CN103417574A - Preparation method of recombinant antimicrobial peptide extracted from small intestine of pig and application thereof - Google Patents
Preparation method of recombinant antimicrobial peptide extracted from small intestine of pig and application thereof Download PDFInfo
- Publication number
- CN103417574A CN103417574A CN2013103430308A CN201310343030A CN103417574A CN 103417574 A CN103417574 A CN 103417574A CN 2013103430308 A CN2013103430308 A CN 2013103430308A CN 201310343030 A CN201310343030 A CN 201310343030A CN 103417574 A CN103417574 A CN 103417574A
- Authority
- CN
- China
- Prior art keywords
- small intestine
- pig small
- time
- composite antibiosis
- pig
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 210000000813 small intestine Anatomy 0.000 title claims abstract description 86
- 238000002360 preparation method Methods 0.000 title claims abstract description 25
- 239000003910 polypeptide antibiotic agent Substances 0.000 title abstract description 35
- 108700042778 Antimicrobial Peptides Proteins 0.000 title abstract description 11
- 102000044503 Antimicrobial Peptides Human genes 0.000 title abstract description 11
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 132
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 55
- 238000001728 nano-filtration Methods 0.000 claims abstract description 35
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 32
- 238000001471 micro-filtration Methods 0.000 claims abstract description 24
- 238000001914 filtration Methods 0.000 claims abstract description 19
- 239000012528 membrane Substances 0.000 claims abstract description 18
- 230000003204 osmotic effect Effects 0.000 claims abstract description 17
- 239000000084 colloidal system Substances 0.000 claims abstract description 16
- 238000007710 freezing Methods 0.000 claims abstract description 11
- 230000008014 freezing Effects 0.000 claims abstract description 11
- 235000013372 meat Nutrition 0.000 claims abstract description 8
- 206010012735 Diarrhoea Diseases 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 81
- 230000003115 biocidal effect Effects 0.000 claims description 74
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 71
- 239000002131 composite material Substances 0.000 claims description 66
- 239000006228 supernatant Substances 0.000 claims description 35
- 238000000034 method Methods 0.000 claims description 28
- 239000008215 water for injection Substances 0.000 claims description 28
- 238000003756 stirring Methods 0.000 claims description 26
- 238000002386 leaching Methods 0.000 claims description 24
- 238000000502 dialysis Methods 0.000 claims description 22
- 239000000126 substance Substances 0.000 claims description 22
- 239000011265 semifinished product Substances 0.000 claims description 21
- 238000004321 preservation Methods 0.000 claims description 16
- 230000001954 sterilising effect Effects 0.000 claims description 16
- 238000010612 desalination reaction Methods 0.000 claims description 12
- 239000002244 precipitate Substances 0.000 claims description 12
- 241000287828 Gallus gallus Species 0.000 claims description 10
- 230000003053 immunization Effects 0.000 claims description 9
- 238000002649 immunization Methods 0.000 claims description 9
- 238000005374 membrane filtration Methods 0.000 claims description 9
- 229960005486 vaccine Drugs 0.000 claims description 9
- 238000004140 cleaning Methods 0.000 claims description 7
- 229960001438 immunostimulant agent Drugs 0.000 claims description 7
- 239000003022 immunostimulating agent Substances 0.000 claims description 7
- 230000003308 immunostimulating effect Effects 0.000 claims description 7
- 230000000968 intestinal effect Effects 0.000 claims description 7
- 239000008367 deionised water Substances 0.000 claims description 6
- 229910021641 deionized water Inorganic materials 0.000 claims description 6
- 239000000706 filtrate Substances 0.000 claims description 6
- 239000000568 immunological adjuvant Substances 0.000 claims description 6
- 244000144977 poultry Species 0.000 claims description 6
- 241000204031 Mycoplasma Species 0.000 claims description 5
- 208000005577 Gastroenteritis Diseases 0.000 claims description 4
- 241000700605 Viruses Species 0.000 claims description 4
- 238000001816 cooling Methods 0.000 claims description 4
- 239000013049 sediment Substances 0.000 claims description 4
- 238000005119 centrifugation Methods 0.000 claims description 2
- 238000003860 storage Methods 0.000 claims description 2
- 108090000623 proteins and genes Proteins 0.000 abstract description 8
- 102000004169 proteins and genes Human genes 0.000 abstract description 8
- 230000000694 effects Effects 0.000 abstract description 6
- 230000036039 immunity Effects 0.000 abstract description 6
- 238000010257 thawing Methods 0.000 abstract description 5
- 239000003242 anti bacterial agent Substances 0.000 abstract description 3
- 229940088710 antibiotic agent Drugs 0.000 abstract description 3
- 238000010438 heat treatment Methods 0.000 abstract description 3
- 238000009835 boiling Methods 0.000 abstract description 2
- 238000005360 mashing Methods 0.000 abstract description 2
- 230000002265 prevention Effects 0.000 abstract description 2
- 241000204022 Mycoplasma gallisepticum Species 0.000 abstract 1
- 230000000249 desinfective effect Effects 0.000 abstract 1
- 230000000091 immunopotentiator Effects 0.000 abstract 1
- 238000012360 testing method Methods 0.000 description 24
- 239000000047 product Substances 0.000 description 17
- 239000012141 concentrate Substances 0.000 description 12
- 238000001514 detection method Methods 0.000 description 12
- 230000036760 body temperature Effects 0.000 description 10
- 230000008569 process Effects 0.000 description 10
- 241000283973 Oryctolagus cuniculus Species 0.000 description 9
- 238000010790 dilution Methods 0.000 description 8
- 239000012895 dilution Substances 0.000 description 8
- 239000002158 endotoxin Substances 0.000 description 8
- 210000004379 membrane Anatomy 0.000 description 8
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 7
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 7
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 7
- 238000009295 crossflow filtration Methods 0.000 description 7
- 239000013558 reference substance Substances 0.000 description 7
- 210000001519 tissue Anatomy 0.000 description 7
- 210000001198 duodenum Anatomy 0.000 description 5
- 238000007689 inspection Methods 0.000 description 5
- 244000005700 microbiome Species 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 5
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 4
- 229920005654 Sephadex Polymers 0.000 description 4
- 239000012507 Sephadex™ Substances 0.000 description 4
- 230000000844 anti-bacterial effect Effects 0.000 description 4
- 230000037396 body weight Effects 0.000 description 4
- 238000005352 clarification Methods 0.000 description 4
- 229910052802 copper Inorganic materials 0.000 description 4
- 239000010949 copper Substances 0.000 description 4
- 239000000385 dialysis solution Substances 0.000 description 4
- 210000003405 ileum Anatomy 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 210000001630 jejunum Anatomy 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 101100244894 Sus scrofa PR39 gene Proteins 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 241000700198 Cavia Species 0.000 description 2
- 208000000059 Dyspnea Diseases 0.000 description 2
- 206010013975 Dyspnoeas Diseases 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 101800001442 Peptide pr Proteins 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 230000004520 agglutination Effects 0.000 description 2
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 description 2
- 230000000840 anti-viral effect Effects 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000005345 coagulation Methods 0.000 description 2
- 230000015271 coagulation Effects 0.000 description 2
- 239000000287 crude extract Substances 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 239000003651 drinking water Substances 0.000 description 2
- 235000020188 drinking water Nutrition 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 210000003746 feather Anatomy 0.000 description 2
- 210000003608 fece Anatomy 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- IXCSERBJSXMMFS-UHFFFAOYSA-N hcl hcl Chemical compound Cl.Cl IXCSERBJSXMMFS-UHFFFAOYSA-N 0.000 description 2
- 210000004347 intestinal mucosa Anatomy 0.000 description 2
- 238000010255 intramuscular injection Methods 0.000 description 2
- 239000007927 intramuscular injection Substances 0.000 description 2
- 238000011031 large-scale manufacturing process Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000002510 pyrogen Substances 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- WXHLLJAMBQLULT-UHFFFAOYSA-N 2-[[6-[4-(2-hydroxyethyl)piperazin-1-yl]-2-methylpyrimidin-4-yl]amino]-n-(2-methyl-6-sulfanylphenyl)-1,3-thiazole-5-carboxamide;hydrate Chemical compound O.C=1C(N2CCN(CCO)CC2)=NC(C)=NC=1NC(S1)=NC=C1C(=O)NC1=C(C)C=CC=C1S WXHLLJAMBQLULT-UHFFFAOYSA-N 0.000 description 1
- 125000003345 AMP group Chemical group 0.000 description 1
- 206010002198 Anaphylactic reaction Diseases 0.000 description 1
- -1 Biogel Substances 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 206010010904 Convulsion Diseases 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- 239000012506 Sephacryl® Substances 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000000172 allergic effect Effects 0.000 description 1
- 230000036783 anaphylactic response Effects 0.000 description 1
- 208000003455 anaphylaxis Diseases 0.000 description 1
- 230000000843 anti-fungal effect Effects 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 238000004500 asepsis Methods 0.000 description 1
- 208000010668 atopic eczema Diseases 0.000 description 1
- 230000006399 behavior Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 230000036461 convulsion Effects 0.000 description 1
- 210000004395 cytoplasmic granule Anatomy 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000002086 displacement chromatography Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 231100000284 endotoxic Toxicity 0.000 description 1
- 230000002346 endotoxic effect Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229920006227 ethylene-grafted-maleic anhydride Polymers 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000005213 imbibition Methods 0.000 description 1
- 230000008799 immune stress Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 1
- 238000005304 joining Methods 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- OIPPWFOQEKKFEE-UHFFFAOYSA-N orcinol Chemical compound CC1=CC(O)=CC(O)=C1 OIPPWFOQEKKFEE-UHFFFAOYSA-N 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 238000002103 osmometry Methods 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000002423 protozoacide Substances 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 230000008313 sensitization Effects 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000008354 sodium chloride injection Substances 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 231100000820 toxicity test Toxicity 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
Images
Landscapes
- Peptides Or Proteins (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses a preparation method of a recombinant antimicrobial peptide extracted from a small intestine of a pig and an application thereof. The preparation method comprises the following steps: mashing, homogenizing and repeatedly freezing and thawing the small intestine tissue of the pig with a meat grinder and a colloid mill, and preliminarily crushing cells; then performing the water boiling heat treatment, and extracting with acetic acid solution to obtain a large amount of antimicrobial peptides; centrifuging to enable the quantity of other proteins to be reduced greatly, filtering the supernate obtained through centrifuging by a tangential flow micro-filtration membrane and a tangential flow ultrafiltration membrane sequentially, adjusting the pH value, filtering the supernate by a tangential flow nanofiltration membrane, concentrating and dialyzing to desalt, and disinfecting after adjusting the osmotic pressure to obtain the recombinant antimicrobial peptide extracted from the small intestine of the pig. According to the invention, the application of the recombinant antimicrobial peptide adopts effects of the antimicrobial peptide on aspects of mucosal immunity, immunopotentiators, novel antibiotics and the like, and the mucosal immunity character of the antimicrobial peptide can play an important role in the prevention and control of transmissible gastroenteristis, epidemic diarrhea and mycoplasma gallisepticum; the antimicrobial peptide replaces traditional antibiotics, and is remarkable in effect.
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 composite antibiosis peptide.
Background technology
Antibacterial peptide is the class antimicrobial that produces in resisting the defense reaction process of pathogenic microorganism of living organism and the small peptide of some malignant cells.Use " antimicrobial peptide " (Antimicrobial peptides, AMPs) or " peptide antibiotic " (peptide antibiotics) in some international literatures.Antibacterial peptide has antibacterial action, and some also has protozoacide, antifungal, antiviral and the effects such as inhibition or killing tumor cell, parasite.It has the characteristics such as molecular weight little (3-10 KD), good water solubility, heat-resisting, non-immunogenicity, can become antibiotic, antiviral and the PTS of a kind of efficient, low toxicity and noresidue harm.
At present a lot of microorganisms have produced extensive drug resistance to traditional antibiotic, and antibacterial peptide has still retained its powerful antibacterial efficacy in the evolution course of having passed through millions of years, and microorganism is difficult for it is developed immunity to drugs.Antibacterial activity and unique antibacterial mechanisms thereof due to the antibacterial peptide broad-spectrum high efficacy, make antibacterial peptide especially noticeable.
To 2003, the Italian Trieste antibacterial peptide data base of university 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 | The 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 |
There is again successively afterwards new Antibacterial Peptide Extracted from Pig Small Intestine to be found, as the 5972Da Antibacterial Peptide Extracted from Pig Small Intestine is found by Ma Weiming.
Due to the intestinal tissue complex structure, foreign protein is of a great variety, and antibacterial peptide usually is present in cytoplasmic granule, and its molecular weight is lower, and the content in tissue is lower, thus separation and purification ratio more difficult.The method of 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 displacement chromatography, gel electrophoresis and anti-phase high pressure liquid chromatography etc.As: someone adopts water proof to boil rear acid extraction, the centrifugal crude extract that obtains, successively through Sephadex G-100 gel column, Sephadex G-25 or Biogel P10 gel filtration column chromatography, the protein respectively molecular size not waited and salt are separately.Preliminary purification gained Antibacterial Peptide Extracted from Pig Small Intestine semifinished product, remove thermal source through the 10kD ultra-filtration centrifuge tube, then do and be further purified through the 3-5KD ultra-filtration centrifuge tube.These method complicated operations, just can obtain antibacterial peptide through separation and purification repeatedly, cost of idleness, and 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 the composite antibiosis peptide.
In membrane separation technique, the filtration of two types is arranged usually: conventional vertical (Normal Flow Filtration, NFF) and the tangential flow filtration (Tangential Flow Filtration, TFF) of filtering.In the vertical filtration of routine, all fluids directly pass through filter membrane, the material accumulating film surface be trapped.Due to the accumulation of these materials, the flow by filter membrane descends rapidly until stop fully, as Fig. 1; And, in tangential flow filtration, fluid flows through filter membrane in cross ground as shown in Figure 2 and Figure 3.
Tangential flow filtration is different from conventional vertical filtration, and the liquid slipstream is crossed the film surface, and the transmembrane pressure that fluid produces, by part solution press filtration film, is held back circulating reflux in system of part.In whole process, liquid, with certain speed Continuous Flow filter membrane surface, is also washed away the filter membrane surface in the time of filtration, makes the film surface not form gel layer, thereby makes the granule in feed liquid can not stop up very soon filter membrane, has kept the stable rate of filtration.
Microorganism is oozed residing osmotic pressure requirement etc., and the osmotic pressure value of isosmotic solution is 280-320mosm/kg.Osmotic pressure value claims hypisotonic solution lower than the solution of this value, as distilled water etc.; Osmotic pressure value claims hyperosmotic solution higher than the solution of this value, the Glucose Liquid as 10% or 50% Glucose Liquid etc.Plasmolysis shrinkage death, in hyperosmotic solution, easily occurs in microorganism; If contrary in hypisotonic solution, the easily imbibition death of breaking.
The patent that application number is 201210305972.2 discloses the preparation method of a kind of Antibacterial Peptide Extracted from Pig Small Intestine PR39, it adopts ultrasonic degradation to discharge the albumen in intestinal mucosa, adopt the detached dowel chromatography to remove most foreign protein, obtain antibacterial peptide, the method has been 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 composite antibiosis peptide, and the method comprises the steps:
(1) the fresh pig small intestinal is rinsed well with deionized water, removed content, go to use the water for injection cleaning down after serous coat and degrease, freezing shredding then, meat grinder just twists 1-3 time, then uses colloid mill homogenate, makes homogenate;
Pig small intestine of the present invention refer to pig duodenum, jejunum or ileum arbitrary section or several sections;
(2) by after described homogenate multigelation 3-6 time, 100 ℃ of water proofs boil 15-25min, then cooling rear cold preservation rapidly, and refrigerated storage temperature is 1-4 ℃, cold preservation time is 0-5 hour;
(3) add the acetic acid solution that concentration is 5.00% in homogenate, stirring and leaching, it is centrifugal for the first time that 4 ℃ of conditions are carried out, by the freezing preservation of centrifugal for the first time supernatant, the acetic acid solution that described concentration is 5.00% is prepared with water for injection, and the volume ratio of described acetic acid solution and homogenate is 1:1;
(4) add the acetic acid solution that concentration is 6.67%-5.00% in centrifugal for the first time precipitate, stirring and leaching, it is centrifugal for the second time that 4 ℃ of conditions are carried out, and by the freezing preservation of centrifugal for the second time supernatant, the acetic acid solution that described concentration is 6.67%-5.00% is prepared with water for injection;
(5) add again the acetic acid solution that concentration is 8.75%-5.63% in centrifugal for the second time precipitate, stirring and leaching, it is centrifugal for the third time that 4 ℃ of conditions are carried out, and obtains centrifugal for the third time supernatant, and the acetic acid solution that described concentration is 8.75%-5.63% is prepared with water for injection;
(6) merge above-mentioned three supernatant, the supernatant that merges, through 0.1,0.22 or 0.45um slipstream micro-filtrate membrane filtration, is collected to microfiltration and seen through thing, obtain clear liquor; Clear liquor, through 8 or 10KD cross-flow ultrafiltration membrane filtration, is collected ultrafiltration and is seen through thing, obtains ultrafiltrate; Regulate the ultrafiltrate pH value to 6.5-7.5, obtain pig small intestine composite antibiosis peptide semifinished product;
(7) by pig small intestine composite antibiosis peptide semifinished product through 1 or 3KD slipstream NF membrane filtering and concentrating and dialysis desalination, collect the nanofiltration trapped substance; Regulate osmotic pressure to 280-320mosm/kg, then, through the sterilizing filter degerming of 0.1um, obtain pig small intestine composite antibiosis peptide.
Further, step (3), (4) described cryogenic temperature are below-20 ℃, 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 ℃; Step (3), (4), (5) described centrifugation time are 15-25min, and centrifuge speed is 6000-8000rpm.
Further, the weight/volume (W:V) of described centrifugal sediment for the first time and acetic acid solution is 1Kg:0.6-1L.
Further, the weight/volume (W:V) of described centrifugal sediment for the second time and acetic acid solution is 1Kg:0.4-0.8L.
The present invention also will protect the pig small intestine composite antibiosis peptide that adopts 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 composite antibiosis peptide, and described pig small intestine composite antibiosis peptide is used as poultry new antibiotic, immunostimulant and immunological adjuvant.
Further, described pig small intestine composite antibiosis peptide separately or with the vaccine combined immunization.
Further, described pig small intestine composite antibiosis peptide 1-6mL separately or with transmissible gastroenteritis of swine-every pig of epidemic diarrhea vaccine 1-3 head part combined immunization.
Further, described pig small intestine composite antibiosis 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 subsequently water proof and boil heat treatment, on the one hand with the further cell lysis of heat treatment mode, the antibacterial peptide in endochylema is discharged as much as possible, make on the other hand the foreign protein degeneration, then can obtain the antibacterial peptide of more amount with the acetic acid solution lixiviate.Centrifugal afterwards, the amount of foreign protein is reduced greatly.Centrifugal gained supernatant is 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 that adopts slipstream to filter step by step, and technique is simple, and production efficiency and antibacterial peptide yield are high.After regulating pH value, concentrated and desalination process employing 1 or the filtration of 3KD slipstream NF membrane, than adopting Rotary Evaporators deacidification desalination or adopting Sephadex G-25, Biogel P10 isogel post filtration chromatography, technique is more efficient, practical, applicable large-scale production.Carrying out that osmotic pressure is adjusted to subsequently etc. oozes (280-320mosm/kg), and isosmotic solution both had been conducive to reduce immunological stress, was conducive to again the combined immunization of gained pig small intestine composite antibiosis peptide and vaccine.Finally by the sterilizing filter degerming of 0.1um, the sterilizing filter than adopting 0.22um, more be conducive to remove the microorganisms such as mycoplasma.
Technique of the present invention is simply efficient, and gained pig small intestine composite antibiosis peptide concentration is high, yield is high.
The purposes of the pig small intestine composite antibiosis peptide that the present invention proposes, use the effect of antibacterial peptide at aspects such as mucosal immunity, immunostimulant, new antibiotics, antibacterial peptide is played to the prevention and control of transmissible gastroenteritis of swine-epidemic diarrhea and the chicken virus mycoplasma disease of important function for mucosal immunity, effect is remarkable.
The accompanying drawing explanation
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail
Fig. 1 is the conventional vertical schematic diagram that filters;
Fig. 2 is tangential flow filtration cleaning effect schematic diagram;
Fig. 3 is the tangential flow filtration schematic diagram.
The specific embodiment
Embodiment 1
1, the preparation of pig small intestine composite antibiosis peptide
(1) by 7.5Kg fresh pig small intestinal (comprising duodenum, jejunum), with deionized water, rinse well, remove content, remove serous coat and degrease, clean with the water for injection cleaning down, freezingly shred, meat grinder just twists 2 times and obtain the pig small intestine colloid;
(2) by above-mentioned pig small intestine colloid through colloid mill homogenate, make tissue homogenate 3.8L, then homogenate is sub-packed in to apyrogenic bottle-40 ℃ freezing, 37 ℃ of flowing water thaw, multigelation 3 times, boil 15min by 100 ℃ of water proofs of homogenate, is cooled to rapidly 4 ℃;
Under (3) 4 ℃ of conditions, the acetic acid solution 3.8L stirring and leaching that is 5.00% by the 3.8L homogenate after freeze thawing and concentration 12 hours, mixture after stirring and leaching carries out centrifugal for the first time in 4 ℃ of condition 8000rpm, centrifugal 15min, obtain supernatant 4.4L for the first time,-20 ℃ of preservations, the acetic acid solution that described concentration is 5.00% is prepared with water for injection;
Under (4) 4 ℃ of conditions, by the acetic acid solution 2.24L(W:V that centrifugal gained precipitate 3.2Kg and concentration are 6.07% for the first time, be 1:0.7) stirring and leaching 12 hours, repeat above-mentioned centrifugal process, obtain supernatant 1.54L for the second time,-20 ℃ of preservations, the acetic acid solution that described concentration is 6.07% is prepared with water for injection;
Under (5) 4 ℃ of conditions, by the acetic acid solution 1.95L(W:V that centrifugal gained precipitate 3.9Kg and concentration are 7.5% for the second time, be 1:0.5) stirring and leaching 12 hours, repeat above-mentioned centrifugal process again, obtain supernatant 1.32L for the third time, the acetic acid solution that described concentration is 7.5% is prepared with water for injection;
(6) merge above-mentioned three centrifugal gained 7.26L supernatant, supernatant, through 0.45um micro-filtrate membrane filtration, 8KD ultrafiltration membrance filter, is collected to ultrafiltration and seen through thing, obtain ultrafiltrate; Regulate ultrafiltrate pH value to 7.0, obtain pig small intestine composite antibiosis peptide semifinished product;
Wherein, a.0.45um microfiltration is by the 7.26L supernatant, through the 0.45um boxlike film slipstream system (Pellicon2Durapore0.45um1.0m of Millipore company
2The film bag) microfiltration clarification (5 times concentrated), obtain microfiltration clear liquor 5.81L, and microfiltration trapped substance 1.45L is 3 times of equal-volumes dialysis clear liquor 4.35L that gets back again, obtains altogether the 10.16L clear liquor.
0.45um boxlike film slipstream system mean parameter is as follows:
B.8KD ultrafiltration is by microfiltration clear liquor 10.16L, through the 8KD boxlike film slipstream system (Pellicon2Biomax8KD0.5m of Millipore company
2The film bag) ultrafiltration (15 times concentrated), ultrafiltration obtains ultrafiltrate 9.48L, and ultrafiltration trapped substance 0.68L is 3 times of equal-volumes dialysis ultrafiltrate 2.04L that gets back again, obtains altogether the 11.52L ultrafiltrate, regulates ultrafiltrate pH value to 7.0, is pig small intestine composite antibiosis peptide semifinished product.
8KD boxlike film slipstream system mean parameter is as follows:
(7) by pig small intestine composite antibiosis peptide semifinished product through 1KD NF membrane filtering and concentrating and dialysis desalination, collect the nanofiltration trapped substance; After the osmotic pressure of the nanofiltration trapped substance of collection is adjusted to 280mosm/kg, through the sterilizing filter degerming of 0.1um, obtain pig small intestine composite antibiosis peptide 8.1L.
Wherein, a.1KD concentrated the and dialysis desalination of nanofiltration is by pig small intestine composite antibiosis peptide semifinished product, through the 1KD boxlike film slipstream system (Pellicon2PLAC1KD2.0m of Millipore company
2The film bag) nanofiltration concentrated (1.3 times concentrated), collect nanofiltration trapped substance 8.86L, the 17.72L water for injection of take is again done 2 times of equal-volume dialysis as dialysis solution, obtains altogether 8.86L nanofiltration concentrate dialysate, and the osmotic pressure of the nanofiltration concentrate dialysate of collection is adjusted to 280mosm/kg;
1KD boxlike film slipstream system mean parameter is as follows:
B.0.1um sterilizing filter degerming is by 8.86L nanofiltration concentrate dialysate, through the degerming of 0.1um sterilizing filter, obtains pig small intestine composite antibiosis peptide 8.1L, and quality inspection is qualified.
Using the pig small intestine composite antibiosis peptide of embodiment 1 preparation as poultry new antibiotic, immunostimulant and immunological adjuvant, pig small intestine composite antibiosis peptide 0.02mL is immune every plumage chicken separately.
2, the quality inspection method of pig small intestine composite antibiosis 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 " appendix, asepsis growth;
(3) pH value and osmometry this product are tested by existing " Chinese veterinary pharmacopoeia " appendix, and pH value should be 6.5-7.5, and osmotic pressure value should be 280-320mosm/kg;
(4) the protein qualitative determination mixes 20% sulfosalicylic acid 2ml and sample 2ml, after reaction, without turbidity and precipitation, reacts negative;
(5) determining content of peptides this product is measured by Forint phenol method, and every milliliter of content of peptides should be not less than 2.0mg;
Forint phenol method wherein
Reagent
Alkaline copper solution solution I is carried out after 5 times of dilutions and the solution II, with the ratio of 50:1, mixes, and is alkaline copper solution after mixing, and this reagent can only be with 1 day, expire.
Phenol solution
Maneuver
1., the preparation of reference substance solution gets the bovine serum albumin reference substance, add water make every milliliter in containing the solution of 250ug.
2., that this product is got in the preparation of need testing solution is appropriate, water carries out suitably dilution as need testing solution.
3., 7 test tubes are got in the preparation of standard curve, precision measures 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 respectively alkaline copper solution 5.0ml, shake up, place 10min under room temperature, respectively add phenol solution 0.5ml, mix immediately, put room temperature and place 30min, then in the wavelength place of 500nm, measure trap; Using simultaneously and do not add solution in standard albumen test tube (0 pipe) as blank, take reference substance solution concentration as abscissa, trap is vertical coordinate, and the drawing standard curve line linearity of going forward side by side returns, and correlation coefficient is not less than 0.99.
4., the algoscopy precision measures need testing solution 1.0ml, the method under the preparation of sighting target directrix curve, from " adding respectively alkaline copper solution ", 0 pipe of take in standard curve, as blank, is measured trap in accordance with the law again.Try to achieve the concentration of need testing solution according to trap from regression equation, and be multiplied by extension rate, obtain the test sample content of peptides.
(6) Ribose concentration mensuration this product is measured by the Ribose concentration algoscopy, and every milliliter of Ribose concentration should be lower than 85 μ g;
The Ribose concentration algoscopy
Reagent
1., 5.00% trichloroacetic acid takes trichloroacetic acid 5g, adding distil water is dissolved into 100ml.
2., 0.1% ferric chloride-hydrochloric acid solution takes ferric chloride (FeCl
36H
2O) 0.5g, the enriching dissolving with hydrochloric acid becomes 500ml, but life-time service.
3., 1%3,5-orcin (orcin) takes 3,5-orcin 1g, is dissolved in 100ml0.1% ferric chloride-hydrochloric acid solution, faces with newly joining.
Operational approach
It is appropriate that the preparation precision of reference substance solution takes D ribose reference substance, with 5.00% trichloroacetic acid, makes every milliliter of solution containing ribose 20ug, shakes up.
It is appropriate that this product is got in the preparation of need testing solution, with 5.00% trichloroacetic acid, carries out suitably dilution as need testing solution.
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 respectively in band plug scale test tube, respectively add 5.00% trichloroacetic acid solution to 2.0ml, add respectively 1%3 again, 5-orcin solution 2.0ml, shake up, put in boiling water bath and accurately heat 30min, be cooled to rapidly room temperature, take No. 0 pipe as blank, according to spectrophotography (see existing " Chinese pharmacopoeia appendix), wavelength place at 650nm measures trap, take concentration as abscissa, trap is vertical coordinate, the drawing standard curve line linearity of going forward side by side returns, correlation coefficient is not less than 0.995.
The algoscopy precision measures need testing solution 2.0ml, and the preparation method of sighting target directrix curve rose from " adding respectively 1%3,5-orcin solution 2.0ml " again, measured trap in accordance with the law.Try to achieve the concentration of need testing solution according to trap from regression equation, and be multiplied by extension rate, obtain the test sample Ribose concentration.
(7) bacterial endotoxin mensuration this product is measured by the detection of bacterial endotoxin method, and every milliliter of endotoxin content should be no more than 10EU;
The detection of bacterial endotoxin method
Adopt the detection of bacterial endotoxin test kit to detect.
Preheating utensil used in 37 ℃ of incubators.
Sodium chloride injection for test sample (endotoxin<0.125EU/ml) is done to 10 times of dilutions, to be measured.
The test sample 0.5ml got after dilution carefully adds sample detection pipe (SPL), and shakes up gently.
The test sample of drawing carefully 0.25ml with pyrogen-free suction pipe from the sample detection pipe adds sample positive detection pipe (PPC) to mix gently 60 seconds, gets final product.
Sample detection pipe after mixing fully and sample positive detection pipe are positioned over to 37 ℃ of cultivation 30min.
Be inverted sample detection pipe and sample positive detection pipe, observe and have or not coagulation.
Sentencing test when the complete coagulation of positive control detector tube (PPC) sets up.
If the 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 in this dilution test sample that endotoxin content is lower than 1.0EU/ml.Calculate endotoxin content in former test sample according to dilution factor.
(8) safety verification
Pyrogen check adopts 3 of body weight 1.7-3.0Kg healthy rabbits (doe should without pregnant), raises separately 2, without abnormal.Survey body temperature once every 30min, survey twice, twice temperature difference and be no more than 0.2 ℃, using the meansigma methods of this body temperature of twice as the normal body temperature of this rabbit.The rabbit of using the same day, normal body temperature is in the scope of 38.0-39.6 ℃, and between each rabbit, the regular using warming therapy is poor is no more than 1 ℃.Syringe, the syringe needle of test use and the vessel that all contact with this product, the pyrogen of should going out.Rabbit is in measuring its normal body temperature 15min, slowly inject prescribed dose (injection volume is per kilogram of body weight 0.5ml) from ear vein and be warmed to approximately this product of 38 ℃, then every the 30min thermometric once, survey altogether 6 times, with the highest normal body temperature that once deducts in 6 body temperature, be the body temperature (when rabbit is heated up as negative value, all in 0 ℃) that this rabbit raises.The temperature that 3 rabbit body temperatures raise all should be lower than 0.6 ℃, and 3 rabbit body temperature rising summations should be lower than 1.4 ℃.
With chicken check, with 10 of 1 age in days SPF chickling, after this product is diluted to 10 times with physiological saline solution, every oral 1ml of chicken, repeat once orally in 24 hours, observes 5.Chickling should all be good for and be lived, and spirit, search for food, drinking-water, feather, feces should be all without extremely.
5 of body weight 18-22g healthy guinea pigs for abnormal toxicity test, every mouse peritoneal injection this product 0.5ml, observe 7.Mice should all be good for and be lived, and spirit, search for food, drinking-water, feather, feces should be all without extremely.
Animal is 6 of body weight 250-350g healthy guinea pigs for irritated check, the next day every each lumbar injection this product 1.0ml, totally three times, carry out sensitization.Then it is divided into to 2 groups, 3 every group, latter the 14th day and the 21st day of injection first, by intramuscular injection this product, 1.0ml was attacked respectively.Observe behavior and the sign of every animal every day.Observe and attack in rear 30min, animal is without symptoms of allergic such as perpendicular hair, dyspnea, tics.In intramuscular injection this product 2 hours, anaphylaxis must not appear.If any perpendicular hair, sneeze, retch, cough in the phenomenons such as 3 sound and dyspnea 2 kinds or two or more continuously, or have a convulsion, the one of shock, the phenomena of mortality, sentence this product against regulation.
Embodiment 2
(1) by 150Kg fresh pig small intestinal (comprising duodenum, jejunum, ileum), with deionized water, rinse well, remove content, remove serous coat and degrease, clean with the water for injection cleaning down, freezingly shred, meat grinder just twists 3 times and obtain the pig small intestine colloid;
(2) by above-mentioned pig small intestine colloid through colloid mill homogenate, make tissue homogenate 80L, then homogenate is sub-packed in to apyrogenic bottle-40 ℃ freezing, 37 ℃ of flowing water thaw, multigelation 5 times boils 20min by 100 ℃ of water proofs of homogenate, is cooled to rapidly 4 ℃;
Under (3) 4 ℃ of conditions, the acetic acid solution 80L stirring and leaching that is 5.00% by the homogenate 80L after freeze thawing and concentration 10 hours, mixture after stirring and leaching carries out centrifugal for the first time in 4 ℃ of condition 8000rpm, centrifugal 25min, obtain supernatant 93L for the first time,-20 ℃ of preservations, the acetic acid solution that described concentration is 5.00% is prepared with water for injection;
Under (4) 4 ℃ of conditions, by the acetic acid solution 53.6L(W:V that centrifugal gained precipitate 67Kg and concentration are 5.63% for the first time, be 1:0.8) stirring and leaching 9 hours, repeat above-mentioned centrifugal process, obtain supernatant 34L for the second time,-20 ℃ of preservations, the acetic acid solution that described concentration is 5.63% is prepared with water for injection;
Under (5) 4 ℃ of conditions, by the acetic acid solution 52L(W:V that centrifugal gained precipitate 86Kg and concentration are 6.67% for the second time, be 1:0.6) stirring and leaching 12 hours, repeat above-mentioned centrifugal process, obtain supernatant 32L for the third time, the acetic acid solution that described concentration is 6.67% is prepared with water for injection;
(6) merge above-mentioned three centrifugal gained 159L supernatant; Supernatant, through 0.22um micro-filtrate membrane filtration, 10KD ultrafiltration membrance filter, is collected to ultrafiltration and seen through thing, obtain ultrafiltrate; Regulate ultrafiltrate pH value to 7.0, make pig small intestine composite antibiosis peptide semifinished product;
Wherein a.0.22um microfiltration is by the 159L supernatant, through the 0.22um boxlike film slipstream system (CUF-50Durapore0.22um5.0m of Millipore company
2The film bag) microfiltration clarification (6 times concentrated), obtain microfiltration clear liquor 133L, and microfiltration trapped substance 26L is 2 times of dialysis clear liquor 52L that gets back again, obtains altogether the 185L clear liquor.
0.22um boxlike film slipstream system mean parameter is as follows:
B.10KD ultrafiltration is by microfiltration clear liquor 185L, through the 10KD boxlike film slipstream system (Pellicon2Biomax10KD2.5m of Millipore company
2The film bag) ultrafiltration (15 times concentrated), ultrafiltration obtains ultrafiltrate 173L, and ultrafiltration trapped substance 12L is 2.5 times of equal-volumes dialysis ultrafiltrate 30L that gets back again, obtains altogether the 203L ultrafiltrate, regulates ultrafiltrate pH value to 7.0, is pig small intestine composite antibiosis peptide semifinished product.
10KD boxlike film slipstream system mean parameter is as follows:
(7) by pig small intestine composite antibiosis peptide semifinished product through 3KD NF membrane filtering and concentrating and dialysis desalination, collect the nanofiltration trapped substance; After the osmotic pressure of the nanofiltration trapped substance of collection is adjusted to 280mosm/kg, through the sterilizing filter degerming of 0.1um, obtain pig small intestine composite antibiosis peptide 100.3L.
Wherein, a.3KD concentrated the and dialysis desalination of nanofiltration is by pig small intestine composite antibiosis peptide semifinished product, through the 3KD boxlike film slipstream system (Pellicon2PLBC3KD2.5m of Millipore company
2The film bag) nanofiltration concentrated (2 times concentrated), collect nanofiltration trapped substance 101.5L, the 101.5L water for injection of take is again done 1 times of equal-volume dialysis as dialysis solution, obtains altogether 101.5L nanofiltration concentrate dialysate, and the osmotic pressure of the nanofiltration concentrate dialysate of collection is adjusted to 280mosm/kg;
3KD boxlike film slipstream system mean parameter is as follows:
B.0.1um sterilizing filter degerming is by 101.5L nanofiltration concentrate dialysate, through the degerming of 0.1um sterilizing filter, obtains pig small intestine composite antibiosis peptide 100.3L, and quality inspection is qualified.
Using the pig small intestine composite antibiosis peptide of embodiment 2 preparations as poultry new antibiotic, immunostimulant and immunological adjuvant, pig small intestine composite antibiosis peptide 4mL and transmissible gastroenteritis of swine-every boar of 2 of epidemic diarrhea vaccines part combined immunization.
Embodiment 3
(1) by 7.5Kg fresh pig small intestinal (comprising duodenum, ileum), with deionized water, rinse well, remove content, remove serous coat and degrease, clean with the water for injection cleaning down, freezingly shred, meat grinder just twists 1 time and obtain the pig small intestine colloid;
(2) by above-mentioned pig small intestine colloid through colloid mill homogenate, make tissue homogenate 3.8L, then homogenate is sub-packed in to apyrogenic bottle-40 ℃ freezing, 37 ℃ of flowing water thaw, multigelation 6 times, boil 25min by 100 ℃ of water proofs of homogenate, is cooled to rapidly 4 ℃;
Under (3) 1 ℃ of conditions, the acetic acid solution 3.80L stirring and leaching that is 5.00% by the 3.8L homogenate after freeze thawing and concentration 8 hours, mixture after stirring and leaching carries out centrifugal for the first time in 4 ℃ of condition 7000rpm, centrifugal 20min, obtain supernatant 4.4L for the first time,-20 ℃ of preservations, the acetic acid solution that described concentration is 5.00% is prepared with water for injection;
Under (4) 1 ℃ of conditions, by the acetic acid solution 1.92L(W:V that centrifugal gained precipitate 3.2Kg and concentration are 6.67% for the first time, be 1:0.6) stirring and leaching 8 hours, repeat above-mentioned centrifugal process, obtain supernatant 1.19L for the second time,-20 ℃ of preservations, the acetic acid solution that described concentration is 6.67% is prepared with water for injection;
Under (5) 1 ℃ of conditions, by the acetic acid solution 1.57L(W:V that centrifugal gained precipitate 3.93Kg and concentration are 8.75% for the second time, be 1:0.4) stirring and leaching 14 hours, repeat again above-mentioned centrifugal process, obtain supernatant 1.03L for the third time, the acetic acid solution that described concentration is 8.75% is prepared with water for injection;
(6) merge above-mentioned three centrifugal gained 6.62L supernatant, supernatant, through 0.1um micro-filtrate membrane filtration, 10KD ultrafiltration membrance filter, is collected to ultrafiltration and seen through thing, obtain ultrafiltrate; Regulate ultrafiltrate pH value to 6.5, make pig small intestine composite antibiosis peptide semifinished product;
Wherein, a.0.1um microfiltration is by the 6.62L supernatant, through the 0.1um boxlike film slipstream system (Pellicon2Durapore0.1um1.0m of Millipore company
2The film bag) microfiltration clarification (5 times concentrated), obtain microfiltration clear liquor 5.30L, and microfiltration trapped substance 1.32L is 3 times of dialysis clear liquor 3.96L that gets back again, obtains altogether the 9.26L clear liquor.
0.1um boxlike film slipstream system mean parameter is as follows:
B.10KD ultrafiltration is by microfiltration clear liquor 9.26L, through the 10KD boxlike film slipstream system (Pellicon2Biomax8KD0.5m of Millipore company
2The film bag) ultrafiltration (15 times concentrated), ultrafiltration obtains ultrafiltrate 8.64L, and ultrafiltration trapped substance 0.62L is 3 times of equal-volumes dialysis ultrafiltrate 1.86L that gets back again, obtains altogether the 10.5L ultrafiltrate, regulates ultrafiltrate pH value to 6.5, is pig small intestine composite antibiosis peptide semifinished product.
10KD boxlike film slipstream system mean parameter is as follows:
(7) by pig small intestine composite antibiosis peptide semifinished product through 1KD NF membrane filtering and concentrating and dialysis desalination, collect the nanofiltration trapped substance; After the osmotic pressure of the nanofiltration trapped substance of collection is adjusted to 290mosm/kg, through the sterilizing filter degerming of 0.1um, obtain pig small intestine composite antibiosis peptide 6.82L.
Wherein, a.1KD concentrated the and dialysis desalination of nanofiltration is by pig small intestine composite antibiosis peptide semifinished product, through the 1KD boxlike film slipstream system (Pellicon2PLAC1KD2.0m of Millipore company
2The film bag) nanofiltration concentrated (1.5 times concentrated), collect nanofiltration trapped substance 7.00L, the 7.00L water for injection of take is again done 1 times of equal-volume dialysis as dialysis solution, obtains altogether 7.00L nanofiltration concentrate dialysate, and the osmotic pressure of the nanofiltration concentrate dialysate of collection is adjusted to 290mosm/kg;
1KD boxlike film slipstream system mean parameter is as follows:
B.0.1um sterilizing filter degerming is by 7.00L nanofiltration concentrate dialysate, through the degerming of 0.1um sterilizing filter, obtains pig small intestine composite antibiosis peptide 6.82L, and quality inspection is qualified.
Using the pig small intestine composite antibiosis peptide of embodiment 3 preparation as poultry new antibiotic, immunostimulant and immunological adjuvant, pig small intestine composite antibiosis 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), with deionized water, rinse well, remove content, remove serous coat and degrease, clean with the water for injection cleaning down, freezingly shred, meat grinder just twists 1 time and obtain the pig small intestine colloid;
(2) by above-mentioned pig small intestine colloid through colloid mill homogenate, make tissue homogenate 3.8L, then homogenate is sub-packed in to apyrogenic bottle-40 ℃ freezing, 37 ℃ of flowing water thaw, multigelation 6 times, boil 25min by 100 ℃ of water proofs of homogenate, is cooled to rapidly 4 ℃;
Under (3) 6 ℃ of conditions, the acetic acid solution 3.8L stirring and leaching that is 5.00% by the 3.8L homogenate after freeze thawing and concentration 14 hours, mixture after stirring and leaching carries out centrifugal for the first time in 4 ℃ of condition 6000rpm, centrifugal 25min, obtain supernatant 4.4L for the first time,-40 ℃ of preservations, the acetic acid solution that described concentration is 5.00% is prepared with water for injection;
Under (4) 6 ℃ of conditions, by the acetic acid solution 1.92L(W:V that centrifugal gained precipitate 3.2Kg and concentration are 6.67% for the first time, be 1:0.6) stirring and leaching 8 hours, repeat above-mentioned centrifugal process, obtain supernatant 1.25L for the second time,-40 ℃ of preservations, the acetic acid solution that described concentration is 6.67% is prepared with water for injection;
Under (5) 6 ℃ of conditions, by the acetic acid solution 2.34L(W:V that centrifugal gained precipitate 3.9Kg and concentration are 6.67% for the second time, be 1:0.6) stirring and leaching 14 hours, repeat again above-mentioned centrifugal process, obtain supernatant 1.50L for the third time, the acetic acid solution that described concentration is 6.67% is prepared with water for injection;
(6) merge above-mentioned three centrifugal gained 7.15L supernatant, supernatant, through 0.22um micro-filtrate membrane filtration, 8KD ultrafiltration membrance filter, is collected to ultrafiltration and seen through thing, obtain ultrafiltrate; Regulate ultrafiltrate pH value to 7.5, make pig small intestine composite antibiosis peptide semifinished product;
Wherein, a.0.22um microfiltration is by the 7.15L supernatant, through the 0.22um boxlike film slipstream system (Pellicon2Durapore0.22um1.0m of Millipore company
2The film bag) microfiltration clarification (4 times concentrated), obtain microfiltration clear liquor 5.36L, and microfiltration trapped substance 1.79L is 3 times of dialysis clear liquor 5.37L that gets back again, obtains altogether the 10.73L clear liquor.
0.22um boxlike film slipstream system mean parameter is as follows:
B.8KD ultrafiltration is by microfiltration clear liquor 10.73L, through the 8KD boxlike film slipstream system (Pellicon2Biomax8KD0.5m of Millipore company
2The film bag) ultrafiltration (16 times concentrated), ultrafiltration obtains ultrafiltrate 10.00L, and ultrafiltration trapped substance 0.73L is 2 times of equal-volumes dialysis ultrafiltrate 1.46L that gets back again, obtains altogether the 11.46L ultrafiltrate, regulates ultrafiltrate pH value to 7.5, is pig small intestine composite antibiosis peptide semifinished product.
8KD boxlike film slipstream system mean parameter is as follows:
(7) by pig small intestine composite antibiosis peptide semifinished product through 1KD NF membrane filtering and concentrating and dialysis desalination, collect the nanofiltration trapped substance; After the osmotic pressure of the nanofiltration trapped substance of collection is adjusted to 310mosm/kg, through the sterilizing filter degerming of 0.1um, obtain pig small intestine composite antibiosis peptide 7.5L.
Wherein, a.1KD concentrated the and dialysis desalination of nanofiltration is by pig small intestine composite antibiosis peptide semifinished product, through the 1KD boxlike film slipstream system (Pellicon2PLAC1KD2.5m of Millipore company
2The film bag) nanofiltration concentrated (1.5 times concentrated), collect nanofiltration trapped substance 7.64L, the 15.28L water for injection of take is again done 2 times of equal-volume dialysis as dialysis solution, obtains altogether 7.64L nanofiltration concentrate dialysate, and the osmotic pressure of the nanofiltration concentrate dialysate of collection is adjusted to 310mosm/kg;
1KD boxlike film slipstream system mean parameter is as follows:
B.0.1um sterilizing filter degerming is by 7.64L nanofiltration concentrate dialysate, through the degerming of 0.1um sterilizing filter, obtains pig small intestine composite antibiosis peptide 7.5L, and quality inspection is qualified.
Using the pig small intestine composite antibiosis peptide of embodiment 4 preparations as poultry new antibiotic, immunostimulant and immunological adjuvant, pig small intestine composite antibiosis peptide 3mL is every pig of immunity separately.
Obviously, the above embodiment of the present invention is only for example of the present invention clearly is 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 can't give all embodiments exhaustive.Every still row in protection scope of the present invention of apparent variation that technical scheme of the present invention extends out or change that belong to.
Claims (9)
1. the preparation method of a pig small intestine composite antibiosis peptide, is characterized in that, comprises and be prepared as follows step:
(1) the fresh pig small intestinal is rinsed well with deionized water, removed content, go to use the water for injection cleaning down after serous coat and degrease, freezing shredding then, meat grinder just twists 1-3 time, then uses colloid mill homogenate, makes homogenate;
(2) by after described homogenate multigelation 3-6 time, 100 ℃ of water proofs boil 15-25min, then cooling rear cold preservation rapidly, and refrigerated storage temperature is 1-4 ℃, cold preservation time is 0-5 hour;
(3) add the acetic acid solution that concentration is 5.00% in homogenate, stirring and leaching, it is centrifugal for the first time that 4 ℃ of conditions are carried out, by the freezing preservation of centrifugal for the first time supernatant, the acetic acid solution that described concentration is 5.00% is prepared with water for injection, and the volume ratio of described acetic acid solution and homogenate is 1:1;
(4) add the acetic acid solution that concentration is 6.67%-5.00% in centrifugal for the first time precipitate, stirring and leaching, it is centrifugal for the second time that 4 ℃ of conditions are carried out, and by the freezing preservation of centrifugal for the second time supernatant, the acetic acid solution that described concentration is 6.67%-5.00% is prepared with water for injection;
(5) add again the acetic acid solution that concentration is 8.75%-5.63% in centrifugal for the second time precipitate, stirring and leaching, it is centrifugal for the third time that 4 ℃ of conditions are carried out, and obtains centrifugal for the third time supernatant, and the acetic acid solution that described concentration is 8.75%-5.63% is prepared with water for injection;
(6) merge above-mentioned three supernatant, the supernatant that merges, through 0.1,0.22 or 0.45um slipstream micro-filtrate membrane filtration, is collected to microfiltration and seen through thing, obtain clear liquor; Clear liquor, through 8 or 10KD cross-flow ultrafiltration membrane filtration, is collected ultrafiltration and is seen through thing, obtains ultrafiltrate; Regulate the ultrafiltrate pH value to 6.5-7.5, obtain pig small intestine composite antibiosis peptide semifinished product;
(7) by pig small intestine composite antibiosis peptide semifinished product through 1 or 3KD slipstream NF membrane filtering and concentrating and dialysis desalination, collect the nanofiltration trapped substance; Regulate osmotic pressure to 280-320mosm/kg, then, through the sterilizing filter degerming of 0.1um, obtain pig small intestine composite antibiosis peptide.
2. the preparation method of pig small intestine composite antibiosis peptide according to claim 1, is characterized in that, step (3), (4) described cryogenic temperature are below-20 ℃, 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 ℃; Step (3), (4), (5) described centrifugation time are 15-25min, and centrifuge speed is 6000-8000rpm.
3. the preparation method of pig small intestine composite antibiosis peptide according to claim 1, is characterized in that, the weight/volume (W:V) of described centrifugal sediment for the first time and acetic acid solution is 1Kg:0.6-1L.
4. the preparation method of pig small intestine composite antibiosis peptide according to claim 1, is characterized in that, the weight/volume (W:V) of described centrifugal sediment for the second time and acetic acid solution is 1Kg:0.4-0.8L.
5. the pig small intestine composite antibiosis peptide obtained as method as described in claim 1-4 any one.
6. the purposes of pig small intestine composite antibiosis peptide as claimed in claim 5, is characterized in that, pig small intestine composite antibiosis peptide is as the purposes of poultry new antibiotic, immunostimulant and immunological adjuvant.
7. the purposes of pig small intestine composite antibiosis peptide according to claim 5, is characterized in that, described pig small intestine composite antibiosis peptide separately or with the vaccine combined immunization.
8. the purposes of pig small intestine composite antibiosis peptide according to claim 5, is characterized in that, described pig small intestine composite antibiosis peptide 1-6mL separately or with transmissible gastroenteritis of swine-every pig of epidemic diarrhea vaccine 1-3 head part combined immunization.
9. the purposes of pig small intestine composite antibiosis peptide according to claim 5, is characterized in that, described pig small intestine composite antibiosis peptide 0.01-0.2mL separately or with the every plumage chicken of chicken virus mycoplasma vaccine 1-3 plumage part combined immunization.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310343030.8A CN103417574B (en) | 2013-08-08 | 2013-08-08 | The Preparation method and use of pig small intestine recombinant antimicrobial peptide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310343030.8A CN103417574B (en) | 2013-08-08 | 2013-08-08 | The Preparation method and use of pig small intestine recombinant antimicrobial peptide |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103417574A true CN103417574A (en) | 2013-12-04 |
| CN103417574B CN103417574B (en) | 2015-10-28 |
Family
ID=49643316
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310343030.8A Expired - Fee Related CN103417574B (en) | 2013-08-08 | 2013-08-08 | The Preparation method and use of pig small intestine recombinant antimicrobial peptide |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103417574B (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN106977595A (en) * | 2017-05-09 | 2017-07-25 | 派生特（福州）生物科技有限公司 | The extracting method of one boar derived antimicrobial peptide |
| 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 |
| CN108837151A (en) * | 2018-09-30 | 2018-11-20 | 派生特（福州）生物科技有限公司 | The preparation method of one boar vaccine diluent |
| CN109206477A (en) * | 2018-09-30 | 2019-01-15 | 派生特（福州）生物科技有限公司 | A kind of rabbit sacculus rotundus antibacterial peptide and preparation method thereof |
| CN109331169A (en) * | 2018-09-30 | 2019-02-15 | 派生特（福州）生物科技有限公司 | A kind of sheep bone peptide oral liquid and preparation method thereof |
| CN109464652A (en) * | 2018-11-28 | 2019-03-15 | 派生特（福州）生物科技有限公司 | A kind of composition and its preparation method and application for poultry diarrhea |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107084939A (en) * | 2017-05-09 | 2017-08-22 | 派生特（福州）生物科技有限公司 | A kind of authentication method of antibacterial peptide |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102816226A (en) * | 2012-08-23 | 2012-12-12 | 郑州后羿制药有限公司 | Preparation method of porcine small intestine antibacterial peptides PR39 |
-
2013
- 2013-08-08 CN CN201310343030.8A patent/CN103417574B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102816226A (en) * | 2012-08-23 | 2012-12-12 | 郑州后羿制药有限公司 | Preparation method of porcine small intestine antibacterial peptides PR39 |
Non-Patent Citations (2)
| Title |
|---|
| 马卫明: "猪小肠抗菌肽分离鉴定及其生物活性研究", 《中国博士学位论文全文数据库》 * |
| 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 (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN106977595A (en) * | 2017-05-09 | 2017-07-25 | 派生特（福州）生物科技有限公司 | The extracting method of one boar derived antimicrobial peptide |
| 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 |
| CN108837151A (en) * | 2018-09-30 | 2018-11-20 | 派生特（福州）生物科技有限公司 | The preparation method of one boar vaccine diluent |
| CN109206477A (en) * | 2018-09-30 | 2019-01-15 | 派生特（福州）生物科技有限公司 | A kind of rabbit sacculus rotundus antibacterial peptide and preparation method thereof |
| CN109331169A (en) * | 2018-09-30 | 2019-02-15 | 派生特（福州）生物科技有限公司 | A kind of sheep bone peptide oral liquid and preparation method thereof |
| CN109464652A (en) * | 2018-11-28 | 2019-03-15 | 派生特（福州）生物科技有限公司 | A kind of composition and its preparation method and application for poultry diarrhea |
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103417574B (en) | 2015-10-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103417574B (en) | The Preparation method and use of pig small intestine recombinant antimicrobial peptide | |
| CN103566370B (en) | Preparation method and application of swine vaccine specific swine spleen transfer factor (TF) | |
| FI74474B (en) | FREQUENCY FOR EXTRACTION OF NYY, UR KLEBSIELLA PNEUMONIAE EXTRAHERADE GLYKOPROTEINER. | |
| CN103316045A (en) | Combined specific transfer factor for livestock and poultry and preparation method of combined specific transfer factor | |
| CN101690813B (en) | Concentrated freeze-dried yolk antibody composite preparation for Newcastle disease and preparation process thereof | |
| CN101362805A (en) | Extraction of astragalus polysaccharide and molecular modification method thereof | |
| CN100484959C (en) | Modified recombinant porcine alpha interferon protein and coding gene and uses thereof | |
| CN102286100B (en) | SEB (staphylococcal enterotoxin B) resisting immune globulin F(ab')2 and preparation method thereof | |
| CN103709247A (en) | Divalent egg yolk antibodies of type I and novel duck hepatitis as well as preparation method and application thereof | |
| CN102198155B (en) | Production method of pig spleen transfer factor injection | |
| CN108676061B (en) | Mannatide and purification method thereof | |
| CN105147716A (en) | Newcastle disease vaccine Yupingfeng polysaccharide immunopotentiator | |
| CN101690812A (en) | Concentrated freeze-dried yolk antibody composite preparation for Newcastle disease-infectious bursal disease and preparation process thereof | |
| CN101475627A (en) | Preparation of transfer factor against swine fever | |
| CN103599534B (en) | Preparation method and use of poultry vaccine-specific pig spleen transfer factor | |
| CN115770251A (en) | Application of polysaccharide in preparing medicine for preventing and/or treating ulcerative colitis | |
| CN103864930A (en) | Preparation and use method of antibody resisting scorpion venom of buthus martensii karsch in China F (ab') 2 | |
| CN101820891A (en) | BCG polysaccharide nucleic acid extractive and preparation method thereof | |
| CN108588034A (en) | A kind of rabbit hemorrhagic disease virus of variation and its application in preparing inactivated vaccine | |
| CN101037469A (en) | Method for extracting small molecule active peptides from scapharca subcrenata | |
| CN103565834A (en) | Preparation method and application of swine vaccine specific cattle spleen transfer factor (TF) | |
| CN105342996A (en) | Preparation method for spleen polypeptide injection | |
| CN100371347C (en) | Active material of polypeptide proteins in bloody clam, preparation method and usage | |
| CN101317857A (en) | Method for extracting goat submaxillary gland biologically active substance and application thereof | |
| CN109745548A (en) | A kind of application of pertussis toxin in treatment cerebral infarction |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| ASS | Succession or assignment of patent right |
Owner name: XU LEI Effective date: 20150505 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20150505 Address after: Fujian Province, Lianjiang County, Fuzhou County, Fuxing Road, Jiangnan County, No. 8, Ling Xin Xintiandi, building 6, 1, 1 storefront Applicant after: FUZHOU PAISHENGTE BIOTECHNOLOGY Co.,Ltd. Applicant after: Xu Lei Address before: Jiangnan Lianjiang County of Fuzhou City, Fujian province 350000 Township 8 Fuxing Road, the new spirit world No. 6 Building 1 layer 1 stores Applicant before: FUZHOU PAISHENGTE BIOTECHNOLOGY Co.,Ltd. |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CP03 | Change of name, title or address |
Address after: Jiangnan Lianjiang County of Fuzhou City, Fujian province 350000 Township 8 Fuxing Road, the new spirit world No. 6 Building 1 layer 1 stores Co-patentee after: Xu Lei Patentee after: PRESENT (FUZHOU) BIO-TECH CO.,LTD. Address before: Fujian Province, Lianjiang County, Fuzhou County, Fuxing Road, Jiangnan County, No. 8, Ling Xin Xintiandi, building 6, 1, 1 storefront Co-patentee before: Xu Lei Patentee before: FUZHOU PAISHENGTE BIOTECHNOLOGY Co.,Ltd. |
|
| CP03 | Change of name, title or address | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20151028 |