CN101690810B - Tetravalent inactivated vaccine adjuvant against Newcastle Disease, infectious bronchitis, egg drop syndrome and bird flu and preparation method thereof - Google Patents
Tetravalent inactivated vaccine adjuvant against Newcastle Disease, infectious bronchitis, egg drop syndrome and bird flu and preparation method thereof Download PDFInfo
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Abstract
The invention provides a tetravalent inactivated vaccine adjuvant against Newcastle Disease, infectious bronchitis, egg drop syndrome and bird flu, which consists of the following components in part by weight: 9.5 parts of Chinese medicinal polysaccharide, 0.5 part of Chinese medicinal saponin and 30 parts of Tween-20, wherein the Chinese medicinal polysaccharide and the Chinese medicinal saponin are all extracted from the following Chinese medicinal raw materials in part by weight: 100 parts of platycodon root, 80 parts of pinellia tuber, 80 parts of baical skullcap root, 80 parts of honeysuckle, 80 parts of weeping forsythiae capsule, 65 parts of dwarf lilyturf tuber, 40 parts of vietnamese sophora root, 50 parts of liquorice, 80 parts of red paeony root, 60 parts of figwort root, 80 parts of white paeony root, 45 parts of fresh rehmannia, 90 part of indigowoad root, 75 parts of tree peony bark and 60 parts of golden thread. The vaccine adjuvant has obvious protection effect on immunogen in the processes of processing, storing and using vaccine, can guide the immunogen to generate specific immunoreactions on a specific organism part, and achieves the aim of integrating channel tropism of Chinese medicines, immunoadjuvant and immunogen protective agent.
Description
Technical field
The present invention relates to a kind of immunological adjuvant, be specifically related to a kind of special immunological adjuvant at newcastle disease, infectious bronchitis, egg drop syndrome, bird flu tetrad inactivated vaccine, and preparation method thereof.
Background technology
The tradition immunological adjuvant is to realize immunogenic reinforced immunological and the adjuvant effect that changes immunization route by enrichment immunogen (as aluminium glue), slow release immunogen (as oil emulsion), activated mononuclear phagocyte submission antigen (as bacillus calmette-guerin vaccine, bacteria lipopolysaccharide) and multiple comprehensive function (as incomplete Freund's adjuvant and complete Freund's adjuvant).
Animal body main which para-immunity that takes place under immunogenic stimulation reacts, and depends primarily on immunological adjuvant and excites which kind of helper T cell (Th).The main inducing cell immunoreation of Th1, it stimulates T cell performance immunoreation by secretion IL-2, IFN-γ, TNF-b; Th2 mainly induces humoral immune reaction, and it stimulates B cell performance immunoreation by secretion IL-4, IL-5, IL-10, produces high-caliber circulation and secreting type antibody.The vaccine-induced immunoreation of aluminium glue type is the Th2 type more than 90%, mainly produces IgG1 and IgA; The vaccine-induced immunoreation of bacillus calmette-guerin vaccine and bacteria lipopolysaccharide type is occupied an leading position for the Th1 type.
Morein proposes immunostimulating complex (ISCOM) first, it is a brand-new immunological adjuvant, the diameter that is made of saponin, cholesterol, phospholipid etc. is spherical cage shape granule (the Cox JC of 30-40nm, SjolanderA, Barr Ig.ISCOMs and other saponin based adjuvants[J] .Adv Drug Deiv Rev, 1998,32 (3): 247-271).This adjuvant is characterised in that by saponin gives mononuclear phagocyte with the immunogen submission efficiently, for immunne response has been established powerful basis.
In recent years, the Chinese medicine immunological adjuvant also emerges in an endless stream, such as propolis immunological adjuvant, ginsenoside's adjuvant (ZL02110578.2), Radix Angelicae Sinensis polysaccharide-lactone complex immunity adjuvant (ZL 94115186.7) or the like, they all are that pharmaceutically active ingredient activation immunity of organism is realized immunoadjuvant function in utilizing.
But above-mentioned these existing immunological adjuvant effects are desirable not enough, and one side is because these immunological adjuvants have universality in similar vaccine, thereby its effect base reason shortage can not activate the immunity of matrix fully to the specific aim of disease specific; On the other hand, these adjuvants often can not be protected immunogenic biological activity effectively in immunogenic processing and making process.
Based on above-mentioned technical background; we will return specific herbal polysaccharide, the Chinese medicine saponin made through principle to add in the immunogen solution with Chinese medicine; not only utilize the hydration shell principle to form hydration shell on the immunogen surface; protected immunogen activity proteic 3 D stereo conformation and biological activity, and can activate the immunologic function of body more targetedly this class disease.
Summary of the invention
The objective of the invention is to: a kind of new immunological adjuvant is provided, and it utilizes Chinese medicine to return through characteristic not only can fully activate immunity of organism at concrete cause of disease, and can more effectively protect immunogenic biological activity.
The present invention also aims to: the preparation method that described immunological adjuvant is provided.
Described goal of the invention is achieved through the following technical solutions:
A kind of newcastle disease, infectious bronchitis, egg drop syndrome, bird flu tetrad inactivated vaccine adjuvant are provided, form: 9.5 parts of herbal polysaccharides, 0.5 part of Chinese medicine saponin, 30 parts of tween 20s by the component of following weight portion.
Described herbal polysaccharide and Chinese medicine saponin all extract from following parts by weight of Chinese traditional medicine raw material:
Radix Platycodonis 100 Rhizoma Pinelliaes 80 Radix Scutellariaes 80 Flos Loniceraes 80 Fructus Forsythiaes 80
Radix Ophiopogonis 65 Radix Sophorae Tonkinensiss, 40 Radix Glycyrrhizaes, 50 Radix Paeoniae Rubra, 80 Radix Scrophulariaes 60
The Radix Paeoniae Alba 80 Radix Rehmanniae 45 Radix Isatidis 90 Cortex Moutans 75 Rhizoma Coptidis 60
Because newcastle, infectious bronchitis, egg drop syndrome, bird flu are the strong acute mixing infectious disease based on the vital organs of the human body functional disorder of lungs, serve as that the main vital organs of the human body of holding concurrently are the heat-clearing and toxic substances removing prescription of assisting so set up to return lung meridian.Wherein: return lung meridian Chinese medicine to comprise Radix Platycodonis, the Rhizoma Pinelliae, Radix Scutellariae, Flos Lonicerae, Fructus Forsythiae, Radix Ophiopogonis and Radix Sophorae Tonkinensis; Return spleen channel Chinese medicine to comprise Radix Glycyrrhizae and Radix Paeoniae Rubra; Return kidney channel Chinese medicine to comprise Radix Scrophulariae; Return Liver Channel Chinese medicine to comprise the Radix Paeoniae Alba and Radix Rehmanniae; GUIXIN comprises Radix Isatidis, Cortex Moutan and Rhizoma Coptidis through Chinese medicine.
Described herbal polysaccharide is the main component of immunological adjuvant of the present invention, and it makes the immunogen preferential hydration in aqueous solution, plays the effect of protein protective agent; By every kind of distinctive returning of herbal polysaccharide, play immunoadjuvant function in animal body through the former immune effect of mode enhance immunity.
Described Chinese medicine saponin is the surface active molecules material, and they all have hydrophilic-lipophilic parents' function.The protein-protective of Chinese medicine saponin and immunoadjuvant function are very strong; Also return through the former immune effect of mode enhance immunity, play immunoadjuvant function with distinctive.
Tween 20 is the stronger surfactant of hydrophilic, and hydrophilic-lipophilic balance value (HLB) is more than 15.0.It mainly acts on is the HLB of balance adjuvant, keeps adjuvant to have good water-solubility.
The present invention also provides the preparation method of described immunological adjuvant, be with herbal polysaccharide, Chinese medicine saponin and tween 20 according to described mixed, newcastle disease of the present invention, infectious bronchitis, egg drop syndrome, bird flu tetrad inactivated vaccine adjuvant are promptly made in homogenate.
Wherein, the extraction of herbal polysaccharide and Chinese medicine saponin can realize by multiple means of the prior art, for example can extract in accordance with the following methods:
The extraction of herbal polysaccharide:
Get described raw material of Chinese medicine in proportion, through decocting, concentrate, ethanol precipitation, washing precipitate, by concentrating and dry, promptly make described herbal polysaccharide again; Or with the water drug-decocting concentrating liquid that obtains in the last method through activated carbon column chromatography, water-washing with alcohol liquid washing then, reconcentration is also dry, promptly make described herbal polysaccharide (Lu Yanhua, Chinese medicine extracts active ingredients isolation technics, Chemical Industry Press, January in 2005 the 1st edition, 295-314).
The extraction of Chinese medicine saponin:
Get described raw material of Chinese medicine in proportion,, reclaim solvent, get the residue cake and make aqueous solution, and then do following two-way extraction and purification with water dissolution or suspendible with ethanol or methanol extraction:
A, add petroleum ether or ether in the aqueous solution of making, lipophilic substance pigment in the aqueous solution and oil are reclaimed, the Chinese medicine saponin is stayed in the aqueous solution;
Add the stronger organic solvent butanols of hydrophilic in b, the aqueous solution that makes toward a step, the material that hydrophilic is stronger in the solution continues to stay in the aqueous solution, and the Chinese medicine saponin is transferred in the organic solvent extraction liquid, collects organic solvent extraction liquid, drying under reduced pressure makes rough Chinese medicine saponin;
C, the rough Chinese medicine saponin that the b step is made are dissolved in ethanol or the methanol, drip the mixed liquor of ether, acetone or ether-acetone (1: 1 volume ratio) more gradually, till separating out from solution to the Chinese medicine saponin, the Chinese medicine saponin drying of going to separate out can make Chinese medicine saponin (Lu Yanhua, Chinese medicine extracts active ingredients isolation technics, Chemical Industry Press, January in 2005 the 1st edition, 172-194).
The present invention also provides the application of described adjuvant in preparation newcastle disease, infectious bronchitis, egg drop syndrome, bird flu tetrad inactivated vaccine, and wherein, described adjuvant shared volume ratio in the vaccine for preparing is 2.0%～7.0%.
Newcastle disease of the present invention, infectious bronchitis, egg drop syndrome, bird flu tetrad inactivated vaccine adjuvant are made up of the two large divisions, and wherein herbal polysaccharide and Chinese medicine saponin are cores, and slave part is a tween 20.The effect of herbal polysaccharide, the existing immunostimulant of Chinese medicine saponin has the protectant effect of immunogen again.The present invention utilizes them to realize that Chinese medicine returns that to close three through, immunological adjuvant and immunogen protective agent be one purpose.
The key character of adjuvant of the present invention is also embodied in returns in the utilization targetedly of theory Chinese medicine.Chinese medicine is returned when being meant the tcm clinical practice medication, the conduction of Chinese medicine by meridians, transfers, and arrives disease institute, brings into play the Chinese medicine utility theory of its preventive and therapeutic effect.The lung that modern combination of Chinese and Western medicine research and clinical practice have progressively confirmed the traditional Chinese medical science is an immunologic barrier, spleen promotes immunologic function, kidney is that immunity effects a permanent cure, liver helps immunity movable, the heart participates in and regulates immunity, the internal organs of immunity theory of immunity theoretical and doctor trained in Western medicine is organically linked up, correctly instructed scientific experiments and the clinical practice of Chinese medicine in immunology.
Chinese medicine is returned through being to be the pharmacological action principle at center with the vital organs of the human body, use do not reach the same goal through Chinese medicine can excite from different angles and the enhancing human body immunity function.Return the Chinese medicine of lung spleen kidney channel can strengthen nonspecific immunity and specific immune, strengthen the phagocytic activity of mononuclear cell system, strengthen lymphocyte transformation, stimulate lymphokines such as T cell differentiation, growth, ripe justacrine IL-1, IL-2 and IFN-γ, stimulate B cell differentiation, growth, ripe justacrine IgM, antibody such as IgG, IgA; Return the Chinese medicine of liver heart channel can regulate immunologic function, correct the disorder of immunologic function by the informational molecule (as neuropeptide, neurotransmitter, hormone, lymphokine, cytokine etc.) of nerve-endocrine-immunity three big systems.
Specifically, lung governing qi belongs to be defended, close fur (element ask the five internal organs give birth to a piece of writing) outward,, and in the presence of juice, lysozyme, IgA etc., bring into play non-special and specific immune function with its biochemical functions by the special construction of mucosa not only to the organic tool barrier action of exopathogen.Lung governing qi does not singly refer to gas exchange, comprises the healthy energy of the traditional Chinese medical science yet, promptly by lung cytophagously engulf, complement C
3, C
6Generation, generation, identification and the processing antigen of interferon, excite, regulate, participate in immunity of organism;
The spleen has the function to transport and transform nutrients, and main system blood is the source of vital function, the foundation of acquired constitution." the spleen acting as the protector of the body " (element asks five infirmity body fluid an other piece of writing).Defend the generation of gas, vigour abundant, the power of healthy energy all depends on the keeping of taste, temper healthy and vigorous.Defend gas, vigour, healthy energy and summarized the partial immunity function of body, spleen is one of immune active material base.From immunologic angle of modern times, the spleen of the traditional Chinese medical science comprises the spleen in the anatomy, also comprises mononuclear phagocyte system, lymph node, the lymphoid tissue that is dispersed in.Mononuclear phagocyte is not only used the nonspecific immunity function, can start the specific immune function simultaneously; They all are the main place that B cell, T cell exist for spleen, lymph node, the lymphoid tissue that is dispersed in, thereby also are the main place of cellular immunization and humoral immunization.Therefore, spleen is the core of immunity;
The kidney being the origin of congenital constitution, the root of life.Kidney storing essence, main bone, spermatogenesis (element ask flaccidity opinion a piece of writing) has important excitation to many immunocytes such as mononuclear cell, B cell, T cell and with the performance of differentiation, growth, maturation and the function of Ia cell;
Liver controlling conveyance and dispersion is transferred the gas reach the whole body, makes vital organs of the human body function happy and obstacle does not take place, thereby has guaranteed the normal activity of body.The liver of the traditional Chinese medical science has comprised the liver of modern medicine, and the phagocyte in the liver can be engulfed the foreign bodies such as microorganism that bring from digestive tract through blood, and processing and submission antigenic determinant and B cell, T cell start immunoreation.Irritability is not smooth, neurological disorders, and the endocrine disturbance weakened the immunity of body, so liver is very important immune link;
Heart governing blood and vessels, main mind (element asks LINGLAN a secret allusion quotation piece of writing).This explanation heart can influence immunologic function by blood constitutent, blood flow circulation, blood vessel endothelium, cerebral cortex, part vegetative nerve, long plexus nervorum etc.
The present invention uses above-mentioned Chinese medicine to return through theory, selects and has prepared the source of specific Chinese medicine preparation as herbal polysaccharide and Chinese medicine saponin, has further strengthened immunological adjuvant exciting and transferring body's immunity.
Compared with prior art, immunological adjuvant of the present invention has following beneficial effect:
1. has the effect of immunogen protective agent
Adjuvant of the present invention is to utilize herbal polysaccharide and the hydrophilic lipophilic Chinese medicine saponin that is rich in hydroxyl; make up multidimensional network space hydrogen bond structure; form false hydration shell on the immunogen surface, realize that by the false hydration shell of this multidimensional network space hydrogen bond adjuvant of the present invention is to immunogenic protective effect.In these two kinds necessary materials, herbal polysaccharide bonds together formation multidimensional network space according to immunogen with hydrogen bond (bond energy 42KJ/mol), for immunogen has been built the mansion shelter as reinforcing bar, cement, brick and tile; The Chinese medicine saponin is just as rivet, and hydrophilic group is with the hydroxyl combination of hydrogen bond and herbal polysaccharide, and lipophilic group firmly is fixed on immunogen in the herbal polysaccharide multidimensional network space with hydrophobic bond and immunogenic hydrophobic group combination; Form stable, the firm false hydration shell of multidimensional network space hydrogen bond like this, protected immunogen to avoid the destruction of external environment better.When immunogen processing and fabricating, the storage time spent, environment's heat is at first accepted and water and motion disorderly in the multidimensional network space that herbal polysaccharide forms, but because the spatial construction features of multidimensional network, cause hydroxyl on the herbal polysaccharide molecule can be at once when dehydration directly to form hydrogen bond, caused whole molecular motion to go to zero with the amino on immunogen surface and hydroxyl etc.The false hydration shell passivation of this multidimensional network space hydrogen bond the immunogen induction of heat energy to external world, alleviated the vibration of the secondary key of immunogen intramolecule and the extension of peptide chain, kept immunogenic three conformations and biological activity.So adjuvant of the present invention has the protectant effect of significant immunogen.Therefore use that immunological adjuvant of the present invention can make that immunogen is stable more, more anti-storage and easier transportation.
Related experiment is as follows:
1, uses newcastle disease, infectious bronchitis, egg drop syndrome, the bird flu tetrad inactivated vaccine of newcastle disease, infectious bronchitis, egg drop syndrome, bird flu tetrad inactivated vaccine and the preparation of conventional oil emulsion of the embodiment of the invention 4 preparations, place 37 ℃ of environment, after preserving January, be applied in next step the clinical trial.
2,, 10 every group, be divided into 4 groups with 40 of 30 age in days SPF chickens.1 group is 37 ℃ of processing of vaccine experimental group of the embodiment of the invention 4 preparations, and every SPF Corium Gallus domesticus is injected the vaccine 0.5ml of the embodiment of the invention 4 preparations of 37 ℃ of processing down; 2 groups is that conventional oil-emulsion vaccine is handled experimental group for 37 ℃, and every SPF Corium Gallus domesticus is injected the conventional oil-emulsion vaccine 0.5ml of 37 ℃ of processing down; 3 groups is the vaccine cryopreservation experimental group of the embodiment of the invention 4 preparations, and every SPF Corium Gallus domesticus is injected 2-8 ℃ of vaccine 0.5ml that preserves the embodiment of the invention 4 preparations down; 4 groups is conventional oil-emulsion vaccine cryopreservation experimental group, and every SPF Corium Gallus domesticus is injected 2-8 ℃ down and preserved conventional oil-emulsion vaccine 0.5ml.After 28 days, blood sampling respectively, separation of serum is measured the HI antibody titer.The results are shown in following table 1.
The vaccine of table 1. adjuvant preparation of the present invention and the comparison of heat-resisting, the anti-storage effect of prior art vaccine
From last table as seen, this experimental result has fully been showed newcastle disease, infectious bronchitis, the egg drop syndrome of adjuvant of the present invention preparation, the heat-resistant quality of bird flu tetrad inactivated vaccine.On the one hand, the vaccine of adjuvant preparation of the present invention still can produce extraordinary immune effect to chicken after placing 37 ℃ of environment to preserve January, with the not obviously difference of product under the cryopreservation; And by behind the conventional oil emulsion vaccine immunity chicken of handling with quadrat method, four kinds of HI antibody titers of newcastle disease, infectious bronchitis, egg drop syndrome, bird flu are compared appearance with the like products of cryopreservation and are descended significantly, have lost the immunoprophylaxis effect.On the other hand, under 2-8 ℃ of cryopreservation, newcastle disease, infectious bronchitis, egg drop syndrome, the bird flu tetrad inactivated vaccine immune effect of adjuvant preparation of the present invention equally obviously exceed conventional oil emulsion newcastle disease, infectious bronchitis, egg drop syndrome, bird flu tetrad inactivated vaccine.Thereby illustrated that adjuvant of the present invention is to immunogenic protective effect and reinforced immunological effect.
2. the immunoadjuvant function that has enhance immunity
The main component of adjuvant of the present invention is Chinese medicine extract herbal polysaccharide and Chinese medicine saponin; return through theory according to aforesaid Chinese medicine; herbal polysaccharide in the immunological adjuvant of the present invention and important saponin can be protected immune organ, increase immune organ weight, strengthen the mononuclear phagocyte function, strengthen the T cell function, strengthen the B cell function, strengthen the NK cell function, increase complement content and strengthen complement activity, induce IL-1, IL-2, IFN, cytokines such as CSF, TNF, excite and regulate immunologic function effectively.So adjuvant of the present invention must embody the immunological adjuvant feature of enhance immunity.
3. adjuvant of the present invention have Chinese medicine return through the special pharmacological effect
Adjuvant of the present invention at first utilizes herbal polysaccharide molecule multidimensional network space to inherit traditional immunological adjuvant enrichment and the immunogenic characteristics of slow release, realizes the characteristics of similar Freund adjuvant simultaneously by the activated mononuclear phagocyte; Secondly also utilize the immunogenic effect of Chinese medicine saponin submission and inherited the characteristics of immunostimulating complex (ISCOM).But adjuvant of the present invention is not pharmacology characteristics of having inherited existing immunological adjuvant simply, but under the Chinese medicine meridian guide of theory, use Chinese medicine return through clinical practice, give the significant traditional Chinese medical science pharmacological action of adjuvant characteristic, strengthened immunogenic immune performance from different angles: what have strengthens engulfing and the submission antigenic action of mononuclear phagocyte from lung meridian, what have directly stimulates T cell and B cell function from spleen channel, have from kidney, liver, heart channel sets out by various neural factors, the hormone factor and immune factor are regulated immunologic function between nerve-endocrine-immunity three big systems, the application that has is comprehensively returned through principle and transferred body's immunological function comprehensively.
More than the 2nd, 3 beneficial effect can further prove by following experiment:
Use newcastle disease, infectious bronchitis, the egg drop syndrome of adjuvant of the present invention preparation, the clinical prevention effect experiment of bird flu tetrad inactivated vaccine:
[being tried the chicken situation]
Tried the chicken group and be to be about to the chicken group that begins to lay eggs, kind is unrestricted, raises once popular and crosses the old epidemic-stricken area that newcastle disease, infectious bronchitis, egg drop syndrome, bird flu infect.
[medical diagnosis on disease standard]
Sick chicken dead in the experiment is carried out pathology analyses diagnosis, make a definite diagnosis standard:
1, newcastle disease
With sick chicken glandular stomach thelorrhagia, muscular stomach cutin membrane bleed bottom, small intestinal island shape hemorrhagic necrosis being arranged is principal character, hold concurrently following any two or multinomial symptom:
(1), the purplish red blackout of sick chicken cockscomb, fervescence, dyspnea gets rid of a rhinorrhea;
(2), wryneck nods, the sagging lower limb paralysis of wing is drawn the yellow green loose stool;
(3), whole body mucosa, serous coat be hemorrhage, lymphonodi caecales is hemorrhage, hemorrhage of rectum;
(4), follicle is hemorrhage, distortion, fallopian tube, metrorrhagia.
2, infectious bronchitis
(1), sick chicken opens one's mouth to breathe, and stretches the neck sneeze, trachea hello sound;
(2), laying rate descends, and produces soft egg, abnormal-shape egg, egg white is thin;
(3), the tunica mucosa tracheae edema, have hyalomucoid, the trachea and bronchus intersection has cheesy obturator;
(4), follicle is hemorrhage breaks, fallopian tube and uterus edema, hemorrhage, renomegaly is kidney as the flower sometimes.
3, egg drop syndrome
(1), laying rate descends product soft egg, no shell egg and abnormal-shape egg;
(2), follicle inflammation, distortion, atrophy, fallopian tube atrophy, uterine mucosa edema.
4, bird flu
With hemorrhage between suppurative hemorrhage, the muscular stomach cutin membrane bleed bottom of the hyperemia of sick chicken trachea, hemorrhage and glandular stomach nipple, glandular stomach and muscular stomach be principal character, hold concurrently following any two or multinomial symptom:
(1), the purplish red blackout of sick chicken cockscomb, fervescence, dyspnea gets rid of a rhinorrhea, face is swollen, draws the yellow green loose stool;
(2), whole body mucosa, serous coat are hemorrhage;
(3), adularescent secretions in the fallopian tube is easily broken in the follicle distortion.
[experiment and observational technique]
Chicken house in the old epidemic-stricken area that newcastle disease, infectious bronchitis, egg drop syndrome, bird flu infect, the laying hen group is divided into three groups, i.e. newcastle disease, infectious bronchitis, egg drop syndrome, bird flu tetrad inactivated vaccine immune group (A), conventional newcastle disease, infectious bronchitis, egg drop syndrome, bird flu tetrad inactivated vaccine immune group (B) and the non-immune group (C) of the embodiment of the invention 4 preparations.Open product in the time of 28 and 14 days laying hen, A, B group uses corresponding vaccine to carry out immunity respectively, and C organizes injecting normal saline, and method is the every chicken muscle injection of a vaccine group 0.5ml tetrad inactivated vaccine, every chicken muscle injection of normal saline group 0.5ml normal saline.Raised then 100 days, and between whole feeding period,, determined whether to cause, and add up disease chicken number, calculate the vaccine immunity protective rate by the following method by newcastle disease, infectious bronchitis, egg drop syndrome and bird flu to the diagnosis of morbidity chicken travel follow-up:
Vaccine immunity protective rate=(immune number-morbidity number)/immune number * 100%
[experimental result]
Experiment finishes back statistical experiment result learns the immune protective rate 88.36% of the newcastle disease of immunological adjuvant preparation of the present invention, infectious bronchitis, egg drop syndrome, bird flu tetrad inactivated vaccine immune group (A), sickness rate 11.64%; And the immune protective rate 83.49% of conventional newcastle disease, infectious bronchitis, egg drop syndrome, bird flu tetrad inactivated vaccine immune group (B), sickness rate 16.51%; The chicken of normal saline group (C) causes morbidity by newcastle disease, infectious bronchitis, egg drop syndrome and bird flu, sickness rate 93.58%.Respectively the protective rate of three experimental grouies is carried out t check in twos by the applying biological statistics; show that fully all there are utmost point significant difference (u＞2.58 each other in they; therefore p＜0.01), proved that newcastle disease, infectious bronchitis, the egg drop syndrome of immunological adjuvant preparation of the present invention, the immune protective effect of bird flu tetrad inactivated vaccine are better than conventional newcastle disease, infectious bronchitis, egg drop syndrome, bird flu tetrad inactivated vaccine.Concrete experimental result sees Table 2.
The immune protective effect of each experimental group of table 2.
Annotate: " * * " all represent and organizes data with other of index and relatively have highly significant difference in the table.
The specific embodiment:
Further specify feature of the present invention below by specific embodiment.
Embodiment 1. produces herbal polysaccharide
The source:
100 kilograms of Radix Platycodoniss, 80 kilograms of the Rhizoma Pinelliaes, 80 kilograms of Radix Scutellariaes, 80 kilograms of Flos Loniceraes, 80 kilograms of Fructus Forsythiaes, 65 kilograms of Radix Ophiopogonis, 40 kilograms of Radix Sophorae Tonkinensiss, 50 kilograms in Radix Glycyrrhizae, 80 kilograms of Radix Paeoniae Rubra, 60 kilograms of Radix Scrophulariaes, 80 kilograms of the Radix Paeoniae Albas, 45 kilograms in the Radix Rehmanniae, 90 kilograms of Radix Isatidis, 75 kilograms of Cortex Moutans, 60 kilograms of Rhizoma Coptidis.Preparation:
A, with after the described Chinese medicinal formulae raw material chopping of embodiment 1, cleaning, fully soak into cold water soak 2 hours to the medicine heart, 12 times the water that adds raw material weight again is heated to the boiling back under 90 ℃ situation, decocts to concentrate simultaneously in 180 minutes under the hot reflux concentration technology to obtain concentrated medicament.
B, with the concentrated medicament of a preparation, again by 10000 rev/mins centrifugal 20 minutes, discard precipitate, obtain supernatant and also concentrate through negative pressure, making it form dry is 25% concentrated medicament.
C, the concentrated medicament of b is added 4-8 95% ethanol doubly, standing over night, discard the ethanol supernatant, the precipitate heating in water bath removes ethanol or vacuum distillation is removed ethanol, be dissolved in water then and by the 100KD membrane filtration, remove macromole impurity such as foreigh protein removing, endotoxin, collect filtered solution and filter by the 1KD NF membrane again, remove organic molecule and impurity such as inorganic molecule and ion, the reverse current dialysis in material dilution back that is trapped 24 hours; Taking-up is concentrated into a little volume, adds 5 times of amount 99% ethanol precipitations, stir, and standing over night, 3000-5000 rev/min is centrifugal 20 minutes; Precipitate is removed ethanol and through spray drying, is herbal polysaccharide.
The preparation of embodiment 2. Chinese medicine saponin
After embodiment 1 described Chinese medicinal formulae raw material chopping, cleaning, with ethanol or methanol extraction, reclaim solvent, get the residue cake and make aqueous solution, and then do two-way extraction and purification with water dissolution or suspendible:
A, add petroleum ether or ether in the aqueous solution, lipophilic substance pigment in the aqueous solution and oil are reclaimed, the Chinese medicine saponin is stayed in the aqueous solution;
Add the stronger organic solvent butanols of hydrophilic in b, the aqueous solution that makes toward a step, the material that hydrophilic is stronger in the solution continues to stay in the aqueous solution, and the Chinese medicine saponin is transferred in the organic solvent extraction liquid, collects organic solvent extraction liquid, drying under reduced pressure makes rough Chinese medicine saponin;
C, the rough Chinese medicine saponin that the b step is made are dissolved in ethanol or the methanol, drip the mixed liquor of ether, acetone or ether-acetone (1: 1) more gradually, and till separating out from solution to the Chinese medicine saponin, the Chinese medicine saponin drying of going to separate out can make the Chinese medicine saponin.
Embodiment 3. adjuvants of the present invention are synthetic
Embodiment 1 herbal polysaccharide, embodiment 2 Chinese medicine saponin and commercial tween 20 are formed and weight kilogram number according to the feature of adjuvant of the present invention, be 9.5 kilograms of herbal polysaccharides, 0.5 kilogram of Chinese medicine saponin, 30 kilograms of mixing of tween 20, newcastle disease of the present invention, infectious bronchitis, egg drop syndrome, bird flu tetrad inactivated vaccine adjuvant are promptly made in 10000 rev/mins of high-speed cutting homogenate.
The application of embodiment 4. adjuvants of the present invention in newcastle disease, infectious bronchitis, egg drop syndrome, the preparation of bird flu tetrad inactivated vaccine
Newcastle disease, infectious bronchitis, egg drop syndrome, the newcastle disease virus La Sota strain of bird flu tetrad inactivated vaccine system, infectious bronchitis virus M 41 strains, bird flu H9N2 hypotype A/chicken/Henan Puyang/2/98 (H9N2) strain (being the HP strain) inoculated into chick embryo is respectively cultivated, the embryo culture of egg drop syndrome virus Z16 strain inoculation duck, collect blastochyle, ultrafiltration and concentration, after the formalin deactivation, add newcastle disease of the present invention, infectious bronchitis, egg drop syndrome, bird flu tetrad inactivated vaccine adjuvant and conventional mineral oil adjuvant mixing and emulsifying are made.
1 seed culture of viruses
The seed culture of viruses that manufacturing this product is used is newcastle disease virus La Sota strain, infectious bronchitis virus M 41 strains, egg drop syndrome virus Z16, bird flu virus H9N2 hypotype A/chicken/HenanPuyang/2/98 (H9N2) strain (being the HP strain); Imitating inspection is newcastle disease virus Beijing strain (CVCCAV1611 strain), bird flu virus H9N2 hypotype HP strain with strong poison.La Sota strain, M41 strain and the strain of newcastle disease virus Beijing (CVCC AV1611 strain) are identified, are preserved and supply by China Veterinary Drugs Supervisory Inst.; All the other effluent south agriculture university fowl diseases institutes are identified, are taken care of and supply.
The 2 vaccine manufacturing and the inspections of semifinished product
2.1 carry out semi-finished product production and check according to Ministry of Agriculture's approval " newcastle disease, infectious bronchitis, egg drop syndrome, bird flu (H9 hypotype) tetrad inactivated vaccine (La Sota strain+M41 strain+Z16 strain+HP strain) are made and the tentative rules of check ".
2.2 the semi-finished product that are up to the standards are concentrated the newcastle disease of the adjuvant of the present invention that is applied, infectious bronchitis, egg drop syndrome, bird flu tetrad inactivated vaccine venom.
2.3 deactivation and deactivation check
Venom is carried out deactivation, carry out the deactivation check by existing " Chinese veterinary drug allusion quotation " appendix, standby after the assay was approved.
The preparation of 3 newcastle diseases, infectious bronchitis, egg drop syndrome, bird flu tetrad inactivated vaccine
3.1 oil phase preparation
By volume part is got 94 parts of injection white oils and 6 parts of Si Ben-80, mixes post-heating, adds aluminium stearate 2 weight portions (promptly adding according to the ratio of 2g/100ml) then in the solution that obtains, with add be stirred to transparent till, autoclaving, the cooling back is standby.
3.2 newcastle disease of the present invention, infectious bronchitis, egg drop syndrome, the preparation of bird flu tetrad inactivated vaccine adjuvant water
Get the embodiment of the invention 3 synthetic immunological adjuvants 4～12% (v/v%), be respectively charged in 4 jars, sterilization, cooling, each 96～88% (v/v%) of deactivation blastochyle that add newcastle disease virus, infectious bronchitis virus, egg drop syndrome virus and bird flu H9N2 subtype virus more respectively, shake well dissolves fully until the present invention, makes 4 kinds of waters respectively.
3.3 emulsifying
By volume part is counted, getting the oil phase of 3.1 steps preparation puts in the oil phase jar for 3 parts, starting motor stirs at a slow speed, slowly each 1 part of 4 kinds of water that add 3.2 step preparations simultaneously respectively, stirred 2-5 minute with 10000r/min again after adding, before stopping stirring, the ratio according to 1g/100ml adds thimerosal solution again, and making thimerosal final weight percent concentration is 0.01%.After the emulsifying, sampling 3-5ml, with 3000r/min centrifugal 15 minutes, should be not stratified.After making newcastle disease, infectious bronchitis, egg drop syndrome and 4 kinds of single Seedlings of bird flu H9N2 hypotype respectively, 4 kinds of single Seedling equal-volumes are fully mixed in emulsion tank, promptly make the tetrad Seedling.
3.4 the quantitative packing of packing seals.
4 product inspections
4.1 character
Outward appearance: yellow-white Emulsion.
Dosage form: water-in-oil type.Get a cleaning suction pipe, draw a small amount of vaccine and drip in cold water, should be the indiffusion of oil droplet shape.
Stability: get vaccine 10ml, be loaded in the centrifuge tube, with the centrifugal 15min of 3000r/min, the water that separate out at the pipe end should be not more than 0.5ml.
Viscosity: with 1ml suction pipe (the end opening internal diameter is 1.2mm, and internal diameter suitable for reading is 2.7mm), draw 25 ℃ of left and right sides vaccine 1ml, make its vertical outflow naturally, record flows out the 0.4ml required time, should be in 8 seconds.
4.2 steriling test
Test by existing " Chinese veterinary drug allusion quotation " appendix, answer asepsis growth.
4.3 safety verification
With 10 of 1-2 monthly age SPF chickens, every muscle or subcutaneous injection vaccine 1ml observed 14, any part or the general reaction that are caused by vaccine should not occur.
4.4 efficacy test
4.4.1 newcastle disease part: adopt serological method to test, when the result is against regulation, can adopt immune counteracting toxic substances method to test.
4.4.1.1 serological method: with 15 of 30-60 age in days SPF chickens, 10 each subcutaneous or intramuscular injection vaccine 20 μ l, 5 compare in addition.Inoculation back 21-28 day, every each blood sampling of chicken, separation of serum carries out the HI antibody titer and measures.The geometrical mean of immune group HI antibody titer should be not less than 1: 16, and the geometrical mean of matched group HI antibody titer should not be higher than 1: 4.
4.4.1.2 immune counteracting toxic substances method: with 15 of 30-60 age in days SPF chickens, 10 each subcutaneous or intramuscular injection vaccine 20 μ l, 5 compare in addition.Inoculation back 21-28 day, every the strong poison of chicken difference intramuscular injection newcastle disease virus Beijing strain (CVCCAV1611 strain) 105.0ELD50 observed 14.Matched group should be all dead, and immune group should be protected 7 at least.
4.4.2 infectious bronchitis of chicken part: with 20 of 14-42 age in days SPF in age chickens, each eye dripping inoculation infectious bronchitis of chicken live vaccine (H120 strain) 1 plumage part, take a blood sample respectively 21-28 day in the inoculation back, separation of serum, respectively inoculate simultaneously this vaccine 0.5ml again, 21-28 day blood sampling respectively again behind the secondary inoculation, separation of serum.Twice serum is made the HI antibody titer respectively to be measured.The geometrical mean that two geometrical means (GMT) of exempting from serum HI antibody titer should firstly be exempted from serum HI antibody titer is high more than 3 times.
4.4.3 chicken egg drop syndrome part: with 10 of 21-42 age in days SPF chickens, every muscle or subcutaneous injection vaccine 0.5ml, other gets 10 of SPF chickens and compares.21-35 took a blood sample respectively after day, and separation of serum is measured the HI antibody titer.Immunity chicken HI antibody titer geometric mean titer should be not less than 1: 128, and contrast chicken HI antibody titer should not be higher than 1: 4.
4.4.4 bird flu part: get 15 of 21～42 age in days SPF chickens, wherein 10 each cervical region subcutaneous injection vaccine 0.3ml do not inoculate for 5 in addition, in contrast, after 21 days, blood sampling respectively, separation of serum is with H9 hypotype HP strain bird flu antigen measuring HI antibody titer.Immunity chicken HI antibody geometric mean titer should be not less than 1: 64, and contrast chicken HI antibody titer should not be higher than 1: 4.
4.5 formaldehyde, thimerosal assay
Test by existing " Chinese veterinary drug allusion quotation " appendix respectively, should meet veterinary biologics general rule regulation.
5 effect and purposes
Be used to prevent newcastle disease, infectious bronchitis, egg drop syndrome, bird flu, the chicken immune phase of growing up is 4 months.
6 usages and consumption
Subcutaneous or the intramuscular injection of cervical region.Open to produce about previous month and plant chicken, laying hen, every 0.5ml.
Claims (5)
1. a newcastle disease, infectious bronchitis, egg drop syndrome, bird flu tetrad inactivated vaccine adjuvant, it is characterized in that: it is made up of the component of following weight portion: 9.5 parts of herbal polysaccharides, 0.5 part of Chinese medicine saponin, 30 parts of tween 20s;
Described herbal polysaccharide and Chinese medicine saponin all extract from following parts by weight of Chinese traditional medicine raw material:
Radix Platycodonis 100 Rhizoma Pinelliaes 80 Radix Scutellariaes 80 Flos Loniceraes 80 Fructus Forsythiaes 80
Radix Ophiopogonis 65 Radix Sophorae Tonkinensiss, 40 Radix Glycyrrhizaes, 50 Radix Paeoniae Rubra, 80 Radix Scrophulariaes 60
The Radix Paeoniae Alba 80 Radix Rehmanniae 45 Radix Isatidis 90 Cortex Moutans 75 Rhizoma Coptidis 60
2. the described vaccine adjuvant of claim 1 is characterized in that, described herbal polysaccharide extracts in accordance with the following methods:
1) with after described raw material of Chinese medicine chopping, cleaning, fully soak into cold water soak 2 hours to the medicine heart, 12 times the water that adds raw material weight again is heated to the boiling back under 90 ℃ situation, decocts to concentrate simultaneously in 180 minutes under the hot reflux concentration technology to obtain concentrated medicament;
2) with the concentrated medicament of step 1) preparation, again by 10000 rev/mins centrifugal 20 minutes, discard precipitate, obtain supernatant and also concentrate through negative pressure, making it form dry is 25% concentrated medicament;
3) with step 2) concentrated medicament that obtains adds 4-8 95% ethanol doubly, standing over night, discard the ethanol supernatant, the precipitate heating in water bath removes ethanol or vacuum distillation is removed ethanol, be dissolved in water then and by the 100KD membrane filtration, remove macromole impurity such as foreigh protein removing, endotoxin, collect filtered solution and filter by the 1KD NF membrane again, remove organic molecule and impurity such as inorganic molecule and ion, the reverse current dialysis in material dilution back that is trapped 24 hours; Taking-up is concentrated into a little volume, adds 5 times of amount 99% ethanol precipitations, stir, and standing over night, 3000-5000 rev/min is centrifugal 20 minutes; Precipitate is removed ethanol and through spray drying, is herbal polysaccharide.
3. the described vaccine adjuvant of claim 1 is characterized in that, described Chinese medicine saponin extracts in accordance with the following methods:
Get described raw material of Chinese medicine in described ratio,, reclaim solvent, get the residue cake and make aqueous solution, and then do following two-way extraction and purification with water dissolution or suspendible with ethanol or methanol extraction:
A, add petroleum ether or ether in the aqueous solution, lipophilic substance pigment in the aqueous solution and oil are reclaimed, the Chinese medicine saponin is stayed in the aqueous solution;
Add the stronger organic solvent butanols of hydrophilic in b, the aqueous solution that makes toward a step, the material that hydrophilic is stronger in the solution continues to stay in the aqueous solution, and the Chinese medicine saponin is transferred in the organic solvent extraction liquid, collects organic solvent extraction liquid, drying under reduced pressure makes rough Chinese medicine saponin;
C, the rough Chinese medicine saponin that the b step is made are dissolved in ethanol or the methanol, drip ether, acetone or volume ratio more gradually and be ether-acetone mixed liquor of 1: 1, till separating out from solution to the Chinese medicine saponin, the Chinese medicine saponin drying of separating out can be made described Chinese medicine saponin.
4. the preparation method of the described newcastle disease of claim 1, infectious bronchitis, egg drop syndrome, bird flu tetrad inactivated vaccine adjuvant, may further comprise the steps: according to described ratio, with described herbal polysaccharide, Chinese medicine saponin and tween 20 mixing homogenate, promptly make newcastle disease of the present invention, infectious bronchitis, egg drop syndrome, bird flu tetrad inactivated vaccine adjuvant.
5. the application of the described vaccine adjuvant of claim 1 in preparation newcastle disease, infectious bronchitis, egg drop syndrome, bird flu tetrad inactivated vaccine, it is characterized in that: described adjuvant shared volume ratio in the vaccine for preparing is 2.0%～7.0%.
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| CN101804162B (en) * | 2010-04-19 | 2011-08-31 | 河南工业大学 | Anti-newcastle disease virus Chinese medicinal composition and preparation method thereof |
| WO2012045198A1 (en) * | 2010-10-09 | 2012-04-12 | 广州白云山和记黄埔中药有限公司 | Use of polysaccharides from radix isatidis in manufacture of medicaments against influenza virus |
| CN102698088B (en) * | 2012-01-05 | 2013-10-30 | 青岛华仁技术孵化器有限公司 | Traditional Chinese medicine composition for treating newcastle disease, feed, preparation method and application |
| CN103288973B (en) * | 2012-03-02 | 2015-12-02 | 北京中安佐际生物科技有限公司 | Banlangen Polysaccharide, Preparation Method And The Use |
| CN103143012B (en) * | 2013-03-26 | 2015-06-10 | 广西大学 | Traditional Chinese medicine composition and applications in preparation of porcine pseudorabies vaccine immunopotentiator |
| CN103908565A (en) * | 2014-04-21 | 2014-07-09 | 张颖 | Traditional Chinese medicinal veterinary granules for preventing and treating atypical newcastle disease and preparation method thereof |
| CN104258123A (en) * | 2014-09-19 | 2015-01-07 | 安徽天安动物药业有限公司 | Traditional Chinese medicine for treating poultry egg drop syndrome |
| CN105796798B (en) * | 2016-03-17 | 2019-08-20 | 长江大学 | A kind of plant extracts of anti-avian infectious bronchitis virus |
| CN111529702B (en) * | 2020-05-27 | 2022-09-09 | 山东绿都生物科技有限公司 | Traditional Chinese medicine composite newcastle disease vaccine and preparation method thereof |
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