 1 times of equal-volume dialysis with 101.5L water for injection for dialysis solution again, obtain 101.5L nanofiltration concentrate dialysate altogether, the osmotic pressure of the nanofiltration concentrate dialysate of collection is adjusted to 280mosm/kg;
3KD boxlike film tangential flow systems mean parameter is as follows:
B.0.1um sterilizing filter is degerming is by 101.5L nanofiltration concentrate dialysate, and degerming through 0.1um sterilizing filter, obtain pig small intestine recombinant antimicrobial peptide 100.3L, quality inspection is qualified.
Pig small intestine recombinant antimicrobial peptide prepared by embodiment 2 as poultry new antibiotic, immunostimulant and immunological adjuvant, pig small intestine recombinant antimicrobial peptide 4mL and transmissible gastroenteritis of swine-epidemic diarrhea vaccine 2 part combined immunization every boar.
Embodiment 3
(1) by 7.5Kg fresh pig small intestinal (comprising duodenum, ileum), clean with deionized water rinsing, remove content, remove serous coat and degrease, clean with water for injection cleaning down, freezingly to shred, meat grinder just twists 1 time and obtain pig small intestine colloid;
(2) by above-mentioned pig small intestine colloid through colloid mill homogenate, obtained tissue homogenate 3.8L, is then sub-packed in-40 DEG C, apyrogenic bottle freezing by homogenate, 37 DEG C of flowing water thaw, homogenate 100 DEG C of water proofs are boiled 25min, are cooled to rapidly 4 DEG C by multigelation 6 times;
Under (3) 1 DEG C of conditions, it is the acetic acid solution 3.80L stirring and leaching 8 hours of 5.00% by the 3.8L homogenate after freeze thawing and concentration, it is centrifugal that mixture after stirring and leaching carries out first time in 4 DEG C of condition 7000rpm, centrifugal 20min, obtain first time supernatant 4.4L,-20 DEG C of preservations, described concentration be 5.00% acetic acid solution water for injection prepare;
Under (4) 1 DEG C of conditions, the acetic acid solution 1.92L(W:V being 6.67% by first time centrifugal gained precipitate 3.2Kg and concentration is 1:0.6) stirring and leaching 8 hours, repeat above-mentioned centrifugal process, obtain second time supernatant 1.19L,-20 DEG C of preservations, described concentration be 6.67% acetic acid solution water for injection prepare;
Under (5) 1 DEG C of conditions, the acetic acid solution 1.57L(W:V being 8.75% by the centrifugal gained precipitate 3.93Kg of second time and concentration is 1:0.4) stirring and leaching 14 hours, repeat above-mentioned centrifugal process again, obtain third time supernatant 1.03L, described concentration be 8.75% acetic acid solution water for injection prepare;
(6) merge above-mentioned three centrifugal gained 6.62L supernatant, by supernatant through 0.1um micro-filtrate membrane filtration, 10KD ultrafiltration membrance filter, collect ultrafiltration through thing, obtain ultrafiltrate; Regulate ultrafiltrate pH value to 6.5, obtained pig small intestine recombinant antimicrobial peptide semifinished product;
Wherein, a.0.1um microfiltration is by 6.62L supernatant, through 0.1um boxlike film tangential flow systems (Millipore company Pellicon2Durapore0.1um1.0m
2film bag) microfiltration clarification (5 times concentrate), obtain microfiltration clear liquor 5.30L, MF retentate 1.32L does 3 times of dialysis again and to get back clear liquor 3.96L, obtains 9.26L clear liquor altogether.
0.1um boxlike film tangential flow systems mean parameter is as follows:
B.10KD ultrafiltration is by microfiltration clear liquor 9.26L, through 10KD boxlike film tangential flow systems (Millipore company Pellicon2Biomax8KD0.5m
2film bag) ultrafiltration (15 times concentrate), ultrafiltration obtains ultrafiltrate 8.64L, and ultrafiltration retentate 0.62L does 3 times of equal-volumes dialysis again and to get back ultrafiltrate 1.86L, obtains 10.5L ultrafiltrate altogether, regulates ultrafiltrate pH value to 6.5, be pig small intestine recombinant antimicrobial peptide semifinished product.
10KD boxlike film tangential flow systems mean parameter is as follows:
(7) pig small intestine recombinant antimicrobial peptide semifinished product is concentrated and dialysis desalination through 1KD nanofiltration membrane, collect nanofiltration retentate; After the osmotic pressure of the nanofiltration retentate of collection is adjusted to 290mosm/kg, the sterilizing filter through 0.1um is degerming, obtains pig small intestine recombinant antimicrobial peptide 6.82L.
Wherein, a.1KD concentrated the and dialysis desalination of nanofiltration is by pig small intestine recombinant antimicrobial peptide semifinished product, through 1KD boxlike film tangential flow systems (Millipore company Pellicon2PLAC1KD2.0m
2film bag) nanofiltration concentrated (1.5 times concentrate), collect nanofiltration retentate 7.00L, do 1 times of equal-volume dialysis with 7.00L water for injection for dialysis solution again, obtain 7.00L nanofiltration concentrate dialysate altogether, the osmotic pressure of the nanofiltration concentrate dialysate of collection is adjusted to 290mosm/kg;
1KD boxlike film tangential flow systems mean parameter is as follows:
B.0.1um sterilizing filter is degerming is by 7.00L nanofiltration concentrate dialysate, and degerming through 0.1um sterilizing filter, obtain pig small intestine recombinant antimicrobial peptide 6.82L, quality inspection is qualified.
Pig small intestine recombinant antimicrobial peptide prepared by embodiment 3 as poultry new antibiotic, immunostimulant and immunological adjuvant, pig small intestine recombinant antimicrobial peptide 0.05mL separately or with the every plumage chicken of chicken virus mycoplasma vaccine 1 plumage part combined immunization.
Embodiment 4
(1) by 7.5Kg fresh pig small intestinal (comprising duodenum, jejunum, ileum), clean with deionized water rinsing, remove content, remove serous coat and degrease, clean with water for injection cleaning down, freezingly to shred, meat grinder just twists 1 time and obtain pig small intestine colloid;
(2) by above-mentioned pig small intestine colloid through colloid mill homogenate, obtained tissue homogenate 3.8L, is then sub-packed in-40 DEG C, apyrogenic bottle freezing by homogenate, 37 DEG C of flowing water thaw, homogenate 100 DEG C of water proofs are boiled 25min, are cooled to rapidly 4 DEG C by multigelation 6 times;
Under (3) 6 DEG C of conditions, it is the acetic acid solution 3.8L stirring and leaching 14 hours of 5.00% by the 3.8L homogenate after freeze thawing and concentration, it is centrifugal that mixture after stirring and leaching carries out first time in 4 DEG C of condition 6000rpm, centrifugal 25min, obtain first time supernatant 4.4L,-40 DEG C of preservations, described concentration be 5.00% acetic acid solution water for injection prepare;
Under (4) 6 DEG C of conditions, the acetic acid solution 1.92L(W:V being 6.67% by first time centrifugal gained precipitate 3.2Kg and concentration is 1:0.6) stirring and leaching 8 hours, repeat above-mentioned centrifugal process, obtain second time supernatant 1.25L,-40 DEG C of preservations, described concentration be 6.67% acetic acid solution water for injection prepare;
Under (5) 6 DEG C of conditions, the acetic acid solution 2.34L(W:V being 6.67% by the centrifugal gained precipitate 3.9Kg of second time and concentration is 1:0.6) stirring and leaching 14 hours, repeat above-mentioned centrifugal process again, obtain third time supernatant 1.50L, described concentration be 6.67% acetic acid solution water for injection prepare;
(6) merge above-mentioned three centrifugal gained 7.15L supernatant, by supernatant through 0.22um micro-filtrate membrane filtration, 8KD ultrafiltration membrance filter, collect ultrafiltration through thing, obtain ultrafiltrate; Regulate ultrafiltrate pH value to 7.5, obtained pig small intestine recombinant antimicrobial peptide semifinished product;
Wherein, a.0.22um microfiltration is by 7.15L supernatant, through 0.22um boxlike film tangential flow systems (Millipore company Pellicon2Durapore0.22um1.0m
2film bag) microfiltration clarification (4 times concentrate), obtain microfiltration clear liquor 5.36L, MF retentate 1.79L does 3 times of dialysis again and to get back clear liquor 5.37L, obtains 10.73L clear liquor altogether.
0.22um boxlike film tangential flow systems mean parameter is as follows:
B.8KD ultrafiltration is by microfiltration clear liquor 10.73L, through 8KD boxlike film tangential flow systems (Millipore company Pellicon2Biomax8KD0.5m
2film bag) ultrafiltration (16 times concentrate), ultrafiltration obtains ultrafiltrate 10.00L, and ultrafiltration retentate 0.73L does 2 times of equal-volumes dialysis again and to get back ultrafiltrate 1.46L, obtains 11.46L ultrafiltrate altogether, regulates ultrafiltrate pH value to 7.5, be pig small intestine recombinant antimicrobial peptide semifinished product.
8KD boxlike film tangential flow systems mean parameter is as follows:
(7) pig small intestine recombinant antimicrobial peptide semifinished product is concentrated and dialysis desalination through 1KD nanofiltration membrane, collect nanofiltration retentate; After the osmotic pressure of the nanofiltration retentate of collection is adjusted to 310mosm/kg, the sterilizing filter through 0.1um is degerming, obtains pig small intestine recombinant antimicrobial peptide 7.5L.
Wherein, a.1KD concentrated the and dialysis desalination of nanofiltration is by pig small intestine recombinant antimicrobial peptide semifinished product, through 1KD boxlike film tangential flow systems (Millipore company Pellicon2PLAC1KD2.5m
2film bag) nanofiltration concentrated (1.5 times concentrate), collect nanofiltration retentate 7.64L, do 2 times of equal-volume dialysis with 15.28L water for injection for dialysis solution again, obtain 7.64L nanofiltration concentrate dialysate altogether, the osmotic pressure of the nanofiltration concentrate dialysate of collection is adjusted to 310mosm/kg;
1KD boxlike film tangential flow systems mean parameter is as follows:
B.0.1um sterilizing filter is degerming is by 7.64L nanofiltration concentrate dialysate, and degerming through 0.1um sterilizing filter, obtain pig small intestine recombinant antimicrobial peptide 7.5L, quality inspection is qualified.
Pig small intestine recombinant antimicrobial peptide embodiment 4 prepared is as poultry new antibiotic, immunostimulant and immunological adjuvant, and pig small intestine recombinant antimicrobial peptide 3mL is immunity every pig separately.
Obviously, the above embodiment of the present invention is only for example of the present invention is clearly described, and is not the restriction to embodiments of the present invention.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here cannot give exhaustive to all embodiments.Every belong to technical scheme of the present invention the apparent change of extending out or variation be still in the row of protection scope of the present invention.
Claims (9)
1. a preparation method for pig small intestine recombinant antimicrobial peptide, is characterized in that, comprises following preparation process:
(1) fresh pig small intestinal is clean with deionized water rinsing, remove content, remove serous coat and degrease after use water for injection cleaning down, then freezingly shred, meat grinder just twists 1-3 time, then uses colloid mill homogenate, makes homogenate;
(2) by after described homogenate multigelation 3-6 time, 100 DEG C of water proofs boil 15-25min, then the rear cold preservation of cooling rapidly, and refrigerated storage temperature is 1-4 DEG C, and cold preservation time is 0-5 hour;
(3) in homogenate, the acetic acid solution that concentration is 5.00% is added, stirring and leaching, it is centrifugal that 4 DEG C of conditions carry out first time, by the supernatant freezen protective centrifugal first time, described concentration be 5.00% acetic acid solution water for injection prepare, the volume ratio of described acetic acid solution and homogenate is 1:1;
(4) in precipitate centrifugal for the first time, the acetic acid solution that concentration is 6.67%-5.00% is added, stirring and leaching, it is centrifugal that 4 DEG C of conditions carry out second time, and by supernatant freezen protective centrifugal for second time, described concentration is that the acetic acid solution water for injection of 6.67%-5.00% is prepared;
(5) in the precipitate that second time is centrifugal, the acetic acid solution that concentration is 8.75%-5.63% is added again, stirring and leaching, it is centrifugal that 4 DEG C of conditions carry out third time, and obtain third time centrifugal supernatant, described concentration is that the acetic acid solution water for injection of 8.75%-5.63% is prepared;
(6) merge above-mentioned three supernatant, by the supernatant that merges through 0.1,0.22 or 0.45um slipstream micro-filtrate membrane filtration, collect MF permeate thing, obtain clear liquor; Clear liquor, through 8 or 10KD cross-flow ultrafiltration membrane filtration, is collected ultrafiltration through thing, is obtained ultrafiltrate; Regulate ultrafiltrate pH value to 6.5-7.5, obtain pig small intestine recombinant antimicrobial peptide semifinished product;
(7) pig small intestine recombinant antimicrobial peptide semifinished product is concentrated and dialysis desalination through 1 or 3KD slipstream nanofiltration membrane, collect nanofiltration retentate; Regulate osmotic pressure to 280-320mosm/kg, more degerming through the sterilizing filter of 0.1um, obtain pig small intestine recombinant antimicrobial peptide.
2. the preparation method of pig small intestine recombinant antimicrobial peptide according to claim 1, is characterized in that, step (3), (4) described cryogenic temperature are less than-20 DEG C, and cooling time is no more than 30 days; Step (3), (4), (5) described stirring and leaching are for stirring 8-14 hour, and temperature is 1-6 DEG C; Step (3), (4), (5) described centrifugation time are 15-25min, and centrifuge speed is 6000-8000rpm.
3. the preparation method of pig small intestine recombinant antimicrobial peptide according to claim 1, is characterized in that, described first time centrifugal sediment and the weight/volume (W:V) of acetic acid solution be 1Kg:0.6-1L.
4. the preparation method of pig small intestine recombinant antimicrobial peptide according to claim 1, is characterized in that, the weight/volume (W:V) of described second time centrifugal sediment and acetic acid solution is 1Kg:0.4-0.8L.
5. the pig small intestine recombinant antimicrobial peptide that method obtains as described in any one of claim 1-4.
6. the purposes of pig small intestine recombinant antimicrobial peptide as claimed in claim 5, it is characterized in that, pig small intestine recombinant antimicrobial peptide is as the purposes of poultry new antibiotic, immunostimulant and immunological adjuvant.
7. the purposes of pig small intestine recombinant antimicrobial peptide according to claim 5, is characterized in that, described pig small intestine recombinant antimicrobial peptide separately or with vaccine combined immunization.
8. the purposes of pig small intestine recombinant antimicrobial peptide according to claim 5, is characterized in that, described pig small intestine recombinant antimicrobial peptide 1-6mL separately or with transmissible gastroenteritis of swine-epidemic diarrhea vaccine 1-3 head part combined immunization every pig.
9. the purposes of pig small intestine recombinant antimicrobial peptide according to claim 5, is characterized in that, described pig small intestine recombinant antimicrobial peptide 0.01-0.2mL separately or with the every plumage chicken of chicken virus mycoplasma vaccine 1-3 plumage part combined immunization.
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| CN106916218A (en) * | 2017-05-09 | 2017-07-04 | 派生特（福州）生物科技有限公司 | A kind of preparation method of Antibacterial Peptide Extracted from Pig Small Intestine |
| 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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| CN109464652B (en) * | 2018-11-28 | 2022-02-01 | 派生特（福州）生物科技有限公司 | Composition for livestock diarrhea and preparation method and application thereof |
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