CN102764434A - Preparation method of animal immunopotentiator and application thereof - Google Patents
Preparation method of animal immunopotentiator and application thereof Download PDFInfo
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- CN102764434A CN102764434A CN2012100574204A CN201210057420A CN102764434A CN 102764434 A CN102764434 A CN 102764434A CN 2012100574204 A CN2012100574204 A CN 2012100574204A CN 201210057420 A CN201210057420 A CN 201210057420A CN 102764434 A CN102764434 A CN 102764434A
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Abstract
The invention relates to a preparation method of an animal immunopotentiator and an application thereof. The technical scheme provided by the invention is as follows: Mycobacterium phlei is subjected to fermentation culture and undergoes processes of inactivation, condensation, purification, homogenization and the like so as to prepare the animal immunopotentiator. The animal immunopotentiator can be individually used for nonspecific enhancement of animal body immunity and also can be used as an adjuvant component for animal vaccines. The method provided by the invention can be used to enhance animal body immunity, raise animal productive performance, and make immune effect of vaccines more reliable, and has direct economic and social benefits.
Description
Technical field
The present invention relates to a kind of method for preparing and application of animal immune potentiator, belong to the veterinary biological product technical field.
Background technology
The expansion day by day of the fast development of livestock and poultry breeding industry, especially intensive farm scale makes that the problem of disease prevention is important further.Vaccination be prophylactic the most basically, effective method.But because a variety of causes, the immune effect of at present a lot of vaccines also is not very good, can not protection completely be provided to animal.In order to improve the immune effect of vaccine; People have carried out a large amount of work; As improving antigenic content through concentrated antigen, improve immunogenicity of antigens, optimize seedling technology, adopt the high-quality adjuvant, optimize the immune quality that immune programme for children or the like improves vaccine, through use immunostimulant, reduce various venomous injurants verify immune injury, good raising condition be provided, adopt rational medication program to strengthen the immunne response ability of animal body.Wherein, researching and developing new and effective animal immune potentiator (adjuvant) is one of main method that improves immune effect.
Adjuvant is claimed immunostimulant or immunomodulator again, is meant prior to antigen or with antigen to inject simultaneously in the animal body, and non-specific change of ability or enhancing body are to one type of material of this antigenic immunne response.The kind of adjuvant is more at present, can be divided into according to the source: inorganic adjuvant, and like aluminium hydroxide, Alumen etc.; Organic adjuvant, microorganism and product thereof such as bacillus pumilis, bordetella pertussis, endotoxin etc.; Synthetic adjuvant is like the double-stranded polynucleotide (double-stranded polyadenylic acid, uridylic acid) of synthetic, levamisole, inosine pranobex etc.; The oil adjuvant is like Freund adjuvant, mineral oil adjuvant etc.In the above-mentioned adjuvant, though being successfully applied to after deliberation of having is human like thymosin and interferon, price is comparatively expensive, can not be used for animal.The used adjuvant of animal use vaccine is main with oily adjuvant, aluminium hydroxide aluminium glue adjuvant still mainly at present; Through prolonging vaccine, also there not be directly the adjuvant of the immunne response ability through enhancing animal body self improve immune effect at the time of staying of injection site performance adjuvant effect.
Mycobacterium phlei (Mycobacterium phlei) is a kind of in the mycobacteria; Wherein mycobacteria muramyldipeptide (MDP) has very strong immunization therapy effect; Mycobacterium phlei polysaccharide MPS is having very good effect (Zhou Liting, Zhao Jingjing etc., mycobacteria polysaccharide immunomodulator progress aspect immunomodulating, the inhibition virus replication; The medicine biotechnology, 2011.18 (2): 176-179).Prove through immunological experiment: after the Mycobacterium phlei of deactivation gets into human body; The T lymphocyte is stimulated; Discharge multiple lymphokine, like IL-2, IL-4, TNF, IFN and MAF, MIF, MCF, MMF etc., these factors act on monokaryon-macrophage system; Make it pathogen to be engulfed, kills and wounds and removes to lesions position gathering, activation; Simultaneously, NKT (NK) cell, also activation of bone-marrow-derived lymphocyte, increase, IgM, IgG increase or are tending towards normal; Get involved the immunologic process of body enduringly, constantly regulate cell and immunity system performance immunologic function, thus enhancing human body immunity ability (Chen Xiaozhi; Utilins Injection; Chinese Journal of New Drugs, 2000,9 (9): 644-645).
Mycobacterium phlei is a kind of non-pathogenic microorganism, does not have toxicity when using separately or using simultaneously with vaccine, enhances strong immunity through the immunne response ability that improves host self, and is safe and reliable.The Mycobacterium phlei fermentation is convenient, with low cost, is convenient to clinical large tracts of land and promotes the use of.
Summary of the invention
The objective of the invention is to prepare a kind of animal immune potentiator; Immunocompetence that on the one hand can nonspecific raising animal; Use its adjuvant composition on the other hand, can make animal more certain, the vaccine immunity of animals level is brought up to a new height with the immune effect of vaccine as vaccine; Reduce disease and take place, have direct economic benefit and social benefit.
The immunostimulant that use the present invention relates to can: 1) enhance immune power, improve survival rate and rate of body weight gain; 2) with animal with the protection antibody generation time of inactivated vaccine 5～7 days in advance, make that the blank phase of animal immune is shorter, produce better immune protective effect;
The most important point of the present invention is that the inactivated avian influenza vaccine (H9 hypotype) of interpolation Mycobacterium phlei can make the time of the animal generation protection antibody of this vaccine of inoculation shift to an earlier date 5～7 days.Because oil emulsion inactivated vaccine generally began to produce antibody in back 10 days in immunity, can detect antibody in 14 days serum, reached the protectiveness level in 21～28 days; And broiler is too fast owing to growth, and immune system is grown imperfection, to antigenic immunne response ability; It is slower to produce antibody, and antibody horizontal is lower, and (Ma Lei etc. brief talk the factor that immunity of meat chickens descends that influences; The poultry science, 2007, (11): 27-28).After the Mycobacterium phlei animal immune potentiator used, the broiler immunity can produce protection antibody in back 14 days, had dwindled the immune blank phase greatly, had reduced the risk that disease takes place in culturing.Select for use Mycobacterium phlei to use vaccine adjuvant among the present invention as animal; Set about from transferring the human body immune function aspect; Strengthened immune effect; Remedied that bird flu does not have live vaccine to do the shortcoming of fundamental immunity in the vaccine immunity, reduced the sickness rate of whole culture-cycle bird flu, had direct economic benefit and social benefit.
Main technical schemes of the present invention is:
A kind of animal immune potentiator of the present invention, main component are the purification goods of Mycobacterium phlei (Mycobacterium phlei) culture;
This animal immune potentiator mainly is with the Mycobacterium phlei fermentation culture, through deactivation, concentrate, operations such as purification, homogenizing are prepared from;
The application of this a kind of animal immune potentiator can be used separately, is used for nonspecific enhancing animal body immunocompetence, and its using dosage is to calculate according to the weight of animals, per kilogram of body weight injection Mycobacterium phlei thalline 1～20mg; This immunostimulant can add animal to mix homogeneously in the inactivated vaccine according to the amount of adding 0.032～0.8mg Mycobacterium phlei thalline in every milliliter of vaccine, carries out immunity according to the routine dose of vaccine.
The specific embodiment
1. bacterium source
The Mycobacterium phlei that the present invention relates to (Mycobacterium phlei) AS 4.1180 bacterial strains are purchased No. 3 China Committee for Culture Collection of Microorganisms of Institute of Microorganism, Academia Sinica common micro-organisms centers in the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Numbering: CGMCC AS 4.1180 (China Committee for Culture Collection of Microorganisms; China's strain catalogue, 1992 editions, China Machine Press; 1992, p285).
2. the method for preparing of animal immune potentiator
The preparation of animal immune potentiator is with being numbered CGMCC AS 4.1180 Mycobacterium phlei (Mycobacterium phlei) fermentation culture, through deactivation, concentrate, operation such as purification processes the Mycobacterium phlei thalline.Concrete preparation process:
(1) seed culture
Place seed tank to cultivate Mycobacterium phlei (Mycobacterium phlei) CGMCC AS 4.1180 strains, 37 ℃ of aerobic culture, pH value are 7.0, and incubation time is 3～4 days, obtain fermentation and use seed liquor;
Culture medium prescription in the seed tank: yeast extract powder 1g～5g, concentrate beerwort (available from Jinan diamylose medicated beer goods and materials company limited) 2ml～10ml, glucose 5g～50g, diammonium phosphate 1g～4g, dipotassium hydrogen phosphate 0.5g～1g, water for injection adds to 1000ml.
(2) fermentation culture
In order to obtain active best Mycobacterium phlei, the logarithmic (log) phase that is chosen in bacterial growth stops fermentation, carries out deactivation, and the present invention has carried out screening test to the incubation time of Mycobacterium phlei.After cultivating 72h in the Mycobacterium phlei liquid medium within, every separated 24h counts with blood counting chamber, measures bacterial concentration at microscopically, and the result sees the following form 1:
The incubation time screening test of table 1 Mycobacterium phlei
| Time (my god) | 3 | 4 | 5 | 6 | 7 | 8 |
| Bacterial concentration (cfu/ml) | 4.0×10 5 | 4.7×10 6 | 5.3×10 7 | 5.5×10 7 | 5.3×10 7 | 4.9×10 6 |
According to above result, preferred incubation time is 5 days.
The fluid medium for preparing is put into seed tank with the seed liquor of being cultivated carry out fermentation culture, 37 ℃ of aerobic culture, pH value are 7.0, and incubation time is 5 days;
Culture medium prescription in the fermentation tank: yeast extract powder 5g～10g; Concentrate beerwort (available from Jinan diamylose medicated beer goods and materials company limited) 5.0g～20g, glucose 20g～100g, diammonium phosphate 1.0g～4.0g; Dipotassium hydrogen phosphate 0.5g～1.0g, water for injection adds to 1000ml.
(3) post processing
With fermentation liquid with formalin (0.1%) deactivation after, centrifugal concentrate, abandoning supernatant; Take off layer thalline with an amount of normal saline resuspension 3 times, the centrifugal impurity of removing is measured moisture; Calculate dry mycelium weight,, process finite concentration (10 μ g/ml, 50 μ g/ml etc.) Mycobacterium phlei injection with an amount of normal saline, the laggard horizontal high voltage homogenizing of 0.2% Tween 80 mix homogeneously; Through packing, promptly obtain animal immune potentiator.
3. the application of animal immune potentiator
(1) Mycobacterium phlei is used for enhance immune power
Use separately, be used for nonspecific enhancing animal body immunocompetence, its using dosage is to calculate according to the weight of animals, per kilogram of body weight injection Mycobacterium phlei thalline 1～20mg.
(2) Mycobacterium phlei is used to increase animal and uses immune effect of vaccine
This immunostimulant uses as the adjuvant composition of animal with inactivated vaccine, and method is that every milliliter of animal is with adding 0.032～0.8mg Mycobacterium phlei thalline and mix homogeneously in the inactivated vaccine semi-finished product.When vaccine uses, carry out immunity, can be used for strengthening the immune effect of animal with vaccine according to the routine dose of vaccine.
The present invention relates to microorganism fungus kind information
The Mycobacterium phlei that the present invention relates to (Mycobacterium phlei) CGMCCAS 4.1180 bacterial strains are purchased No. 3 China Committee for Culture Collection of Microorganisms of Institute of Microorganism, Academia Sinica common micro-organisms centers in the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Numbering: CGMCCAS 4.1180 (China Committee for Culture Collection of Microorganisms; China's strain catalogue, 1992 editions, China Machine Press; 1992, p285);
NDV La Sota strain (CVCC AV1615), NDV Beijing strain (CVCC AV1611), avian infectious bronchitis virus M41 strain (CVCC AV1511), capital 911 strains of egg drop syndrome virus, rabbit hemorrhagic disease virus CVCC AV34; All available from No. 8 China Veterinery Drug Inspection Office in the South Street ,Zhongguangcun ,Beijing Haidian District; China veterinary microorganism culture presevation administrative center (China Veterinery Drug Inspection Office, Chinese veterinary microorganism culture presevation administrative center, Chinese veterinary's strain catalogue; Second edition; Scientia Agricultura Sinica technology publishing house, 2002, p136,142,143,155);
This virus of mink enteritis virus (CGMCC No.4331, the application number that the applicant's December in 2010 proposed on the 1st is the microorganism fungus kind that relates in the 201010566499.4 application for a patent for invention cases) has been delivered Chinese microbial preservation committee common micro-organisms center preservation in No. 3 Institute of Microorganism, Academia Sinica of No. 1 institute of Chaoyang District Beijing North Star West Road on November 11st, 2010.
Advantage of the present invention
The present invention utilizes Mycobacterium phlei to prepare a kind of animal immune potentiator.This immunostimulant can be used for nonspecific raising animal body immunity, also can be used for improving the immune effect of animal with vaccine, thereby reduces disease incidence rate, has direct economic benefit and social benefit.
Embodiment
Following examples further specify the present invention, but not as limitation of the present invention.
Embodiment 1
---the preparation of Mycobacterium phlei injection
(1) seed culture
Place seed tank to cultivate Mycobacterium phlei CGMCC AS 4.1180 strains, 37 ℃ of aerobic culture, pH value are 7.0, and incubation time is 3～4 days, obtain fermentation and use seed liquor;
Culture medium prescription in the seed tank: yeast extract powder 1g～5g; Concentrate beerwort (available from Jinan diamylose medicated beer goods and materials company limited) 2ml～10ml, glucose 5g～50g, diammonium phosphate 1g～4g; Dipotassium hydrogen phosphate 0.5g～1.0g, water for injection adds to 1000ml.
(2) fermentation culture
In order to obtain active best Mycobacterium phlei, the logarithmic (log) phase that is chosen in bacterial growth stops fermentation, carries out deactivation, and the present invention has carried out screening test to the incubation time of Mycobacterium phlei.After cultivating 72h in the Mycobacterium phlei liquid medium within, every separated 24h counts with blood counting chamber, measures bacterial concentration at microscopically.
The fluid medium for preparing is put into seed tank with the seed liquor of being cultivated carry out fermentation culture, 37 ℃ of aerobic culture, pH value are 7.0, and incubation time is 5 days;
Culture medium prescription in the fermentation tank: yeast extract powder 5g～10g; Concentrate beerwort (available from Jinan diamylose medicated beer goods and materials company limited) 5.0g～20g, glucose 20g～100g, diammonium phosphate 1.0g～4.0g; Dipotassium hydrogen phosphate 0.5g～1.0g, water for injection adds to 1000ml.
(3) post processing
With fermentation liquid with formalin (final concentration is 0.1%) deactivation after, centrifugal concentrate, abandoning supernatant; Take off layer thalline with an amount of normal saline resuspension 3 times, the centrifugal impurity of removing is measured moisture; Calculate dry mycelium weight,, process finite concentration (10 μ g/ml, 50 μ g/ml etc.) Mycobacterium phlei injection with an amount of normal saline, the laggard horizontal high voltage homogenizing of 0.2% Tween 80 mix homogeneously; Through packing, promptly obtain animal immune potentiator.
Embodiment 2
7 aa broiler chicken, injection Mycobacterium phlei injection is in the Mycobacterium phlei thalline; 0.1mg/ plumage (being equivalent to 1mg/kg), the various vaccine immunities of stipulating according to normal immune programme for children simultaneously are when 45 ages in days are delivered for sale; The test group average weight is 2.53kg, and meat feed ratio is 1: 1.93, and the matched group average weight is 2.39kg; Meat feed ratio is 1: 2.08, and the fertility performance of test group obviously is superior to matched group.
Embodiment 3
28 age in days cockerels, injection Mycobacterium phlei injection is in the Mycobacterium phlei thalline; 1mg/ plumage (being equivalent to the 5mg/kg body weight); Various vaccines (the comprising the newcastle inactivated vaccine) immunity of stipulating according to normal immune programme for children simultaneously, (CVCC AV1611 is available from No. 8 China Veterinery Drug Inspection Office in the South Street ,Zhongguangcun ,Beijing Haidian District with the strong malicious Beijing strain of newcastle disease during 49 ages in days; China veterinary microorganism culture presevation administrative center) counteracting toxic substances; Protective rate is 97% (29/30), and the matched group protective rate is 83% (25/30), and test group has improved 11% than matched group protective rate.
Embodiment 4
60 age in days rabbits, injection Mycobacterium phlei injection is in the Mycobacterium phlei thalline; 20mg/ is (being equivalent to the 20mg/kg body weight) only, carries out immunity according to normal immune programme for children (comprising the rabbit hemorrhagic disease inactivated vaccine) simultaneously, and 81 ages in days are with rabbit hemorrhagic disease virus CVCC AV34 strain counteracting toxic substances (rabbit hemorrhagic disease virus CVCC AV34; Available from No. 8 China Veterinery Drug Inspection Office in the South Street ,Zhongguangcun ,Beijing Haidian District, Chinese veterinary microorganism culture presevation administrative center, Chinese veterinary's strain catalogue; Second edition, Scientia Agricultura Sinica technology publishing house, 2002; P142,155); Protective rate is 95% (19/20), and the matched group protective rate is 80% (16/20), and test group improves 15% than matched group protective rate.
Embodiment 5
2 the monthly age mink; Injection contains the mink viral enteritis inactivated vaccine (application number of the applicant's December in 2010 proposition on the 1st is a kind of mink viral enteritis inactivated vaccine of 201010566499.4 and preparation method thereof) of 0.032mg/ml Mycobacterium phlei; (this virus was delivered Chinese microbial preservation committee common micro-organisms preservation center preservation in No. 3 Institute of Microorganism, Academia Sinica of No. 1 institute of Chaoyang District Beijing North Star West Road to immunity back 3 all water cats panleucopenia virus on November 11st, 2010; Preserving number: CGMCC No.4331) attack; Protective rate is 95% (19/20); The matched group protective rate of not injecting Mycobacterium phlei is 80% (16/20), and test group improves protective rate 15% than matched group.
Embodiment 6
Produce commercial inactivated avian influenza vaccine (H9 hypotype, LG1 strain), production authentication code: veterinary drug new word (2007) 150252081 with the inventor; The ratio that contains 0.32mg Mycobacterium phlei thalline according to every milliliter of vaccine adds the Mycobacterium phlei thalline, behind the mixing, injects 7 aa broiler chicken; 14 days hind wing venous blood collections, separation of serum is measured antibody; Test group H9 subtype avian influenza average antibody is 4.62log2, and protective rate is 83.3% (25/30); And immunity do not contain the matched group of the inactivated avian influenza vaccine (H9 hypotype, LG1 strain) of Mycobacterium phlei, average antibody is 3.53log2, protective rate is 60.0% (18/30), test group has improved 23.3% than the protective rate of matched group.
Embodiment 7
The brown laying hen of 110 age in days Luo Man; Inoculation contains the newcastle, infectious bronchitis of chicken, egg drop syndrome triple inactivated vaccine of 0.8mg/ml Mycobacterium phlei (when the process of preparation commodity newcastle, infectious bronchitis of chicken, egg drop syndrome triple inactivated vaccine prepares the antigen water; The ratio that contains 0.32mg Mycobacterium phlei thalline in every milliliter of vaccine adds the Mycobacterium phlei thalline, carries out emulsifying again), the immunoprotection phase reached more than 12 months; Longer more than 3 months than the matched group immunoprotection phase; Whole laying period is without booster immunization, reduced effectively that immunity inoculation causes stress, have direct economic benefit.
Claims (4)
1. animal immune potentiator; It is characterized in that this immunostimulant main component is the purification goods of Mycobacterium phlei (Mycobacterium phlei) culture that is numbered AS 4.1180; This Mycobacterium phlei is available from No. 3 Institute of Microorganism, Academia Sinica China common micro-organisms preservation administrative centers in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, numbering: CGMCC AS 4.1180.
2. the method for preparing of the said a kind of animal immune potentiator of claim 1 is characterized in that being numbered CGMCC AS 4.1180 Mycobacterium phlei (Mycobacterium phlei) fermentation culture, through deactivation, concentrate, operations such as purification, homogenizing are prepared from.
3. the application of the said a kind of animal immune potentiator of claim 1; It is characterized in that and to use separately; Be used for nonspecific enhancing animal body immunocompetence; Its using dosage is to calculate according to the weight of animals, per kilogram of body weight injection Mycobacterium phlei (Mycobacterium phlei) thalline 1～20mg.
4. the application of the said a kind of animal immune potentiator of claim 1; It is characterized in that this immunostimulant can add animal to mix homogeneously in the inactivated vaccine according to the amount of adding 0.032～0.8mg Mycobacterium phlei (Mycobacterium phlei) thalline in every milliliter of vaccine, carry out immunity according to the routine dose of vaccine.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103396958A (en) * | 2013-07-15 | 2013-11-20 | 广东温氏食品集团股份有限公司 | Preparation method of mycobacterium phlei and immune enhancer thereof |
Citations (3)
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| CN1442151A (en) * | 2003-04-04 | 2003-09-17 | 张素琴 | Mackefulan immune liquid |
| CN101518549A (en) * | 2008-02-26 | 2009-09-02 | 中国人民解放军总医院第二附属医院 | Research and application of Mycobacterium extract |
| CN102206590A (en) * | 2010-12-03 | 2011-10-05 | 张素琴 | Preparation methods of Mycobacterium plei Sq-1 and composite microecological preparation thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1442151A (en) * | 2003-04-04 | 2003-09-17 | 张素琴 | Mackefulan immune liquid |
| CN101518549A (en) * | 2008-02-26 | 2009-09-02 | 中国人民解放军总医院第二附属医院 | Research and application of Mycobacterium extract |
| CN102206590A (en) * | 2010-12-03 | 2011-10-05 | 张素琴 | Preparation methods of Mycobacterium plei Sq-1 and composite microecological preparation thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103396958A (en) * | 2013-07-15 | 2013-11-20 | 广东温氏食品集团股份有限公司 | Preparation method of mycobacterium phlei and immune enhancer thereof |
| CN103396958B (en) * | 2013-07-15 | 2015-02-25 | 广东温氏食品集团股份有限公司 | Preparation method of mycobacterium phlei and immune enhancer thereof |
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Application publication date: 20121107 |