CN103007280B - Piglet colibacillosis lyophilized serum immunoglobulin preparation and preparation process thereof - Google Patents
Piglet colibacillosis lyophilized serum immunoglobulin preparation and preparation process thereof Download PDFInfo
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- CN103007280B CN103007280B CN201310019656.3A CN201310019656A CN103007280B CN 103007280 B CN103007280 B CN 103007280B CN 201310019656 A CN201310019656 A CN 201310019656A CN 103007280 B CN103007280 B CN 103007280B
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Abstract
The invention provides a piglet colibacillosis lyophilized serum immunoglobulin preparation. The piglet colibacillosis lyophilized serum immunoglobulin preparation is composed of following components in parts by weight: 5.0-55.0 parts of saccharicterpenin, 0.005-0.01 parts of hyoscyamine, 55.0-95.0 parts of serum immunoglobulin. The piglet colibacillosis lyophilized serum immunoglobulin preparation disclosed by the invention has higher stability, can inhibit and kill colon bacillus more sustainably and more sufficiently, and has more remarkable anti-infection effects, so that the purpose of preventing and treating diarrhea of the piglet due to the colon bacillus can be realized more easily. Meanwhile, the piglet colibacillosis lyophilized serum immunoglobulin preparation is not only a pure natural environment-friendly type green and anti-infection drug without medicine residue, public hazard, drug tolerance and side effect, but is also an excellent medicine for replacing various harmful antibiotic medicines.
Description
Technical field
The invention belongs to field of biological pharmacy, be specifically related to a kind of colibacillosis of piglet freeze-dry blood serum immunoglobulin preparation and preparation technology thereof.
Background technology
Escherichia coli of piglets diarrhoea morbidity urgency, case fatality rate are up to 90%.Once piglet morbidity, just has little time treatment with regard to large quantities of death.Penicillin finds 80 for many years, has developed successively a large amount of antibiotic and synthetic chemical drugs.The invention of these medicines and application, decline to a great extent clinical common bacterial infection and case fatality rate, also comprises escherichia coli of piglets diarrhoeal diseases.But along with the frequent use of these medicines and long-term, high-dose use, antibacterial has obtained drug resistance, the drug resistant gene being present in bacterial chromosome, plasmid and transposon just can be by modes such as conversion, transduction, combination and swivel bases, resistance energy genetic transmission is arrived to other sensitive organism, make it to become drug resistance strain, brought serious threat to the further prevention and control infectious disease of the mankind.China's animal husbandry develops on an unprecedented scale, and livestock products total amount occupy first of the whole world for many years always.Wherein, the year number of animals raised of pig reaches 1,000,000,000 bulls.Having stimulated thus veterinary drug market to reach unprecedented develops on an unprecedented scale.The main uses of veterinary drug is control poultry infectious disease, accounts for more than 60%, according to estimation year, applies anti-infectives more than 10,000 tons.Along with the continuous growth of China's population, the quality of livestock products enters international market, has further strengthened Production of Livestock and Poultry scale, has given the further prosperous development of veterinary drug., use antibiotic and synthetic chemical drugs to cause in animal body long-term, heavy dose ofly and produced the antagonistic pathogenic bacterium of medicine, and then polluted livestock products; These residues of veterinary drug, in animal products, are passed to people by food chain simultaneously, cause human diseases.These facts have caused that people's extreme fears fear and great attention.Whole world national governments formulated a series of in Production of Livestock and Poultry forbidding and limit the use of antibiotic and government decree antibiotic, antiviral chemical drugs, the livestock products of the residual and cause of disease of refusal import pastille, have directly suppressed the development in veterinary drug market.The medicine that is applied at present anti-escherichia coli of piglets diarrhoea has various bacillus coli vaccines, antibiotic, chemosynthesis medicine, Chinese medicine and preparation thereof and immune globulin Pseudobulbus Bletillae (Rhizoma Bletillae) serum immune globulin preparation and other anti-infective biotic factor preparations.
(1) bacillus coli vaccine
At present, mainly contain colibacillosis of piglet tervalence inactivated vaccine, escherichia coli K88, K99,987P trivalent gene vaccine, K88-ST1-LTB tervalence inactivated vaccine, K88ac-ST1-LTB trivalent gene engineering vaccine etc.These vaccines are mainly used in preventing piglet colon bacillus diarrhoea (yellow scour of piglet and Hakuri), for in-pig immunity, make newborn piglet obtain passive immunity by breast milk.But, due to sow body constitution and the reason such as piglet is weak, cause passive immunity failure, and cause the outburst of escherichia coli of piglets diarrhoea in many cases.
(2) antibiotic
This class anti-infectives is mainly the metabolite being produced by Institute of Micro-biology such as antibacterial, fungus, actinomycetes, and artificial imitated synthetic or semisynthetic antibiotics.They are strong to the inhibition of pathogenic microorganism or killing action, has a broad antifungal spectrum, and good effect, is widely used in human and animal's diseases prevention and treatment.But, their toxic and side effects are large, as very easily there is anaphylaxis in natural penicillin (benzylpenicillin), semisynthetic penicillin (ampicillin, amoxicillin), cephalosporin etc., streptomycin, amikacin, gentamycin, micronomicin, kanamycin etc. very easily cause kidney and the 8th pair of cranial nerve lesions and the effect of disabling to fetus and infant, oxytetracycline, tetracycline, chlortetracycline, polymyxin E, bacitracin etc. cause liver, renal damage, and lincomycin, chloromycetin etc. also has harmful effect (referring to Chinese Pharmacopoeia) to hematopoietic function.
(3) chemosynthesis medicine
This class anti-infectives mainly comprises sulfonamides, quinolones, furans etc.Their has a broad antifungal spectrum, stable in properties, determined curative effect, easy to use.But, there is toxic and side effects large, easily residue in body, cause liver, kidney etc. to organize irreversible infringement (referring to Chinese Pharmacopoeia).
Above two class medicines are except above-mentioned shortcoming, and their common drawbacks also have interference body normal flora, destroy body microecological balance, cause microorganism species imbalance displacement, cause superinfection and other pathological changes; Moreover heavy dose of Reusability can cause drug resistance, reduce therapeutic effect, make the responsive rate of its anti-infective drop to 10-15%, and induce out highly pathogenic bacterial strain.So countries in the world have been formulated a series of policies one after another, forbid using this two classes medicine in animal produces, extend their medical life-spans to human diseases, but this is a way of trying to stop water from boiling by skimming it off and pouring it back.
(4) Chinese medicine and preparation thereof
Chinese medicine belongs to natural drug, has the feature of harmless advantage, and strengthening vital QI to eliminate pathogenic factors, integral body are adjusted and has no drug resistance, and are that antibiotics and chemosynthesis medicine are incomparable, thereby have caused the extensive attention of western developed country.At present, on market, also there are many anti-infective Chinese medicine preparation as Radix Isatidis electuary, SHUANGHUANGLIAN KOUFUYE, CHUANXINLIAN ZHUSHEYE etc.Although these medicine antibacterial spectrums are wide, because drug effect is slow, effect is weak, consumption is large, difficult quality is stable so that use the factors such as inconvenient, therefore in clinical practice, be greatly limited.
(5) immunoglobulin and serum immune globulin preparation
This class anti-infectives is from special animal serum and serum immune globulin.Their speed of actions are fast, and curative effect is accurate, and high specificity, but antibiotic faciostenosis are invalid to mixed infection and secondary infection, sometimes have to by above-mentioned three class medicine auxiliary treatment; Moreover they belong to reactive protein, necessary cryopreservation is easy to deactivation in storing and use procedure, loses medical value.Therefore, these anti-infectious preparations are difficult to operation and have lost commodity meaning on drug market, and then have had influence on clinical practice.
(6) other anti-infective biotic factor preparations
Such as interferon, cytokine etc., due to expensive, be difficult to be accepted extensively by clinical, as for use more difficult implementation in zoonosis.
21 century will be the century of one " green ".The exploitation of green product is that the Chinese government takes " organizational behavior " for problems such as health, environment, resources, is the contribution that global economy and social sustainable development are made.Green product will become the leading trend of consumption.Along with the enhancing of global environmental consciousness, the transformation of people's values, uphold nature, focuses on safely, pursues healthy thought and will first affect people's consuming behavior.The transformation of consumption idea and consuming behavior directly affects again International Trade Policy, makes " green barrier " become new Main Trade barrier.National governments, the especially ,Deng of Europe, the United States developed country have arrived and have been close to overcritical stage the regulation of the pesticide in food, Residual Veterinary Medicines and other poisonous and harmful substances content standard.From 1992, Japan implemented new " inspection of food hygiene system " to import poultry, and its health test major part is higher than world's general standard, and the residual of the antibacterial such as antibiotic, sulfanilamide and pesticide stopped in requirement completely.The countries such as Germany, Britain, France, Australia have also formulated the residue criterion of all kinds of veterinary drugs separately in different food products.So market is in strong birth of calling a kind of pure natural, no drug residue, nuisanceless, the environment-friendly anti-infectives that has no drug resistance, have no side effect.
Summary of the invention
The object of the invention is to overcome the shortcoming of prior art, a kind of colibacillosis of piglet freeze-dry blood serum immunoglobulin preparation is proposed, said preparation has higher stability, more fully lasting to colibacillary inhibitory or killing effect, infection effect is more remarkable, and is a kind of pure natural, no drug residue, nuisanceless, the environment-friendly anti-infectives that has no drug resistance, have no side effect.
The present invention also provides the preparation method of described preparation.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
A kind of colibacillosis of piglet freeze-dry blood serum immunoglobulin preparation is provided, and it is comprised of the component of following weight portion: saccharicterpenin 5.0 ~ 55.0, hyoscyamine 0.005 ~ 0.01, serum immune globulin 55.0 ~ 95.0.
The preferred colibacillosis of piglet freeze-dry blood serum of the present invention immunoglobulin preparation is comprised of the component of following weight portion:
Saccharicterpenin 10.0 ~ 35.0, hyoscyamine 0.005 ~ 0.01, serum immune globulin 60.0 ~ 90.0.
The most preferred colibacillosis of piglet freeze-dry blood serum of the present invention immunoglobulin preparation is comprised of the component of following weight portion:
Saccharicterpenin 12.0, hyoscyamine 0.008, serum immune globulin 88.0.
In described saccharicterpenin, by weight percentage, contents of saccharide >=30%, triterpenoid saponin content >=30%; Described saccharicterpenin can be the pure natural plant extract that the various saccharides that extract from plant and triterpenoid saponin form; In the preferred saccharicterpenin of the present invention, saccharide and triterpenoid saponin are all to extract from the Chinese crude drug of following weight portion: the Rhizoma Atractylodis Macrocephalae 30 ~ 50, Rhizoma Atractylodis 20 ~ 40, the Radix Aucklandiae 15 ~ 20, Rhizoma Alismatis 10 ~ 15.
Described saccharide extracting method can be several different methods of the prior art, can be also following extracting method preferred for this invention:
A, ratio is got the Rhizoma Atractylodis Macrocephalae 30 ~ 50, Rhizoma Atractylodis 20 ~ 40, the Radix Aucklandiae 15 ~ 20 and the rear chopping of Rhizoma Alismatis 10 ~ 15, is cleaned by weight, with cold water soak 2 hours to the medicine heart, fully soak into, the water that adds again 12 times of Chinese medicine gross weights, after being heated to seethe with excitement, the in the situation that of 90 ℃, under hot reflux concentration technology, decoct 180 minutes concentrated concentrated medicament I that obtain simultaneously;
B, concentrated medicament prepared by step a, through 10000 revs/min centrifugal 20 minutes, discard precipitate, obtain supernatant also concentrated through negative pressure, make it form the concentrated medicament II that dry is 25%;
C, the concentrated medicament II that step b is obtained adds doubly 95% the ethanol of (volume) of 4-8, standing over night, abandoning supernatant, precipitate is dissolved in water and passes through 100KD membrane filtration after removing ethanol, except foreigh protein removing, the macromole impurity such as endotoxin, collecting filtered solution filters by 1KD NF membrane again, remove the impurity such as organic molecule and inorganic molecule and ion, after the material dilution being trapped, reverse current dialysis is 24 hours, then taking out and being concentrated into relative density is 1.01 ~ 1.18 grams per milliliters, add again 5 times (volumes) and measure 99% ethanol precipitation, stir, standing over night, 3000-5000 rev/min centrifugal 20 minutes, precipitate is removed ethanol dry through spraying, can make.
Described triterpenoid saponin extracting method can be several different methods of the prior art, can be also following extracting method preferred for this invention:
I. ratio is got the Rhizoma Atractylodis Macrocephalae 30 ~ 50, Rhizoma Atractylodis 20 ~ 40, the Radix Aucklandiae 15 ~ 20 and the rear chopping of Rhizoma Alismatis 10 ~ 15, is cleaned by weight, with ethanol or methanol extraction, reclaims solvent, gets residue cake and makes aqueous solution with water dissolution or suspendible;
In ii, the aqueous solution made toward step I, add petroleum ether or ether, the lipophilic substance pigment in aqueous solution and oil are reclaimed, triterpenoid saponin is stayed in aqueous solution;
In iii, the aqueous solution that makes toward ii step, add the organic solvent butanols that hydrophilic is stronger, the material that in solution, hydrophilic is stronger continues to stay in aqueous solution, and triterpenoid saponin is transferred in organic solvent extraction liquid, collects organic solvent extraction liquid, drying under reduced pressure, makes rough triterpenoid saponin;
Iv, the rough triterpenoid saponin that iii step is made are dissolved in ethanol or methanol, then drip gradually the mixed liquor of ether, acetone or ether-acetone (volume 1:1), till separating out, the triterpenoid saponin of separating out are dried and can be made to triterpenoid saponin from solution.
In formulation components of the present invention, hyoscyamine can be the existing various product that hyoscyamine is main component of take; The hyoscyamine that the present invention preferably extracts from Flos Daturae.
The preferred hyoscyamine of the present invention can extract by several different methods from Flos Daturae, preferred extracting method is as follows: by weight, get 1 part of Flos Daturae, ethanol percolation with 30 parts of pH2 ~ 3, get percolate and reclaim solvent evaporated, alcohol extractum is added to hydro-thermal molten, adjust pH value to 2 ~ 3, filter, get acid solution, add defat with petroleum ether, again obtain aqueous acid, ammonia adjust pH to 10, with chloroform extraction, chloroform extraction liquid is taken out, reclaim chloroform, getting the remaining liquid of extraction is added on cation exchange resin column, first with water elution remove impurity, use again 0.5% ~ 15% acid solution eluting, collect eluent, add alkali neutralization, through desalting processing, obtain hyoscyamine.
Serum immune globulin in preparation of the present invention can be in prior art through piglet colibacillosis tervalence inactivated vaccine (ETEC K88 K99 987P) (the 40th page of People's Republic of China's veterinary biologics quality standard " version 2001 ") repeatedly the height after the healthy growing and fattening pigs of reinforced immunological exempt from synthesis in porcine blood serum.Serum immune globulin of the present invention preferably makes in accordance with the following methods:
1. according to conventional method, prepare the high porcine blood serum of exempting from of piglet colon bacillus
The healthy growing and fattening pigs of 1.1 no-special pathogens are in the isolated rearing of clean plant;
3 immunity are carried out in the healthy growing and fattening pigs of 1.2 pairs of step 1.1 raisings, each to every pig injection piglet colibacillosis tervalence inactivated vaccine (ETEC K88 K99 987P), injection volume is respectively 5 ~ 8 parts, 5 parts and 10 parts, each immune interval week age;
1.3 immune latter 21 days for the third time, collect porcine blood serum, serum immune globulin in serum is carried out to immunology detection, the micro-agglutination titer of the serum immune globulin of ETEC K88 K99 987P be 1:64 ~ 1:128 above after, this porcine blood serum is the high porcine blood serum of exempting from of piglet colon bacillus, and collection storage is standby under 16 ℃ of conditions, if detect defective, according to immunizing dose for the third time again immunity, until detect qualified;
2. according to conventional method, gather Sanguis sus domestica, separation of serum the serum immune globulin of purifying.
Described separation and the concrete grammar of purification can be with reference to the chief editors' such as the Chinese Academy of Agricultural Sciences < < veterinary microbiology > >, Chinese agriculture publishing house, within 1998, December is the 1st edition, the 656th page.
Colibacillosis of piglet freeze-dry blood serum immunoglobulin preparation provided by the invention, on the basis combining in modern molecular immunology, modern endocrine neurological and Chinese medicine, utilize intermolecular hydrogen bonding structural theory and receptor theory, after saccharicterpenin, hyoscyamine, serum immune globulin three are mixed, through cold drying, obtain.
In the present invention's formula, saccharicterpenin is one of main component, is a kind of material that is rich in hydroxyl, can build each other multidimensional network space hydrogen bond structure, on serum immune globulin surface, forms false hydration shell.The existence of the false hydration shell of hydrogen bond has directly affected surface structure, conformation, nature and function of serum immune globulin molecule etc.By the false hydration shell of this multidimensional network space hydrogen bond, realize the protective effect of saccharicterpenin to serum immune globulin, be mainly reflected in the performance that has improved the induction degeneration such as serum immune globulin opposing high temperature, ultraviolet; Saccharicterpenin also strengthens immunogenic immune effect by distinctive mode in animal body, plays the effect of immunostimulant.Studies have shown that, the consumption of saccharicterpenin in pharmaceutical formulation of the present invention should be 5.0 ~ 55.0.The saccharicterpenin of the preparation that the Chinese medicine formula that the present invention adopts extracts; owing to having adopted the theory of Chinese Traditional Medicine " dialectical treating "; by the Rhizoma Alismatis of the Rhizoma Atractylodis of the Rhizoma Atractylodis Macrocephalae of spleen invigorating monarch drug, dampness ministerial drug, damp eliminating accessory drugs with regulate the flow of vital energy and make the Radix Aucklandiae compatibility of medicine form prescription to make; thereby possessing on the basis of protein protective agent effect, more have and strengthen the diarrhoea effect that this preparation for treating colibacillosis causes.
Serum immune globulin is one of second main component during the present invention fills a prescription.It is the magistery during the present invention fills a prescription, and directly for pathogenic microorganism, plays inhibitory or killing effect.Studies have shown that, the consumption of serum immune globulin in colibacillosis of piglet freeze-dry blood serum immunoglobulin preparation formula should be 55.0 ~ 95.0.
Hyoscyamine is the 3rd main component during the present invention fills a prescription, it is the magistery during the present invention fills a prescription, it is a kind of Plant Secondary Metabolites, it can combine with the acetylcholinergic receptor of intestinal wall in intestine of young pigs, appropriateness reduces intestinal peristalsis promoting frequency, saccharicterpenin and the serum immune globulin escherichia coli fully and in intestinal are interacted, but realize, effectively kill the object that escherichia coli reach control escherichia coli of piglets diarrhoea.Studies have shown that, the consumption of hyoscyamine in pharmaceutical formulation of the present invention should be 0.005 ~ 0.01.
The preparation method of the colibacillosis of piglet freeze-dry blood serum immunoglobulin preparation described in the present invention also provides, comprise the following steps: according to weight ratio meter, saccharicterpenin 5.0 ~ 55.0, serum immune globulin 55.0 ~ 95.0 and hyoscyamine 0.005 ~ 0.01 are mixed, add sterile deionized water dilution, after homogenate, emulsifying, filtration, adopt lyophilization, make.
The preparation method of described colibacillosis of piglet freeze-dry blood serum immunoglobulin preparation, preferably include following steps: according to weight ratio meter, by saccharicterpenin 5.0 ~ 55.0, serum immune globulin 55.0 ~ 95.0 and hyoscyamine 0.005 ~ 0.01 mix, add sterile deionized water dilution, the micro-agglutination titer of the serum immune globulin of ETEC K88 K99 987P in every ml soln is reached >=more than 1:512, then be placed in vacuum freeze drier, at-40 ℃, lyophilization is 2 ~ 8 hours, 3 ℃ of left and right of intensification per hour then, through the vacuum dryings to 25 of 12 ~ 18 hours ℃, complete dry, sealing, in 25 ℃ of environment, keep 3 ~ 5 hours, be finished product.
The quality standard of colibacillosis of piglet freeze-dry blood serum immunoglobulin preparation of the present invention is as follows:
[physical behavior] this product is brown Sponge Porosity agglomerate, easily departs from bottle wall, dissolves rapidly after adding diluent.
[steriling test] undertaken by People's Republic of China's veterinary drug allusion quotation requirement, answers asepsis growth.
[safety verification] gets bottled preparation of lyophilizing, with normal saline reduction, be diluted to every milliliter of 1 part, select 5 of 18 ~ 22 grams of white mice of body weight, 0.2 milliliter of each this preparation diluent for oral administration, 2 of 1.5 ~ 2.0 kilograms of rabbit of body weight, 10 milliliters of each this preparation diluents for oral administration, 2 of 350 ~ 400 grams of Cavia porcelluss of body weight, 5 milliliters of each this preparation diluents for oral administration, observe 10, all should be good for work.
[efficacy test] by 2 ~ 4 kilograms of body weight with source without 7 of the pigs of swine escherichia coli K88 K99 987P serum immune globulin, divide 2 groups: first group 4, by oral preparation diluent of every 1 part (with safety verification item), simultaneously oral piglet colon bacillus is 1 milliliter, 1 milliliter of 2 ~ 300,000,000 bacterium.Second group 3, only oral piglet colon bacillus is 1 milliliter, and 1 milliliter of 2 ~ 300,000,000 bacterium in contrast.
Contrast pig should be after oral piglet colon bacillus body temperature rise in 2 ~ 8 hours, present typical coliform diarrhea in piglets symptom, and in 1 ~ 3 day, having 2 at least, to die from acute coliform diarrhea in piglets sick thereupon.4 pigs of first group, observe 5 ~ 10, and at least strong 3 alive is qualified.
The preparation of [titration] test sample serum immune globulin: precision measures 1 milliliter of preparation diluent (with safety verification item), puts into centrifuge tube, then adds chloroform 4ml, fully mix, after standing 1 hour, 5000 revs/min, centrifugal 30 minutes, get supernatant and get final product.
With the serum immune globulin of micro-agglutination (MAT method) check ETEC K88, K99, the 987P Ying≤1:128 that tires.
MAT method: ETEC K88, K99, the preparation of 987P standard coagulation antigen: application LB culture medium culturing K88, K99,987P reference culture, results bacterium liquid is counted rear deactivation, with sterile saline cyclic washing 3 times (centrifugal 5 minutes of 3000rpm), the bacterial sediment of last centrifugal acquisition, according to the dilution of thalline count results, be 2,000,000,000 thalline/mL, as standard coagulation antigen, carry out micro-coagulation experiment.
Test sample according to the form below dilutes and operates.
The micro-agglutination plate that vibrates, makes testing sample evenly mix with standard antigen, then micro-agglutination plate is placed in to 37 ℃ of effects observed result after 4-6 hour.
Result judges, take highly diluted multiple the tiring as this pili serum immune globulin of serum immune globulin of 50% somatic agglutination.
[effect and purposes] is for prevention and treatment yellow and white dysentery of piglet.
[usage and consumption]
1,1 ~ 2 part of oral preparation diluent (with safety verification item) in prevention consumption newborn piglet 1 hour.
2, every 1 part of therapeutic dose, consumption is multiplicable first, and 2 ~ 3 times on the 1st, 1 course for the treatment of on the 3rd.
[storage] room temperature, cool place, dry, ventilate, 36 months effect duration.
Compared with prior art, colibacillosis of piglet freeze-dry blood serum immunoglobulin preparation of the present invention has following several respects beneficial effect:
One, stability significantly improves, the processing of the preparation of being more convenient for, storage and transportation
Preparation of the present invention is the product that saccharicterpenin and serum immune globulin organically combine by molecular separating force (being mainly Van der Waals force and hydrogen bond etc.), is the product that the non-specific immunity of the specific immune of serum immune globulin and saccharicterpenin and they are ideally combined the opsonic action of body self resistance against diseases.Serum immune globulin and saccharicterpenin structure connection, have complementary functions, bring out the best in each other.In preparation of the present invention, described saccharicterpenin is a kind of material that is rich in hydroxyl, and they can build multidimensional network space hydrogen bond structure each other, on serum immune globulin surface, forms false hydration shell.The existence of the false hydration shell of hydrogen bond has directly affected surface structure, conformation, nature and function of serum immune globulin molecule etc.By the false hydration shell of this multidimensional network space hydrogen bond structure, realize the protective effect of saccharicterpenin to serum immune globulin, be mainly reflected in the performance that has improved serum immune globulin opposing high temperature, induced degeneration.Saccharicterpenin is bonded together and forms multidimensional network space with hydrogen bond (hydrogen bonding); for serum immune globulin has been built mansion shelter; serum immune globulin is placed in multidimensional network space with hydrogen bond; form so stable, the firm false hydration shell of multidimensional network space hydrogen bond, protected well serum immune globulin to avoid the destruction of external environment (high temperature or ultraviolet).When serum immune globulin is at processing and fabricating, when being dried and storing and transporting, first the multidimensional network space that saccharicterpenin forms is accepted the effect of the factors such as environment's heat and is moved disorderly, but the construction features due to multidimensional network space, cause hydroxyl on saccharicterpenin molecule can be at once when dehydration directly to form hydrogen bond with the amino on serum immune globulin surface and hydroxyl etc., caused whole molecular motion to go to zero, the vibration of the secondary key of serum immune globulin intramolecule and the extension of peptide chain have been alleviated, three conformations and the biological activity that have kept serum immune globulin.So; saccharicterpenin has the feature of protein protective agent; in the course of processing of preparation, safeguarded the level Four stereochemical structure form of serum immune globulin; overcome the shortcoming that serum immune globulin must be preserved, transport and use in low temperature environment; therefore expand most effectively the range of application of serum immune globulin in clinical practice, greatly strengthened the market potential ability of antibody preparation.In preparation of the present invention, saccharicterpenin has following experiment for card to the effect of improving of serum immune globulin stability:
The experiment of saccharicterpenin protection serum immune globulin thermal denaturation:
(1) method
1 method of inspection: micro-coagulation experiment (MAT) method
The preparation of serum immune globulin solution: height is exempted to porcine blood serum sterilization, broken shell, separation of serum, precision measures serum 1g, puts into centrifuge tube, adds sterile saline 1ml, mixes; Add chloroform 4ml, fully concussion mixes, and after standing 1 hour, 5000 revs/min, centrifugal 30 minutes, gets supernatant and obtains serum immune globulin solution again.
ETEC K88, K99, the preparation of 987P standard coagulation antigen: application LB culture medium culturing K88, K99,987P reference culture, results bacterium liquid is counted rear deactivation, with sterile saline cyclic washing 3 times (centrifugal 5 minutes of 3000rpm), the bacterial sediment of last centrifugal acquisition, according to the dilution of thalline count results, be 3,000,000,000 thalline/mL, as standard coagulation antigen, carry out micro-coagulation experiment.
Test sample serum immune globulin solution is pressed table 1 and is diluted and operate.
The dilution operation table of table 1 test sample serum immune globulin solution
The micro-agglutination plate that vibrates, makes testing sample evenly mix with standard antigen, then micro-agglutination plate is placed in to 37 ℃ of effects observed result after 4 ~ 6 hours.
Result judges, what the highly diluted multiple of antibody of 50% somatic agglutination of take was this fimbriae antibody tires.
2 serum immune globulin preparations
K88, K99,987P escherichia coli inactivated vaccine head are exempted to 300 of the blue brown healthy growing and fattening pigs in sea of 113 ages in days, intramuscular injection, every 0.5ml; Every 7 days two, exempt from intramuscular injection, every 0.5ml; Every 7 days three, exempt from again intramuscular injection, every 1.0ml.Three exempt to start for latter 21 days to get the microagglutination test (MAT) that egg carries out serum immune globulin and K88, K99,987P antigen, when polyclonal serum IgM AT tires while reaching 6 (MAT Xiao Jia ㏒ 2 represent, below identical), are high-immunity egg.Collect high-immunity egg, sterilization, broken shell, separation, obtains hyper-immune serum, according to acidifying water---and ultrafiltration integrated technique, by defat, precipitation, lyophilization, make serum immune globulin, through MAT, measure, every milliliter during containing 200mg serum immune globulin, it is 9 that the MAT of polyclonal serum immunoglobulin tires.
The mensuration of 3 serum immune globulin hot propertys
1. the MAT titration of variable concentrations serum immune globulin solution
Get serum immune globulin and be dissolved in normal saline and make every milliliter containing the serial serum immune globulin solution for 5mg, 10mg, 20mg, 30mg, 40mg and 50mg, and carry out MAT with K88, K99,987P antigen.
2. the MAT that serum immune globulin solution heats after different time under 75 ℃ of conditions tires
In order further to get the relation of serum immune globulin thermal denaturation and heat time heating time clear, we are on the basis of above-mentioned experiment, serum immune globulin is diluted in to the solution that normal saline is made 5mg/ml, at 75 ℃ of Water Unders, bathe after heating 0,5,10,15,20,25,30,35,40,45min, drop into immediately in frozen water coolingly, then carry out MAT with K88, K99,987P antigen.
The change of 4 saccharicterpenin to polyclonal serum immunoglobulin hot property
1. saccharicterpenin impact on serum immune globulin hot property in normal saline
The saccharicterpenin of getting the embodiment of the present invention 1 preparation is dissolved in the serum immune globulin solution of 5mg/ml of normal saline dilution, makes saccharicterpenin concentration to 80mg/ml; The serum immune globulin solution of getting again 5mg/ml contrasts, and heats respectively 0,15,30, after 45min, at once puts into frozen water cooling under 75 ℃ of conditions, then tries MAT with K88, K99,987P antigen.
2. the serum immune globulin hot property in the special serum immune globulin preparation of saccharicterpenin coupling changes
The saccharicterpenin of the embodiment of the present invention 1 preparation is mixed by patent formulation with serum immune globulin, after homogenate, be sub-packed in cillin bottle, be placed in vacuum freeze drier dry.Vacuum freezing condition: 1, be chilled in advance-40 ℃; 2, lyophilization 2 ~ 8 hours at-40 ℃, 3 ℃ of left and right of intensification per hour then, the vacuum drying to 25 ℃ through 12 ~ 18 hours; 3, add a cover, sealing keeps 3 ~ 5 hours in 25 ℃ of environment, makes escherichia coli of piglets serum immune globulin preparation.This preparation of accurate weighing, is 5mg/ml with normal saline dilution, heats 0,15, after 30min, carries out MAT with K88, K99,987P antigen under 75 ℃ of conditions.
(2) results and analysis
The hot property situation of 1 serum immune globulin extract
1. variable concentrations serum immune globulin solution and the MAT that heats after 30min under 75 ℃ of conditions thereof tire
In serum immune globulin solution, serum immune globulin concentration is higher, its MAT tires higher, in every ml physiological saline Immunoglobulins in Serum when 5 ~ 50mg, every increase 1mg serum immune globulin, MAT tires to be increased more than 0.09, is strong positive correlation (R=0.971).When the solution of variable concentrations is heated to 30min under 75 ℃ of conditions, its corresponding MAT tires and declines 2 ~ 3, the biological activity that is to say serum immune globulin declines more than 75.0 ~ 87.5%, also can find out that serum immune globulin concentration is higher larger to hot resistant function simultaneously.Concrete outcome is in Table 2.
The different serum immune globulin solution of table 2 and 75 ℃ of heating the (㏒ 2 that tires of the MAT after 30min)
When 2. serum immune globulin concentration is 5mg/ml, the MAT heating after different time under 75 ℃ of conditions tires
Under 75 ℃ of conditions, the normal saline solution that is 5mg/ml by serum immune globulin concentration, heating 0 ~ 45min, the MAT of its serum immune globulin tires and significantly declines, be every prolongation heat time heating time 1min, the MAT of serum immune globulin tires and declines 0.07, and both are strong negative correlation (R=-0.976), in Table 3.
Table 3 serum immune globulin concentration is 5mg/ml at the MAT of the 75 ℃ of heating different times (㏒ 2 that tires)
The impact of 2 saccharicterpenin on serum immune globulin hot property
1. saccharicterpenin impact on serum immune globulin hot property in normal saline
Serum immune globulin is dissolved in respectively to the saccharicterpenin mixed solution of normal saline and 80mg/ml, make the concentration of serum immune globulin to 5mg/ml, under 75 ℃ of conditions, heat after 0 ~ 30min, in the solution of serum immune globulin, the MAT of serum immune globulin tires and declines rapidly; In the saccharicterpenin normal saline solution of serum immune globulin, MAT tires and does not change.Result shows, in serum immune globulin normal saline solution, adds saccharicterpenin, has broken the thermal denaturation rule in normal saline solution, has effectively protected the biological activity of serum immune globulin.So saccharicterpenin has very significant thermal denaturation resistant effect to serum immune globulin in normal saline, in Table 4.
The hot property result comparison of table 4 serum immune globulin in saccharicterpenin solution and normal saline
2. the serum immune globulin hot property situation in escherichia coli of piglets serum immune globulin preparation of the present invention
By common escherichia coli of piglets freeze-dry blood serum immunoglobulin B(in the escherichia coli of piglets freeze-dry blood serum immunoglobulin preparation A of the embodiment of the present invention 1 preparation and prior art, be simple serum immune globulin) be mixed with the normal saline solution of 5mg/ml concentration, under 75 ℃ of conditions, heat 0,15, after 30min, detect MAT and tire 3, heat the biological activity of the serum immune globulin in preparation of the present invention without any change.This illustrates in preparation of the present invention, and saccharicterpenin has good thermal denaturation resistant effect to serum immune globulin.Detailed results is as table 5.
The MAT (㏒ 2 that tires after table 5 escherichia coli of piglets freeze-dry blood serum of the present invention immunoglobulin preparation A and the common escherichia coli of piglets freeze-dry blood serum of prior art immunoglobulin B heat different time under 75 ℃ of conditions) result and analytical table thereof
(3) conclusion and discussion
Under 75 ℃ of conditions, heating, especially easily there is thermal denaturation in serum immune globulin, thereby causes inactivation.This fact has obtained further checking again in this experiment.On this basis, we have stoped the thermal denaturation of serum immune globulin under 75 ℃ of heating conditions by the saccharicterpenin of development effectively, have guaranteed the biological activity of serum immune globulin.Utilize saccharicterpenin by the refining escherichia coli of piglets freeze-dry blood serum immunoglobulin preparation forming of coupling serum immune globulin processing technique, there is powerful thermal denaturation resistant function, effectively solved the thermal denaturation Pinch technology difficult problem of serum immune globulin preparation when processing, store, transporting and using, for large-scale industrial production and the Market Operation of serum immune globulin preparation have been established sturdy technical foundation.
Two, more fully lasting to colibacillary inhibitory or killing effect
Hyoscyamine in preparation of the present invention can combine with the acetylcholinergic receptor of intestinal wall in intestine of young pigs, appropriateness reduces intestinal peristalsis promoting frequency, serum immune globulin is extended piglet gastral action time, therefore can interact by the escherichia coli fully and in intestinal, but effectively kill the object that escherichia coli reach control escherichia coli of piglets diarrhoea, have following experiment for card:
In this preparation, hyoscyamine extends in this preparation serum immune globulin in the piglet experiment of gastral action time:
(1) experimental animal
Come into being and do not feed Beestings health piglet, kind is Du * large * long three way cross market pig.
(2) method
1 dispensing design and sampling
Select 12 of newborn piglets, be divided into 2 groups, 5 every group.1 group when 10 hour age every piglet by the oral cavity 4ml concentration of feeding, be the preparation of the embodiment of the present invention 1 of 100mg/ml, 2 groups when 10 hour age every piglet by the oral cavity 4ml concentration of feeding, be the simple serum immune globulin of 100mg/ml; The contents samples of measuring respectively the stomach of getting test piglet after feeding for 0,4,6,8,12 hour, 12 fat intestinal, jejunum, ileum, large intestine, the ELISA that measures respectively purification freeze-dry blood serum immunoglobulin wherein tires.
2ELISA measures
1) reagent preparation
1. be coated with buffer (carbonate buffer solution of 0.05mol/LPH9.6)
Na
2cO
3(anhydrous) 1.50g
NaHCO
3 2.98g
With deionized water, be settled to 1000ml
2. cleaning mixture (phosphate buffer-polysorbas20 Tween-20 of PH7.4)
NaH
2PO
4 0.2g NaCl 8.0g
Na
2HPO
4 12H
2O 2.9g KCl 0.2g
If Tween-20 0.5ml(does not add as PBS liquid) with deionized water, be settled to 1000ml
3. for diluent, cleaning mixture is prepared 1% hyclone albumin soln.
4. phosphate-citrate buffer solution of PH5.0
First liquid: citric acid 19.2g is settled to 1000ml with deionized water
Second liquid: Na
2hPO
412H
2o 71.39g is settled to 1000ml with deionized water
Used time gets first liquid 28.0ml second liquid 22.0ml and is settled to 100ml with deionized water
5. substrate reagent is got in phosphate-citrate buffer solution that o-phenylenediamine 10mg is dissolved in 25mlPH5.0, adds 30%H
2o
20.4ml, fully mixes.(during use, prepare temporarily, can not place for a long time)
6. stop buffer (2mol/LH
2sO
4) dense H
2sO
4(95 ~ 98%) 22.2ml is slowly dissolved in water, and ceaselessly stirs heat radiation, in order to avoid produce a large amount of heat in the short time, sets off an explosion.After cooling, be settled to 200ml.
7. stop buffer 2M H
2sO
4solution (concentrated sulphuric acid 22.2ml, sterile deionized water 177.8ml)
8. the anti-pig IgG peroxidase of rabbit (rb Anti-Chicken IgG A9046) is provided by Sigma company.
9. polyclone IgY sterling (I4881 ~ 10mg is purchased in the anti-preparation of the anti-pig IgG two of rabbit, Chicken-IgG, U.S. sigma company) and prepare its oil seepage, antigenic content 0.5mg/ml, give rabbit immunity three times, each one week interval time, after three immunity, gather serum one week time, with two-way agar diffusion, survey anti-the tiring of the anti-pig IgG two of rabbit and reach 1:4(antigenic content 0.125mg/ml), be placed in-20 ℃ of preservations.
2) ELISA titration method
1. coated sensitization flat board is got the polystyrene board of clean dry, with coating buffer, the anti-pig IgG two of rabbit is resisted to 30ug/ml, and 150ul/ hole, builds, and puts 4 ℃ and spends the night.
2. next day is sealed in washing, removes Ag solution in hole, with cleaning mixture, washes 3 ~ 4 times, and each 3 ~ 4min, dries.Add 10% defatted milk powder solution (the PBS liquid preparation of pH7.4 for defatted milk powder), every hole is filled it up with, and 37 ℃ act on 1 hour.
3. washing, adds sample to be checked to remove hole inner sealing liquid, with cleaning mixture, washes 3 ~ 4 times, and each 3 ~ 4min, dries.Sample is done to different multiples dilution with diluent.150ul/ hole, 37 ℃ act on 1 hour.
4. washing, adds the anti-pig IgG peroxidase of enzyme mark rabbit to remove solution in hole, with cleaning mixture, washes 3 ~ 4 times, and each 3 ~ 4min, dries.The anti-pig IgG peroxidase of rabbit that adds 1:800,150ul/ hole, 37 ℃ act on 1 hour.
5. washing, adds substrate reagent to remove solution in hole, with cleaning mixture, washes 3 ~ 4 times, and each 3 ~ 4min, dries.Add substrate reagent, 150ul/ hole, 37 ℃ of effect 10 ~ 15min.Be placed in dark place, note change color simultaneously, extremely positive hole color all occurs, and obvious, can not overresponse.
6., after cessation reaction chromogenic reaction finishes, add stop buffer, 100ul/ hole, cessation reaction at once.
7., after mensuration is opened instrument, first preheating, then be take air as blank once a little, at 492nm, measures and record sample and control wells light absorption value.
8. interpretation of result OD survey/OD the moon >=2.1 are positive
(3) result
10 hour age piglet by the oral cavity 5ml concentration of feeding, be the preparation of the embodiment of the present invention 1 of 100mg/ml and simple serum immune globulin respectively, measure respectively after feeding 0,4,6,8,12 hour serum immune globulin distribution results in gastrointestinal tract in Table 6.
The preparation of table 6 embodiment of the present invention 1/simple serum immune globulin detects information slip in digestive tract
Table 6 result proves, simple serum immune globulin can detect with each section of intestinal for 6 hours after Piglet by Oral under one's belt, within 8 hours, just can't check serum immune globulin under one's belt with each section of intestinal.But the serum immune globulin in the preparation of the embodiment of the present invention 1 can detect with each section of intestinal for 8 hours after Piglet by Oral under one's belt, within 12 hours, also can detect at each section of intestinal.This explanation, because the hyoscyamine in preparation of the present invention is to gastral effect, has weakened the spastic wriggling of the intestinal being caused by colitoxin, causes serum immune globulin in preparation of the present invention to extend the action time in intestinal.Therefore serum immune globulin can interact by the escherichia coli fully and in intestinal, but effectively kills the object that escherichia coli reach control escherichia coli of piglets diarrhoea.
Three, infection effect significantly and comprehensively
The product that preparation of the present invention ideally combines the non-specific immunity of the specific immune of serum immune globulin and saccharicterpenin and they to the opsonic action of body self resistance against diseases.The cytokines such as saccharicterpenin can be protected immune organ, increases immune organ weight, strengthens Function of mononuclear phagocyte, strengthens T cell function, strengthens B cell function, strengthens NK cell function, increases complement content and strengthen complement activity, induces IL-1, IL-2, IFN, CSF, TNF; But the special pathogenic microorganism that kills of serum immune globulin.So said preparation both infects for protopathy pathogenic microorganism, again for secondary and mixing cause pathogeny imcrobe infection; Both pathogenic microbe killing, excited again autoimmunity and whole disease-resistant function; Both treating both the principal and secondary aspects of a disease, quick and durable again.Therefore, this preparation has embodied the combination of tcm theory and modern immunology principle all sidedly, is a kind of anti-infectives that effectively excites and regulate the new high efficiency of body's immunity.The significant infection effect of preparation of the present invention has following experiment for card:
Colibacillosis of piglet freeze-dry blood serum immunoglobulin preparation treatment experiment
1) tested pig situation
Tested swinery is the piglet group of age in days in puerperal 5 ~ 30, and kind is unrestricted, raises the old epidemic-stricken area in once popular colibacillosis excessively infection.
2) colibacillosis of piglet diagnosis and treatment criterion
1. Infection in Piglets escherichia coli after being ill, main manifestations is piglet diarrhea clinically.Therefore, the difform feces of the different formation of piglet feces water content of take is judgment basis, determines piglet diarrhea scoring, in Table 7:
Table 7, piglet diarrhea standards of grading
2. piglet diarrhea curative effect determinate standard is in Table 8.
Table 8, piglet diarrhea curative effect determinate standard
3) experiment and observational technique
The pig farm in the old epidemic-stricken area of infecting at colibacillosis of piglet, is divided into 4 groups by morbidity piglet, i.e. this preparation group (A) of the embodiment of the present invention 1 preparation, serum immune globulin group (B), enrofloxacin group (C) and the fragrant group (D) of faling apart that connects.After piglet generation colibacillosis dysentery, each group is treated by corresponding Experimental agents description, during treating, morbidity piglet travel follow-up is observed, determine therapeutic effect, and statistics recovery from illness number, significant figure and invalid number, calculate by the following method a healing number and cure rate that each is organized:
Cure number=recovery from illness number+significant figure
Cure rate=(recovery from illness number+significant figure)/morbidity number * 100%
Experimental result shows, uses the colibacillosis of piglet freeze-dry blood serum immunoglobulin preparation treatment escherichia coli of piglets diarrhoea of the embodiment of the present invention 1 to improve cure rate more than 7% than other common drug.Refer to following table 9:
Table 9. colibacillosis of piglet freeze-dry blood serum of the present invention immunoglobulin preparation
Effect and analytic statistics table thereof with other common drug treatment escherichia coli of piglets diarrhoea
* compares with other 3 groups, and difference is (P<0.01) extremely significantly.
The specific embodiment
Below by specific embodiment, further illustrate feature of the present invention.
Embodiment 1:
This preparation for zymad microorganism be escherichia coli of piglets (ETEC K88 K99 987P).
Composition and the parts by weight of the colibacillosis of piglet freeze-dry blood serum immunoglobulin preparation of the present embodiment are as follows:
Saccharicterpenin 12.0
Hyoscyamine 0.008
Serum immune globulin 88.0
The present embodiment preparation is prepared by the following method:
One, prepare saccharicterpenin
(1) saccharide extracts:
A, ratio is got the Rhizoma Atractylodis Macrocephalae 40, Rhizoma Atractylodis 30, the Radix Aucklandiae 20 and the rear chopping of Rhizoma Alismatis 10, is cleaned by weight, with cold water soak 2 hours to the medicine heart, fully soak into, the water that adds again 12 times of Chinese medicine gross weights, after being heated to seethe with excitement, the in the situation that of 90 ℃, under hot reflux concentration technology, decoct 180 minutes concentrated concentrated medicament I that obtain simultaneously;
B, concentrated medicament prepared by step a, through 10000 revs/min centrifugal 20 minutes, discard precipitate, obtain supernatant also concentrated through negative pressure, make it form the concentrated medicament II that dry is 25%;
C, the concentrated medicament II that step b is obtained adds 95% the ethanol of 5 times (volumes), standing over night, abandoning supernatant, precipitate is dissolved in water and passes through 100KD membrane filtration after removing ethanol, except foreigh protein removing, the macromole impurity such as endotoxin, collecting filtered solution filters by 1KD NF membrane again, remove the impurity such as organic molecule and inorganic molecule and ion, after the material dilution being trapped, reverse current dialysis is 24 hours, then taking out and being concentrated into relative density is 1.01～1.18 grams per milliliters, add again 5 times of amounts (volume), 99% ethanol precipitation, stir, standing over night, 3000-5000 rev/min centrifugal 20 minutes, precipitate is removed ethanol dry through spraying, makes saccharide.
(2) triterpenoid saponin extracts:
I. ratio is got the Rhizoma Atractylodis Macrocephalae 40, Rhizoma Atractylodis 30, the Radix Aucklandiae 20 and the rear chopping of Rhizoma Alismatis 10, is cleaned by weight, with ethanol or methanol extraction, reclaims solvent, gets residue cake and makes aqueous solution with water dissolution or suspendible, and then do following two-way extraction and purification:
Ii, in the aqueous solution of making, add petroleum ether or ether, the lipophilic substance pigment in aqueous solution and oil are reclaimed, triterpenoid saponin is stayed in aqueous solution;
In iii, the aqueous solution that makes toward ii step, add the organic solvent butanols that hydrophilic is stronger, the material that in solution, hydrophilic is stronger continues to stay in aqueous solution, and triterpenoid saponin is transferred in organic solvent extraction liquid, collects organic solvent extraction liquid, drying under reduced pressure, makes rough triterpenoid saponin;
Iv, the rough triterpenoid saponin that iii step is made are dissolved in ethanol or methanol, drip gradually again the mixed liquor of ether, acetone or ether-acetone (volume ratio 1:1), till separating out from solution to triterpenoid saponin, by the dry triterpenoid saponin that makes of the triterpenoid saponin of separating out.
(3) sugar step () and step (two) being made and triterpenoid saponin mix and make saccharicterpenin, make wherein saccharide percentage by weight >=30%, triterpenoid saponin percentage by weight >=30%.
Two, prepare hyoscyamine
From Flos Daturae, extract hyoscyamine, extracting method is as follows: get 1 part of Flos Daturae, with the ethanol percolation of 30 parts of pH2 ~ 3, get percolate and reclaim solvent evaporated, alcohol extractum is added to hydro-thermal molten, adjust pH value 2 ~ 3, filter, get acid solution, add defat with petroleum ether, again obtain aqueous acid, ammonia is transferred pH value 10, with chloroform extraction, chloroform extraction liquid is taken out, reclaim chloroform, get the remaining liquid of extraction and be added on cation exchange resin column, first with water elution remove impurity, use again 0.5% ~ 15% acid solution eluting, collect eluent, add alkali neutralization, through desalting processing, obtain hyoscyamine.
Three, prepare serum immune globulin, step is as follows:
1. according to conventional method, prepare the high porcine blood serum of exempting from of piglet colon bacillus
The healthy growing and fattening pigs of 1.1 no-special pathogens are in the isolated rearing of clean plant;
3 immunity are carried out in the healthy growing and fattening pigs of 1.2 pairs of step 1.1 raisings, each to every pig injection piglet colibacillosis tervalence inactivated vaccine (ETEC K88 K99 987P), injection volume is respectively 5 ~ 8 parts, 5 parts and 10 parts, each immune interval week age;
1.3 immune latter 21 days for the third time, collect porcine blood serum, serum immune globulin in serum is carried out to immunology detection, the micro-agglutination titer of the serum immune globulin of ETEC K88 K99 987P be 1:64 ~ 1:128 above after, this porcine blood serum is the high porcine blood serum of exempting from of piglet colon bacillus, and collection storage is standby under 16 ℃ of conditions, if detect defective, according to immunizing dose for the third time again immunity, until detect qualified;
2. according to conventional method, gather Sanguis sus domestica, separation of serum and purify and obtain serum immune globulin.The separated concrete grammar with purifying is with reference to the chief editors' such as the Chinese Academy of Agricultural Sciences < < veterinary microbiology > >, Chinese agriculture publishing house, within 1998, December is the 1st edition, the 656th page.
Four, prepare colibacillosis of piglet freeze-dry blood serum immunoglobulin preparation
According to the weight ratio of said components, saccharicterpenin prepared by step 1, serum immune globulin prepared by hyoscyamine prepared by step 2 and step 3 mixes, add sterile deionized water dilution, the micro-agglutination titer of the serum immune globulin of ETEC K88 K99 987P in every ml soln is reached >=more than 1:512, then be placed in vacuum freeze drier, at-40 ℃, lyophilization is 2 ~ 8 hours, 3 ℃ of left and right of intensification per hour then, through the vacuum dryings to 25 of 12 ~ 18 hours ℃, complete dry, sealing, in 25 ℃ of environment, keep 3 ~ 5 hours, be finished product.
Quality standard:
[physical behavior] this product is brown Sponge Porosity agglomerate, is easy to bottle wall and departs from, and dissolves rapidly after adding diluent.
[steriling test] undertaken by People's Republic of China's veterinary drug allusion quotation requirement, answers asepsis growth.
[safety verification] gets bottled preparation of lyophilizing, with normal saline reduction, be diluted to every milliliter of 1 part, select 5 of 18 ~ 22 grams of white mice of body weight, 0.2 milliliter of each this preparation diluent for oral administration, 2 of 1.5 ~ 2.0 kilograms of rabbit of body weight, 10 milliliters of each this preparation diluents for oral administration, 2 of 350 ~ 400 grams of Cavia porcelluss of body weight, 5 milliliters of each this preparation diluents for oral administration, observe 10, all should be good for work.
[efficacy test] by 2 ~ 4 kilograms of body weight with source without 7 of the pigs of swine escherichia coli K88 K99 987P serum immune globulin, divide 2 groups: first group 4, by oral preparation diluent of every 1 part (with safety verification item), simultaneously oral piglet colon bacillus is 1 milliliter, 1 milliliter of 2 ~ 300,000,000 bacterium.Second group 3, only oral piglet colon bacillus is 1 milliliter, and 1 milliliter of 2 ~ 300,000,000 bacterium in contrast.
Contrast pig should be after oral piglet colon bacillus body temperature rise in 2 ~ 8 hours, present typical coliform diarrhea in piglets symptom, and in 1 ~ 3 day, having 2 at least, to die from acute coliform diarrhea in piglets sick thereupon.4 pigs of first group, observe 5 ~ 10, and at least strong 3 alive is qualified.
The preparation of [titration] test sample serum immune globulin: precision measures 1 milliliter of preparation diluent (with safety verification item), puts into centrifuge tube, then adds chloroform 4ml, fully mix, after standing 1 hour, 5000 revs/min, centrifugal 30 minutes, get supernatant and get final product.
With the serum immune globulin of micro-agglutination (MAT method) check ETEC K88, K99, the 987P Ying≤1:128 that tires.
MAT method: ETEC K88, K99, the preparation of 987P standard coagulation antigen: application LB culture medium culturing K88, K99,987P reference culture, results bacterium liquid is counted rear deactivation, with sterile saline cyclic washing 3 times (centrifugal 5 minutes of 3000rpm), the bacterial sediment of last centrifugal acquisition, according to the dilution of thalline count results, be 2,000,000,000 thalline/mL, as standard coagulation antigen, carry out micro-coagulation experiment.
Test sample according to the form below dilutes and operates.
The micro-agglutination plate that vibrates, makes testing sample evenly mix with standard antigen, then micro-agglutination plate is placed in to 37 ℃ of effects observed result after 4 ~ 6 hours.
Result judges, take highly diluted multiple the tiring as this pili serum immune globulin of serum immune globulin of 50% somatic agglutination.
[effect and purposes] is for prevention and treatment yellow and white dysentery of piglet.
[usage and consumption]
1,1 ~ 2 part of oral preparation diluent (with safety verification item) in prevention consumption newborn piglet 1 hour.
2, every 1 part of therapeutic dose, consumption is multiplicable first, and 2 ~ 3 times on the 1st, 1 course for the treatment of on the 3rd.
[storage] room temperature, cool place, dry, ventilate, 36 months effect duration.
Embodiment 2:
This preparation for zymad microorganism be escherichia coli of piglets (ETEC K88 K99 987P).
Composition and the parts by weight of the colibacillosis of piglet freeze-dry blood serum immunoglobulin preparation of the present embodiment are as follows:
Saccharicterpenin 55
Hyoscyamine 0.005
Serum immune globulin 55.0
Preparation method is substantially with embodiment 1, and wherein difference is only: the saccharide in saccharicterpenin and triterpenoid saponin all extract the Chinese medicine from following weight ratio: the Rhizoma Atractylodis Macrocephalae 30, Rhizoma Atractylodis 40, the Radix Aucklandiae 15 and Rhizoma Alismatis 15.
Quality standard is with embodiment 1.
Embodiment 3:
This preparation for zymad microorganism be escherichia coli of piglets (ETEC K88 K99 987P).
Composition and the parts by weight of the colibacillosis of piglet freeze-dry blood serum immunoglobulin preparation of the present embodiment are as follows:
Saccharicterpenin 30
Hyoscyamine 0.0055
Serum immune globulin 70
Preparation method is substantially with embodiment 1, and wherein difference is only: the saccharide in saccharicterpenin and triterpenoid saponin all extract the Chinese medicine from following weight ratio: the Rhizoma Atractylodis Macrocephalae 50, Rhizoma Atractylodis 20, the Radix Aucklandiae 15 and Rhizoma Alismatis 15.
Quality standard is with embodiment 1.
Embodiment 4:
This preparation for zymad microorganism be escherichia coli of piglets (ETEC K88 K99 987P).
Composition and the parts by weight of the colibacillosis of piglet freeze-dry blood serum immunoglobulin preparation of the present embodiment are as follows:
Saccharicterpenin 5
Hyoscyamine 0.0035
Serum immune globulin 95
Preparation method is substantially with embodiment 1, and wherein difference is only: the saccharide in saccharicterpenin and triterpenoid saponin all extract the Chinese medicine from following weight ratio: the Rhizoma Atractylodis Macrocephalae 30, Rhizoma Atractylodis 20, the Radix Aucklandiae 15 and Rhizoma Alismatis 10.
Quality standard is with embodiment 1.
Embodiment 5:
This preparation for zymad microorganism be escherichia coli of piglets (ETEC K88 K99 987P).
Composition and the parts by weight of the colibacillosis of piglet freeze-dry blood serum immunoglobulin preparation of the present embodiment are as follows:
Saccharicterpenin 45
Hyoscyamine 0.0085
Serum immune globulin 65
Preparation method is substantially with embodiment 1, and wherein difference is only: the saccharide in saccharicterpenin and triterpenoid saponin all extract the Chinese medicine from following weight ratio: the Rhizoma Atractylodis Macrocephalae 50, Rhizoma Atractylodis 40, the Radix Aucklandiae 20 and Rhizoma Alismatis 15.
Quality standard is with embodiment 1.
Embodiment 6:
This preparation for zymad microorganism be escherichia coli of piglets (ETEC K88 K99 987P).
Composition and the parts by weight of the colibacillosis of piglet freeze-dry blood serum immunoglobulin preparation of the present embodiment are as follows:
Saccharicterpenin 45
Hyoscyamine 0.0085
Serum immune globulin 65
Saccharicterpenin in the present embodiment is existing commercially available prod, can be from following supplier:
There is Bang Shifu bio tech ltd, Beijing (phone 010-59741700), Mu Nong bio tech ltd, Shanghai (phone 021-61373406), peace lattice Ruide (China) Bioisystech Co., Ltd (phone 0371-65607365) etc.
Hyoscyamine in the present embodiment is existing commercially available prod, can be from following supplier:
There is the gloomy medical technological development company limited of Beijing Mel (phone 010-80485714), Suzhou Yacoo Chemical Reagent Corporation (phone 0512-81665519), Sichuan Province's Chinese medicine center for standard (phone 028-85377358) etc.
Other raw materials and preparation method are with embodiment 1.
Quality standard is with embodiment 1.
Claims (9)
1. a colibacillosis of piglet freeze-dry blood serum immunoglobulin preparation, it is comprised of the component of following weight portion: saccharicterpenin 5.0～55.0, hyoscyamine 0.005～0.01, serum immune globulin 55.0～95.0; In described saccharicterpenin, by weight percentage, contents of saccharide >=30%, triterpenoid saponin content >=30%; Described saccharide and triterpenoid saponin are all to extract from the Chinese crude drug of following weight portion: the Rhizoma Atractylodis Macrocephalae 30～50, Rhizoma Atractylodis 20～40, the Radix Aucklandiae 15～20, Rhizoma Alismatis 10～15.
2. colibacillosis of piglet freeze-dry blood serum immunoglobulin preparation claimed in claim 1, is characterized in that, it is comprised of the component of following weight portion: saccharicterpenin 10.0～35.0, hyoscyamine 0.005～0.01, serum immune globulin 60.0～90.0.
3. colibacillosis of piglet freeze-dry blood serum immunoglobulin preparation claimed in claim 1, is characterized in that, described saccharide obtains according to following extracting method:
A, ratio is got the Rhizoma Atractylodis Macrocephalae 30～50, Rhizoma Atractylodis 20～40, the Radix Aucklandiae 15～20 and the rear chopping of Rhizoma Alismatis 10～15, is cleaned by weight, with cold water soak 2 hours to the medicine heart, fully soak into, the water that adds again 12 times of Chinese medicine gross weights, after being heated to seethe with excitement, the in the situation that of 90 ℃, under hot reflux concentration technology, decoct 180 minutes concentrated concentrated medicament I that obtain simultaneously;
B, concentrated medicament prepared by step a, through 10000 revs/min centrifugal 20 minutes, discard precipitate, obtain supernatant also concentrated through negative pressure, make it form the concentrated medicament II that dry is 25%;
C, the concentrated medicament II that step b is obtained adds 95% ethanol of 4-8 times of volume, standing over night, abandoning supernatant, precipitate is dissolved in water and passes through 100KD membrane filtration after removing ethanol, except foreigh protein removing, the macromole impurity such as endotoxin, collecting filtered solution filters by 1KD NF membrane again, remove the impurity such as organic molecule and inorganic molecule and ion, after the material dilution being trapped, reverse current dialysis is 24 hours, then taking out and being concentrated into relative density is 1.01～1.18 grams per milliliters, the 99% ethanol precipitation that adds again 5 times of volumes, stir, standing over night, 3000-5000 rev/min centrifugal 20 minutes, precipitate is removed ethanol dry through spraying, can make.
4. colibacillosis of piglet freeze-dry blood serum immunoglobulin preparation claimed in claim 1, is characterized in that, described triterpenoid saponin obtains according to following extracting method:
I. ratio is got the Rhizoma Atractylodis Macrocephalae 30～50, Rhizoma Atractylodis 20～40, the Radix Aucklandiae 15～20 and the rear chopping of Rhizoma Alismatis 10～15, is cleaned by weight, with ethanol or methanol extraction, reclaims solvent, gets residue cake and makes aqueous solution with water dissolution or suspendible;
In ii, the aqueous solution made toward step I, add petroleum ether or ether, the lipophilic substance pigment in aqueous solution and oil are reclaimed, triterpenoid saponin is stayed in aqueous solution;
In iii, the aqueous solution that makes toward ii step, add the organic solvent butanols that hydrophilic is stronger, the material that in solution, hydrophilic is stronger continues to stay in aqueous solution, and triterpenoid saponin is transferred in organic solvent extraction liquid, collects organic solvent extraction liquid, drying under reduced pressure, makes rough triterpenoid saponin;
Iv, the rough triterpenoid saponin that iii step is made are dissolved in ethanol or methanol, then drip gradually the mixed liquor of ether, acetone or ether-acetone equal-volume ratio, till separating out, the triterpenoid saponin of separating out are dried and can be made to triterpenoid saponin from solution.
5. colibacillosis of piglet freeze-dry blood serum immunoglobulin preparation claimed in claim 1, is characterized in that, described hyoscyamine is the hyoscyamine extracting from Flos Daturae.
6. colibacillosis of piglet freeze-dry blood serum immunoglobulin preparation claimed in claim 5, it is characterized in that, the described method of extracting hyoscyamine from Flos Daturae is as follows: by weight, get 1 part of Flos Daturae, ethanol percolation with 30 parts of pH2～3, get percolate and reclaim solvent evaporated, alcohol extractum is added to hydro-thermal molten, adjust pH value to 2～3, filter, get acid solution, add defat with petroleum ether, again obtain aqueous acid, ammonia adjust pH to 10, with chloroform extraction, chloroform extraction liquid is taken out, reclaim chloroform, getting the remaining liquid of extraction is added on cation exchange resin column, first with water elution remove impurity, use again 0.5%～15% acid solution eluting, collect eluent, add alkali neutralization, through desalting processing, obtain hyoscyamine.
7. colibacillosis of piglet freeze-dry blood serum immunoglobulin preparation claimed in claim 1, is characterized in that, described serum immune globulin makes in accordance with the following methods:
1) according to conventional method, prepare the high porcine blood serum of exempting from of piglet colon bacillus
1.1) the healthy growing and fattening pigs of no-special pathogen are in the isolated rearing of clean plant;
1.2) to step 1.1) the healthy growing and fattening pigs of raising carry out 3 immunity, each to every pig injection piglet colibacillosis tervalence inactivated vaccine (ETEC K88K99987P), injection volume is respectively 5～8 parts, 5 parts and 10 parts, each immune interval week age;
1.3) immune latter 21 days for the third time, collect porcine blood serum, serum immune globulin in serum is carried out to immunology detection, the micro-agglutination titer of the serum immune globulin of ETEC K88K99987P be 1:64～1:128 above after, this porcine blood serum is the high porcine blood serum of exempting from of piglet colon bacillus, and collection storage is standby under 16 ℃ of conditions, if detect defective, according to immunizing dose for the third time again immunity, until detect qualified;
2) according to conventional method, gather Sanguis sus domestica, separation of serum the serum immune globulin of purifying.
8. the preparation method of colibacillosis of piglet freeze-dry blood serum immunoglobulin preparation claimed in claim 1, comprise the following steps: according to weight ratio meter, saccharicterpenin 5.0～55.0, serum immune globulin 55.0～95.0 and hyoscyamine 0.005～0.01 are mixed, add sterile deionized water dilution, after homogenate, emulsifying, filtration, adopt lyophilization, make.
9. preparation method claimed in claim 8, it is characterized in that, comprise the following steps: according to weight ratio meter, by saccharicterpenin 5.0～55.0, serum immune globulin 55.0～95.0 and hyoscyamine 0.005～0.01 mix, add sterile deionized water dilution, the micro-agglutination titer of the serum immune globulin of ETEC K88K99987P in every ml soln is reached >=more than 1:512, then be placed in vacuum freeze drier, at-40 ℃, lyophilization is 2～8 hours, 3 ℃ of left and right of intensification per hour then, through the vacuum dryings to 25 of 12～18 hours ℃, complete dry, sealing, in 25 ℃ of environment, keep 3～5 hours, be finished product.
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