CN102205117B - Dairy cow mammitis staphylococcus aureus-Escherichia coli bigeminy polyvalent vaccine and preparation method thereof - Google Patents
Dairy cow mammitis staphylococcus aureus-Escherichia coli bigeminy polyvalent vaccine and preparation method thereof Download PDFInfo
- Publication number
- CN102205117B CN102205117B CN 201110130527 CN201110130527A CN102205117B CN 102205117 B CN102205117 B CN 102205117B CN 201110130527 CN201110130527 CN 201110130527 CN 201110130527 A CN201110130527 A CN 201110130527A CN 102205117 B CN102205117 B CN 102205117B
- Authority
- CN
- China
- Prior art keywords
- cgmcc
- staphylococcus aureus
- preparation
- antigen
- supernatant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention relates to a dairy cow mammitis staphylococcus aureus-Escherichia coli bigeminy polyvalent vaccine and a preparation method thereof. The vaccine comprises the following components in part by volume: 4 parts of antigens and 6 parts of Montanide ISA70MVG immune oil adjuvant, wherein the antigens comprise a, a combination of any two or more than two of antigens of thallus polysaccharide and protein mixtures of staphylococcus aureus subsp. aureus CGMCC NO.4595, 4596, 4597 and 4598, b, a combination of any two or more than two of toxin antigens of CGMCC NO.4595, 4596, 4597 and 4598, c, a thallus protein antigen of Escherichia coli CGMCC NO. 4594 and d, a toxin antigen of CGMCC NO. 4594. The preparation method comprises the following steps of: preparing strains; preparing the thallus antigens; preparing the toxin antigens; and mixing the thallus antigens, the toxin antigens and the Montanide ISA70MVG immune oil adjuvant according to the volume ratio to obtain the vaccine. The invention has the advantages that: 1, the coverage area of vaccine strain antigens is wide, and the immunogenicity is high; 2, the effect is obvious; and 3, the safety of the vaccine is high.
Description
Technical field
The invention belongs to biovaccine and preparation field thereof, particularly relate to a kind of dairy cow mammitis staphylococcus aureus-Escherichia coli bigeminy polyvalent vaccine and preparation method thereof.
Background technology
Mammitis of cow is the most important infectious disease that affects milk crop and quality.According to domestic and international research, the main pathogen of mammitis of cow is divided infectiousness cause of disease and environment cause of disease two classes.The infectiousness cause of disease mainly comprises staphylococcus aureus, streptococcus agalactiae and streptococcus dysgalactiae; The environment cause of disease mainly contains the antibacterial of colon bacillus, streptococcus uberis and other enterobacteriaceae.Foreign study shows, at the Staphylococcus Aureus Isolated from Cow Mastitis separation rate, is 15-50%, but by this microbial mastitis cure rate, is only 25% in lactation period, thereby the harm of the mastitis due to such pathogen infection is maximum; Although the separation rate at the Isolated from Bovine Mastitis streptococcus intermedius reaches 50%, and reaches 70-90% by this microbial mastitis cure rate.In developed country, due to advanced feeding and management technology, strict sterilization and superior sanitary condition, above-mentioned disease ratio obviously descends, and the mastitis caused by colon bacillus is occupied advantage in some areas.The preliminary survey of China and my district (Xinjiang region) shows, staphylococcus aureus and escherichia coli are also the main cause of diseases of current my district's mammitis of cow.
For the control of mammitis of cow, external main adopt early diagnosis, colony house environment and breast sterilization, immunoprophylaxis and antibiotic prophylaxis and treatment.But, in recent years along with the raising of food drug residue level monitoring in the enhancing of consumer safety consciousness and international trade, various countries have banned use of antibiotic to carry out the mammitis of cow prevention, in addition, strengthen because the life-time service antibiotic causes resistance, the treatment cost of mammitis of cow is sharply increased, therapeutic effect is not obvious, and, due to appearance and the diffusion of fastbacteria, also threaten mankind itself's safety and Health.Thereby develop prevention mammitis of cow vaccine, prevent that by immunoprophylaxis system has become the emphasis of this disease study on prevention.
At present, some successes have been obtained abroad in the research and development of mastitis vaccine: the multi-vaccine of the usury for escherichia coli mastitis (Golicure-J5) of the mammitis of cow multiple vaccines that the StaphVAX staphylococcus pod membrane multivalence Seedling of U.S. Nabi biomedical company (Nabi Biopharmaceutical), general general company (U.S.PanProbe. Ltd) develop and the development of California, USA university.In addition, the states such as Australia, Israel, Germany, Argentina, Canada, also for cause of disease special in national mammitis of cow, have developed corresponding vaccine and have entered field test.The mammitis of cow vaccine technologies route that above country develops and method mainly contain following several: (1) is for unit price or the polyvalent vaccine of single cause of disease: 1. staphylococcus aureus mastitis vaccine: the Watson of Australian CSIRO has filtered out a strain and has had the representational bacterial strain of better antigen from the isolated staphylococcus aureus of national Isolated from Bovine Mastitis, and having filtered out the endotrophic culture medium of plan, the outer pod membrane of polysaccharide and the toxin that make this bacterium have important immunogenicity and pathogenic meaning are expressed fully.He extracts respectively the outer pod membrane of this granulose and toxin adds it in full cell of deactivation, has selected suitable adjuvant to obtain immune effect (1993) preferably.The people such as U.S. USDA immunity and epidemic disease control laboratory O ' Brien have prepared the staphylococcus aureus polysaccharide by the main serotype P8 of national isolated staphylococcus aureus, P5, P336 type respectively, and it is upper to be connected to rEPA, then is combined and prepares vaccine with the poly microsphere.High-titer LgG2 antibody and effective anti-Staphylococcus aureus infection immunity (2000) have been produced with this vaccine immunity cattle.The people such as G.Leitner of the Kimiron of Israel MARD veterinary institute have separated the staphylococcus aureus of three strain different biological characteristics and virulence from the ill cattle of this country, it is cultivated respectively by certain processing, be combined with adjuvant in proportion and prepare the staphylococcus aureus polyvalent vaccine, with this vaccine immunity protective rate, reach 70%(2003); 2. Escherichia coli mastitis vaccine: the people such as Hagan of California, USA university suffer from cattle and to have isolated the escherichia coli variant that a strain has incomplete O polysaccharide from mammitis of cow, and with this bacterial strain, exposed cAg prepares vaccine can protect the colibacillary infection of various serotype (1992) in clinical practice; (2) for the multiple vaccines of many cause of diseases, because mammitis of cow cause of disease kind is various, development and exploitation multiple vaccines have very large application potential, and the development of at present external multiple vaccines is in progress.But up to now, yet there are no formal product development success and the report of clinical practice.(3) surperficial polysaccharide serotype is the key character of staphylococcus aureus.Studies confirm that, can produce the cross immunity protection between phase homopolysaccharide serological type strain.Staphylococcus aureus has 11 kinds of serotypes, and the advantage serotype of the staphylococcus aureus separated in mammitis of cow is capsular polysaccharide 5 types (CP5), capsular polysaccharide 8 types (CP8) and adventitia polysaccharide 336 types (336PS type).Country variant is not identical with regional Major Epidemic serotype.The 212 strain cattle source property staphylococcus aureus typings confirmations that Poutrel(1988) France separated, 70% is CP5 and CP8; The staphylococcus aureus typing that the U.S. separates its cattle farm of 44% finds, what CP5 accounted for 51%, CP8 and failed typing accounts for respectively 18% and 31%; But the typing of 336PS is not all carried out in the research of these two countries.Korea S Hong-Ryul Han etc. carries out the Serotypes discovery to national 107 strain Bovine Mastitis Caused by Staphylococcus aureus in 2000, and 336 types account for 46.7%, and CP5 and CP8 are 12.1%, and CP5/336 is 17.8%, and CP5/336 is 6.5%, and typing is not 4.7%.Accordingly, extract respectively CP5, CP8,336PS the staphylococcus aureus that the people such as O ' Brien separate from the U.S., by itself and the nontoxic restructuring exotoxin A of Pseudomonas aeruginosa (rEPA) coupling, be combined with the poly microsphere again and make staphylococcus aureus subunit vaccine immunity milch cow, confirm that it can effectively resist anti-Staphylococcus aureus and infect.
At home, Zhang Jie in 2006 etc. carry out Serotypes to the 56 strain staphylococcus aureuses that obtain in China's clinic mastitis case and find that 336 types account for 67.9%, 5 type and account for 7.1%, 8 type and account for 3.6%; King in 2010 steps on the 117 strain staphylococcus aureus typings that separate in Deng Dui Xinjiang, peak and shows, CP5 accounts for 10.36%, CP8 and accounts for 13.68%, 336 type and account for 64.96%, and the typing bacterial strain does not account for 11.11%.
The antigen of the Bovine Mastitis Caused by Staphylococcus aureus the present invention relates to, escherichia coli bigeminy multivalent inactivated vaccine comprised the advantage serotype of China's Staphylococcus Aureus Isolated from Cow Mastitis, contain again the former escherichia coli of main environment sexually transmitted disease (STD), the intramammary infection that can effectively prevent China to be caused by above-mentioned cause of disease.
Summary of the invention
the objective of the invention is:
In order to solve the actual production problem of mammitis of cow immunoprophylaxis in China's milk cattle cultivating, according to the situation of China's mammitis of cow cause of disease, the invention provides a kind of prevention dairy cow mammitis staphylococcus aureus-Escherichia coli bigeminy polyvalent vaccine and preparation method thereof.
1,
the technical scheme that the present invention solves the problems of the technologies described above is: (1) dairy cow mammitis staphylococcus aureus-Escherichia coli bigeminy polyvalent vaccine, described vaccine by volume portion rate comprises 4 parts of antigens, described antigen comprises a, any 2 kinds or combination of more than two kinds in the golden yellow subspecies Staphylococcus of staphylococcus aureus aureus subsp.aureusCGMCC NO.4595,4596,4597,4598 mycelial polysaccharides, protein mixture antigen; B, any 2 kinds or combination of more than two kinds in CGMCC NO.4595,4596,4597,4598 toxin antigens; C, the tropina antigen of colon bacillus Escherichia coliCGMCC NO.4594; D, the toxin antigen of CGMCC NO.4594; With the e of 6 parts, the oily adjuvant of Montanide ISA70M VG immunity forms.
Be in described 4 parts of antigens, in CGMCC NO.4595,4596,4597,4598 mycelial polysaccharides, protein mixture antigen, any 2 kinds or combination of more than two kinds are containing 50%; CGMCC NO.4594 tropina antigen is containing 12.5%; In CGMCC NO.4595,4596,4597,4598 toxin antigens, any 2 kinds or combination of more than two kinds are containing 12.5%; CGMCC NO.4594 toxin antigen is containing 25%.
(2) preparation method of above-mentioned dairy cow mammitis staphylococcus aureus-Escherichia coli bigeminy polyvalent vaccine has following steps:
(1) preparation of CGMCC NO.4595,4596,4597,4598 strains and CGMCC NO.4594 strain;
(2) preparation of the tropina antigen of CGMCC NO.4595,4596,4597,4598 mycelial polysaccharides, proteantigen and CGMCC NO.4594;
(3) preparation of the toxin antigen of CGMCC NO.4595,4596,4597,4598 toxin antigen and CGMCC NO.4594;
(4) by the toxin antigen of the tropina antigen of any 2 kinds or combination of more than two kinds in the CGMCC NO.4595,4596,4597 of any 2 kinds or combination of more than two kinds in the CGMCC NO.4595,4596,4597 of above-mentioned acquisition, 4598 mycelial polysaccharides proteantigen and acquisition, 4598 toxin antigen and CGMCC NO.4594 and CGMCC NO.4594 and the oily adjuvant of Montanide ISA70M VG immunity by above-mentioned volume parts than mixing, make vaccine.
Can prepare according to following steps by any one in described CGMCC NO.4595,4596,4597,4598 strains:
(1) a small amount of strain of picking under aseptic condition, rule containing on 7% Sheep Blood flat board, places it in incubator and cultivate, and temperature is controlled under 37 ℃ and cultivates 24-28h;
(2) the single bacterium colony of picking from the strain of cultivating, be inoculated in improvement taxi driver brother rival subculture base test tube, is placed in the jolting incubator and cultivates 20h, and jolting incubator temperature remains on 37 ℃, and jolting speed keeps 170rpm;
(3) from test tube, get in Colombia's fluid medium triangular flask that culture is inoculated into improvement, inoculum concentration, for cultivating 1% of dosage, places it in the jolting incubator and cultivates 20h, and the jolting incubator keeps 37 ℃, and the jolting revolution keeps 170rpm;
(4) to the culture microscopy without miscellaneous bacteria after, be placed in 4 ℃ of refrigerators standby.
According to following steps, prepared by described CGMCC NO.4594 strain:
(1) a small amount of strain of picking under aseptic condition is rule on LB solid culture flat board, places it in incubator and cultivates, and temperature is controlled under 37 ℃ and cultivates 24h;
(2) under aseptic condition, the single bacterium colony of picking from the strain of cultivating, be inoculated in LB fluid medium test tube, is placed in the jolting incubator and cultivates 20h, and jolting incubator temperature remains on 37 ℃, and the jolting revolution keeps 170rpm;
(3) get culture from test tube and be inoculated in LB fluid medium triangular flask, inoculum concentration, for cultivating 1% of dosage, places it in the jolting incubator and cultivates 20h, and the jolting incubator keeps 37 ℃, and the jolting incubator keeps 170rpm;
(4) to the culture microscopy without miscellaneous bacteria after, be placed in 4 ℃ of refrigerators standby.
In described CGMCC NO.4595,4596,4597,4598 can prepare according to following steps by any one mycelial polysaccharides, proteantigen:
(1) from the strain prepared, use painting bacterium rod bacterium liquid to be seeded in uniformly on Colombia's solid culture flat board of improvement, bacterium liquid is placed in incubator and cultivates after absorbing, and the incubator temperature remains on 37 ℃, cultivates 24h;
(2) scraping lawn on the culture plate from cultivating, be adjusted into weight in wet base 30g/L with phosphate buffered saline, with the histiocyte pressure-even pulp crusher, processes bacteria suspension, collects the supernatant of bacteria suspension;
(3) supernatant of acquisition is cooled to 4 ℃, the centrifugal 15min of 8000 X g, regather supernatant, add the DNase of 25mg and the RNase of 75mg to every 1000ml in supernatant, place it in incubator and hatch 2h under 37 ℃ of conditions, be cooled to 4 ℃ and add again 10mMPMSF2ml, each 4ml of 10mM TLCK and TPCK;
(4) subsequently supernatant is measured to mycelial polysaccharides, proteantigen concentration by the Braedford method, with phosphate buffered saline be adjusted into 15g/L(±-0.3g/L), then the membrane filtration that is successively 0.8um and 0.45um with aperture, put-20 ℃ standby.
According to following steps, prepared by described CGMCC NO.4594 tropina antigen:
(1) from the strain of preparation, getting culture is seeded in the fermentation tank that the LB fluid medium is housed in 1% ratio, and the amount of LB fluid medium is no more than 40% of fermentation tank volume, in temperature, be that 37 ℃, pressure are 0.05MPa afterwards, turnover volume of air per minute, than under 1:1 and 170rpm stirring condition, is cultivated 16h;
(2) culture obtained from cultivating is 4 ℃ in temperature, and the centrifugal 30min of 8000 X g collects the bacterial sediment thing, with phosphate buffered saline, is adjusted into weight in wet base 30g/L; Process bacteria suspension with the histiocyte pressure-even pulp crusher, collect the supernatant of bacteria suspension;
(3) supernatant of acquisition is cooled to 4 ℃, the centrifugal 15min of 8000 X g, regather supernatant, add the DNase of 25mg and the RNase of 75mg to every 1000ml in supernatant, place it in incubator and hatch 2h under 37 ℃ of conditions, be cooled to 4 ℃ and add again 10mMPMSF2ml, each 4ml of 10mM TLCK and TPCK;
(4) subsequently supernatant is measured to mycelial polysaccharides, proteantigen concentration by the Braedford method, with phosphate buffered saline be adjusted into 15g/L(±-0.3g/L), then the membrane filtration that is successively 0.8um and 0.45um with aperture, put-20 ℃ standby.
Can prepare according to following steps by any one in described CGMCC NO.4595,4596,4597,4598 verticillium toxin antigens:
(1) from strain, getting bacterium liquid is seeded in the fermentation tank of Colombia's fluid medium that improvement is housed in 1% ratio, cultivate base unit weight and be no more than 40% of fermentation tank volume, in temperature, be that 37 ℃, pressure are 0.05MPa afterwards, turnover volume of air per minute, than under 1:1 and 170rpm stirring condition, is cultivated 20h;
(2) by culture, in temperature, be 4 ℃, the centrifugal 30min of 8000 X g, collect supernatant;
(3) add formaldehyde in supernatant, make the ultimate density of formaldehyde be controlled at 0.15%, temperature deactivation 48h or be deactivation 24h under 25 ℃ of conditions in temperature under 4 ℃ of conditions, in inactivation process, fully stir 2-3 time every day;
(4) 20 times of the ultrafiltration and concentration membrance concentration that deactivation liquid is 10KDa by the molecular retention amount, put-20 ℃ standby.
According to following steps, prepared by described CGMCC NO.4594 toxin antigen:
(1) from the strain of preparation, getting bacterium liquid is seeded in the fermentation tank that the LB fluid medium is housed in 1% ratio, cultivate base unit weight and be no more than 40% of fermentation tank volume, in temperature, be that 37 ℃, pressure are 0.05MPa afterwards, turnover volume of air per minute, than under 1:1 and 170rpm stirring condition, is cultivated 24h;
(2) by culture, in temperature, be 4 ℃, the centrifugal 30min of 8000 X g, collect supernatant;
(3) add formaldehyde in supernatant, make the ultimate density of formaldehyde be controlled at 0.15%, temperature deactivation 48h or be deactivation 24h under 25 ℃ of conditions in temperature under 4 ℃ of conditions, in inactivation process, fully stir 2-3 time every day;
(4) 20 times of the ultrafiltration and concentration membrance concentration that deactivation liquid is 10KDa by the molecular retention amount, put-20 ℃ standby.
The above improvement taxi driver brother rival subculture base, be by Colombia's culture medium, adds 1.5% sodium chloride, after sterilizing, is chilled to 55-60 ℃, then add the milk surum of 10% calf serum and 10% to mix after cooling to make.
The above bacterial strain for the proprietary program application all carries out and preservation at China Committee for Culture Collection of Microorganisms's common micro-organisms center, and has provided the survival proof.Specific as follows: (1) deposit number: CGMCC NO.4594, Classification And Nomenclature: colon bacillus, Latin title: Escherichia coli, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date: 2011 02 yuan 21 days; (2) deposit number: CGMCC NO.4595, Classification And Nomenclature: the golden yellow subspecies of staphylococcus aureus, Latin title: Staphylococcus aureus subsp.aureus, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date: 2011 02 yuan 21 days; (3) deposit number: CGMCC NO.4596, Classification And Nomenclature: the golden yellow subspecies of staphylococcus aureus, Latin title: Staphylococcus aureus subsp.aureus, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date: 2011 02 yuan 21 days; (4) deposit number: CGMCC NO.4597, Classification And Nomenclature: the golden yellow subspecies of staphylococcus aureus, Latin title: Staphylococcus aureus subsp.aureus, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date: 2011 02 yuan 21 days; (5) deposit number: CGMCC NO.4598, Classification And Nomenclature: the golden yellow subspecies of staphylococcus aureus, Latin title: Staphylococcus aureus subsp.aureus, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date: 2011 02 yuan 21 days.Wherein, the material number of the golden yellow subspecies Staphylococcus of staphylococcus aureus aureus subsp.aureusCGMCC NO.4595 is: XJMPAN10; Described CGMCC NO.4596, material number is: XJMP50; Described CGMCC NO.4597 material number is: XJMP69; Described CGMCC NO.4598 material number is: XJMP92.Described colon bacillus Escherichia coliCGMCC NO.4594 material number: XJMEY9.In above-mentioned: PMSF: Phenylmethanesulfonyl fluoride; TLCK:N-tosyl-1B chloromethylation ketone; TPCK: tosylphenylalanine chloromethyl ketone; DNase: deoxyribonuclease; RNase: endoribonuclease; LB: basal medium; The oily adjuvant of Montanide ISA70M VG immunity is the commodity that France match BIC Corp produces; The Braedford method is general protein determination method.
beneficial effect of the present invention
1, vaccine strains antigen broad covered area, immunogenicity is good.The strain that the present invention adopts is on the basis of the different local mammitis of cow Etiology surveys to the whole nation, the antigenicity filtered out is good, the bacterial strain that virulence is strong, above-mentioned staphylococcus aureus is carried capsular polysaccharide 5 types (CP5), 8 types (CP8), adventitia polysaccharide 336 types (336PS) or carries two kinds in above-mentioned polysaccharide antigen simultaneously; These bacterial strains produce α, β toxin or produce two kinds of toxin simultaneously.Above-mentioned colon bacillus is produced the ST toxin and is had extensive antigen representativeness.Processing by thalline and toxin, obtain vaccine antigen, and by screening and optimizing vaccine adjuvant, the bigeminy multivalent inactivated vaccine of preparation.
2, successful.Use vaccine of the present invention to greatly reduce milch cow due to the risk that infects staphylococcus aureus or colon bacillus and occur clinic mastitis.Clinical challenge test shows, the probability of this vaccine immunity Niu Fasheng staphylococcus aureus or the clinical mastitis of colon bacillus type is lower more than 70% than the contrast cattle; The field test demonstration, the probability of immune cattle generation latent mammitis is lower by 30% than the contrast cattle, and the daily yielding of immune cattle is higher more than 8% than contrasting cattle.
3, vaccine safety is high
Adjuvant is mineral oil adjuvant Montanide ISA70M VG, uses high-shearing dispersion emulsifying machine emulsifying.The oil adjuvant killed vaccine prepared after emulsifying carries out respectively steriling test, viscosity check and stability test by version " People's Republic of China's veterinary biologics quality standard " appendix in 2005.In test, choosing is apart from respectively 5 of the health gestation holstein cows of term 30 days and 60 days, and every incidence or intragluteal injection vaccine 5ml, observe 30 days.Annotate cow appetite after Seedling, body temperature is normal, without miscarriage, occurs, and annotates the Seedling position and occurs that diameter is no more than the lump of 3cm and disappear 3 weeks in after injection, all notes Seedling cows in normal expected date of confinement calving and calf growth normally, prove that this batch of vaccine is qualified products.
The specific embodiment
Below by specific embodiment, be that the invention will be further described:
Embodiment 1: dairy cow mammitis staphylococcus aureus-Escherichia coli bigeminy polyvalent vaccine, vaccine by volume portion rate comprises 4 parts of antigens, described antigen comprises a, the golden yellow subspecies Staphylococcus of staphylococcus aureus aureus subsp.aureusCGMCC NO.4595,4596,4597,4598 mycelial polysaccharides, the combination of protein mixture antigen; B, the combination of CGMCC NO.4595,4596,4597,4598 toxin antigens; C, the tropina antigen of colon bacillus Escherichia coliCGMCC NO.4594; D, the toxin antigen of CGMCC NO.4594; With the e of 6 parts, the composition of the oily adjuvant of Montanide ISA70M VG immunity.
In described 4 parts of antigens, the combination of CGMCC NO.4595,4596,4597,4598 mycelial polysaccharides, protein mixture antigen is containing 50%; CGMCC NO.4594 tropina antigen is containing 12.5%; The combination of CGMCC NO.4595,4596,4597,4598 toxin antigens is containing 12.5%; CGMCC NO.4594 toxin antigen is containing 25%.
The preparation method of above-mentioned dairy cow mammitis staphylococcus aureus-Escherichia coli bigeminy polyvalent vaccine has following steps:
(1) preparation of CGMCC NO.4595,4596,4597,4598 strains and CGMCC NO.4594 strain;
(2) preparation of the tropina antigen of CGMCC NO.4595,4596,4597,4598 mycelial polysaccharides, proteantigen and CGMCC NO.4594;
(3) preparation of the toxin antigen of CGMCC NO.4595,4596,4597,4598 toxin antigen and CGMCC NO.4594;
(4) by the toxin antigen of the tropina antigen of the combination in the CGMCC NO.4595,4596,4597 of the combination in the CGMCC NO.4595,4596,4597 of above-mentioned acquisition, 4598 mycelial polysaccharides proteantigen and acquisition, 4598 toxin antigen and CGMCC NO.4594 and CGMCC NO.4594 and the oily adjuvant of Montanide ISA70M VG immunity by above-mentioned volume parts than mixing, make vaccine.
Can prepare according to following steps by any one in described CGMCC NO.4595,4596,4597,4598 strains:
(1) a small amount of strain of picking under aseptic condition, rule containing on 7% Sheep Blood flat board, places it in incubator and cultivate, and temperature is controlled under 37 ℃ and cultivates 24-28h;
(2) the single bacterium colony of picking from the strain of cultivating, be inoculated in improvement taxi driver brother rival subculture base test tube, is placed in the jolting incubator and cultivates 20h, and jolting incubator temperature remains on 37 ℃, and the jolting rotating speed keeps 170rpm;
(3) from test tube, get in Colombia's fluid medium triangular flask that culture is inoculated into improvement, inoculum concentration, for cultivating 1% of dosage, places it in the jolting incubator and cultivates 20h, and the jolting incubator keeps 37 ℃, and the jolting revolution keeps 170rpm;
(4) to the culture microscopy without miscellaneous bacteria after, be placed in 4 ℃ of refrigerators.
According to following steps, prepared by described CGMCC NO.4594 strain:
(1) a small amount of strain of picking under aseptic condition is rule on LB solid culture flat board, places it in incubator and cultivates, and temperature is controlled under 37 ℃ and cultivates 24h;
(2) under aseptic condition, the single bacterium colony of picking from the strain of cultivating, be inoculated in LB fluid medium test tube, is placed in the jolting incubator and cultivates 20h, and jolting incubator temperature remains on 37 ℃, and the jolting revolution keeps 170rpm;
(3) get culture from test tube and be inoculated in LB fluid medium triangular flask, inoculum concentration, for cultivating 1% of dosage, places it in the jolting incubator and cultivates 20h, and the jolting incubator keeps 37 ℃, and the jolting rotating speed keeps 170rpm;
(4) to the culture microscopy without miscellaneous bacteria after, be placed in 4 ℃ of refrigerators standby.
In described CGMCC NO.4595,4596,4597,4598 can prepare according to following steps by any one mycelial polysaccharides, proteantigen:
(1) from the strain prepared, use painting bacterium rod bacterium liquid to be seeded in uniformly on Colombia's solid culture flat board of improvement, bacterium liquid is placed in incubator and cultivates after absorbing, and the incubator temperature remains on 37 ℃, cultivates 24h;
(2) scraping lawn on the culture plate from cultivating, be adjusted into weight in wet base 30g/L with phosphate buffered saline, with the histiocyte pressure-even pulp crusher, processes bacteria suspension, collects the supernatant of bacteria suspension;
(3) supernatant of acquisition is cooled to 4 ℃, the centrifugal 15min of 8000 X g, regather supernatant, add the DNase of 25mg and the RNase of 75mg to every 1000ml in supernatant, place it in incubator and hatch 2h under 37 ℃ of conditions, be cooled to 4 ℃ and add again 10mMPMSF2ml, each 4ml of 10mM TLCK and TPCK;
(4) subsequently supernatant is measured to mycelial polysaccharides, proteantigen concentration by the Braedford method, with phosphate buffered saline be adjusted into 15g/L(±-0.3g/L), then the membrane filtration that is successively 0.8um and 0.45um with aperture, put-20 ℃ standby.
According to following steps, prepared by described CGMCC NO.4594 tropina antigen:
(1) from the strain of preparation, getting culture is seeded in the fermentation tank that the LB fluid medium is housed in 1% ratio, and the amount of LB fluid medium is no more than 40% of fermentation tank volume, in temperature, be that 37 ℃, pressure are 0.05MPa afterwards, turnover volume of air per minute, than under 1:1 and 170rpm stirring condition, is cultivated 16h;
(2) culture obtained from cultivating is 4 ℃ in temperature, and the centrifugal 30min of 8000 X g collects the bacterial sediment thing, with phosphate buffered saline, is adjusted into weight in wet base 30g/L;
(3) supernatant of acquisition is cooled to 4 ℃, the centrifugal 15min of 8000 X g, regather supernatant, add the DNase of 25mg and the RNase of 75mg to every 1000ml in supernatant, place it in incubator and hatch 2h under 37 ℃ of conditions, be cooled to 4 ℃ and add again 10mMPMSF2ml, each 4ml of 10mM TLCK and TPCK;
(4) subsequently supernatant is measured to mycelial polysaccharides, proteantigen concentration by the Braedford method, with phosphate buffered saline be adjusted into 15g/L(±-0.3g/L), then the membrane filtration that is successively 0.8um and 0.45um with aperture, put-20 ℃ standby.
Can prepare according to following steps by any one in described CGMCC NO.4595,4596,4597,4598 verticillium toxin antigens:
(1) from strain, getting bacterium liquid is seeded in the fermentation tank of Colombia's fluid medium that improvement is housed in 1% ratio, cultivate base unit weight and be no more than 40% of fermentation tank volume, in temperature, be that 37 ℃, pressure are 0.05MPa afterwards, turnover volume of air per minute, than under 1:1 and 170rpm stirring condition, is cultivated 20h;
(2) by culture, in temperature, be 4 ℃, the centrifugal 30min of 8000 X g, collect supernatant;
(3) add formaldehyde in supernatant, make the ultimate density of formaldehyde be controlled at 0.15%, temperature deactivation 48h or be deactivation 24h under 25 ℃ of conditions in temperature under 4 ℃ of conditions, in inactivation process, fully stir 2-3 time every day;
(4) 20 times of the ultrafiltration and concentration membrance concentration that deactivation liquid is 10KDa by the molecular retention amount, put-20 ℃ standby.
According to following steps, prepared by described CGMCC NO.4594 toxin antigen:
(1) from the strain of preparation, getting bacterium liquid is seeded in the fermentation tank that the LB fluid medium is housed in 1% ratio, cultivate base unit weight and be no more than 40% of fermentation tank volume, in temperature, be that 37 ℃, pressure are 0.05MPa afterwards, turnover volume of air per minute, than under 1:1 and 170rpm stirring condition, is cultivated 24h;
(2) by culture, in temperature, be 4 ℃, the centrifugal 30min of 8000 X g, collect supernatant;
(3) add formaldehyde in supernatant, make the ultimate density of formaldehyde be controlled at 0.15%, temperature deactivation 48h or be deactivation 24h under 25 ℃ of conditions in temperature under 4 ℃ of conditions, in inactivation process, fully stir 2-3 time every day;
(4) 20 times of the ultrafiltration and concentration membrance concentration that deactivation liquid is 10KDa by the molecular retention amount, put-20 ℃ standby.
Described improvement taxi driver brother rival subculture base, be by Colombia's culture medium, adds 1.5% sodium chloride, after sterilizing, is chilled to 55-60 ℃, adds the milk surum of 10% calf serum and 10% to mix after cooling and make.
The polysaccharide, albumen and the toxin antigen that contain 5 strain bacterium in the dairy cow mammitis staphylococcus aureus-Escherichia coli bigeminy polyvalent vaccine of preparation.Wherein, the material number XJMPAN10 of the golden yellow subspecies Staphylococcus of staphylococcus aureus aureus subsp.aureusCGMCC NO.4595; Described CGMCC NO.4596, material number XJMP50; Described CGMCC NO.4597 material number XJMP69; Described CGMCC NO.4598 material number XJMP92.Colon bacillus Escherichia coliCGMCC NO.4594 material number XJMEY9.4 strain staphylococcus aureuses are cultivated toxin antigen, and it is Montanide ISA70M VG that 1 strain colon bacillus is cultivated the toxin antigen, the mineral oil adjuvant that produce, use high-shearing dispersion emulsifying machine emulsifying.The oil adjuvant killed vaccine prepared after emulsifying carries out respectively steriling test, viscosity check and stability test by version " People's Republic of China's veterinary biologics quality standard " appendix in 2005.
Embodiment 2: dairy cow mammitis staphylococcus aureus-Escherichia coli bigeminy polyvalent vaccine, vaccine by volume portion rate comprises 4 parts of antigens, described antigen comprises a, the combination of any 2 kinds in the golden yellow subspecies Staphylococcus of staphylococcus aureus aureus subsp.aureusCGMCC NO.4595,4596,4597,4598 mycelial polysaccharides, protein mixture antigen; B, the combination of any 2 kinds in CGMCC NO.4595,4596,4597,4598 toxin antigens; C, the tropina antigen of colon bacillus Escherichia coliCGMCC NO.4594; D, the toxin antigen of CGMCC NO.4594; With the e of 6 parts, the composition of the oily adjuvant of Montanide ISA70M VG immunity.
In described 4 parts of antigens, in CGMCC NO.4595,4596,4597,4598 mycelial polysaccharides, protein mixture antigen, the combination of any 2 kinds is containing 50%; CGMCC NO.4594 tropina antigen is containing 12.5%; In CGMCC NO.4595,4596,4597,4598 toxin antigens, the combination of any 2 kinds is containing 12.5%; CGMCC NO.4594 toxin antigen is containing 25%.
Bovine Mastitis Caused by Staphylococcus aureus-colon bacillus bigeminy vaccine preparation method is identical with the preparation method in embodiment 1.
Embodiment 3: dairy cow mammitis staphylococcus aureus-Escherichia coli bigeminy polyvalent vaccine, vaccine by volume portion rate comprises 4 parts of antigens, described antigen comprises a, the combination of any 3 kinds in the golden yellow subspecies Staphylococcus of staphylococcus aureus aureus subsp.aureusCGMCC NO.4595,4596,4597,4598 mycelial polysaccharides, protein mixture antigen; B, the combination of any 3 kinds in CGMCC NO.4595,4596,4597,4598 toxin antigens; C, the tropina antigen of colon bacillus Escherichia coliCGMCC NO.4594; D, the toxin antigen of CGMCC NO.4594; With the e of 6 parts, the oily adjuvant of Montanide ISA70M VG immunity forms.
In described 4 parts of antigens, in CGMCC NO.4595,4596,4597,4598 mycelial polysaccharides, protein mixture antigen, the combination of any 3 kinds is containing 50%; CGMCC NO.4594 tropina antigen is containing 12.5%; The combination of CGMCC NO.4595,4596,4597,4598 toxin antigens is containing 12.5%; CGMCC NO.4594 toxin antigen is containing 25%.
The dairy cow mammitis staphylococcus aureus-Escherichia coli bigeminy polyvalent vaccine preparation method is identical with the preparation method in embodiment 1.
Practical application test and the detection of this vaccine
Safety detects: choosing is apart from respectively 5 of the health gestation holstein cows of term 30 days and 60 days, every incidence or intragluteal injection vaccine 5ml, observe 30 days, the vaccine injection position allows to occur that diameter is no more than the 3cm lump, inject in latter 20 days and disappear, do not have the general reaction or the miscarriage that are caused by vaccine injection to occur, the vaccine that this batch is described is qualified products.
Efficacy test: in 30 3 parity, apart from term 40-60 days, the conceived milch cow of He Sitan, minute A, B, C, D, five groups of E, every group of 6 cow heads, wherein respectively organize 3 and within first 40～60 days, carry out immunity for the first time in childbirth, divide and within puerperium 30-40 days, carry out immunity for the second time.Immune musculi colli vaccinate 3ml for the first time, immune 2ml for the second time, other 3 cow heads are cattle in contrast, and two exempt from 60 days, carry out protest test.6 cattle of A group are used staphylococcus aureus XJMPAN10, XJMP69, XJMP50, in XJMP92 tetra-strain bacterium, counteracting toxic substances is carried out in any strain, the counteracting toxic substances bacterium is cultivated the 37 ℃ of cultivation 20h of Colombia's fluid medium that use improvement, centrifugal collection thalline, phosphate buffer (PBS, pH7.0-7.5) dilution is 60cfu/mL, counteracting toxic substances dosage is 5mL(300cfu/ breast district), every cattle selects breast district, same position to carry out counteracting toxic substances, the counteracting toxic substances method is for being used the lactogenesis pin that 5mL bacterium liquid is injected in newborn pond, and massage 1-2min, after counteracting toxic substances, observe 7 days, counteracting toxic substances breast district occurred that milk is abnormal or breast is red in 7 days, swollen, heat, parts such as pain or follow the General Symptoms cattle that is defined as falling ill, B organizes 6 cattle and uses in XJMPPAN10, XJMP69, XJMP50, XJMP92 tetra-strain bacterium other any strain of bacterial strain of removing the use of A group to carry out counteracting toxic substances, and the cultivation of counteracting toxic substances bacterium, counteracting toxic substances dosage, the district's selection of counteracting toxic substances breast, counteracting toxic substances method and morbidity judgement are organized consistent with A, 6 cattle of C group are used the staphylococcus aureus of clinical separation to carry out counteracting toxic substances, and the cultivation of counteracting toxic substances bacterium, counteracting toxic substances dosage, the district's selection of counteracting toxic substances breast, counteracting toxic substances method and morbidity judgement are consistent with the A group, 6 cattle of D group are used colon bacillus XJMEY9 to carry out counteracting toxic substances, the counteracting toxic substances bacterium is cultivated and uses 37 ℃ of LB fluid mediums to cultivate 20h, counteracting toxic substances dosage is 2000cfu/ breast district, every cattle selects breast district, same position to carry out counteracting toxic substances, the counteracting toxic substances method is for being used the lactogenesis pin that 5mL bacterium liquid is injected in newborn pond, and massage 1-2min, observe the part such as counteracting toxic substances breast district occurs in 7 days that milk is abnormal or breast is red, swollen, hot, pain or follow the General Symptoms cattle that is defined as falling ill after counteracting toxic substances 7 days, 6 cattle of E group are used the colon bacillus of separating from the mammitis of cow pathological material of disease to carry out counteracting toxic substances, and the cultivation of counteracting toxic substances bacterium, counteracting toxic substances dosage, the district's selection of counteracting toxic substances breast, counteracting toxic substances method and morbidity judgement are consistent with the D group.
Calculate immune protective rate according to following formula, in A, B, C, D, five test group of E, it is qualified products that five group immune protective rates all are not less than 50% vaccine.Immune protective rate (%)=(matched group Incidence-immune group Incidence)/matched group Incidence * 100%.
1, the clinical challenge test example of dairy cow mammitis staphylococcus aureus-Escherichia coli bigeminy polyvalent vaccine
(1) counteracting toxic substances method
In 30 3 parity, apart from the conceived milch cow of the term He Sitan of 40～60 days, minute A, B, C, D, five groups of E, every group of 6 cow heads, wherein respectively organize 3 and within first 40～60 days, carry out immunity for the first time in childbirth, divides and within puerperium 30-40 days, carry out immunity for the second time.Immune musculi colli vaccinate 3ml for the first time, immune 2ml for the second time, other 3 cow heads are cattle in contrast, and two exempt from 60 days, carry out protest test.
6 cattle of A group are used staphylococcus aureus XJMP 69 to carry out counteracting toxic substances, the counteracting toxic substances bacterium is cultivated and uses 37 ℃ of Colombia's fluid mediums to cultivate 20h, centrifugal collection thalline, phosphate buffer (PBS, pH7.0-7.5) dilution is 60cfu/mL, counteracting toxic substances dosage is 5mL(300cfu/ breast district), every cattle selects breast district, same position to carry out counteracting toxic substances, the counteracting toxic substances method is for being used the lactogenesis pin that 5mL bacterium liquid is injected in newborn pond, and massage 1-2min, after counteracting toxic substances, observe 7 days, counteracting toxic substances breast district occurred that milk is abnormal or breast is red in 7 days, swollen, heat, parts such as pain or follow the General Symptoms cattle that is defined as falling ill,
6 cattle of B group are used No. 92 bacterium of XJMP to carry out counteracting toxic substances, and the cultivation of counteracting toxic substances bacterium, counteracting toxic substances dosage, the district's selection of counteracting toxic substances breast, counteracting toxic substances method and morbidity judgement are consistent with the A group;
6 cattle of C group are used the staphylococcus aureus that is numbered XJMP 52 of clinical separation to carry out counteracting toxic substances, and the cultivation of counteracting toxic substances bacterium, counteracting toxic substances dosage, the district's selection of counteracting toxic substances breast, counteracting toxic substances method and morbidity judgement are consistent with the A group;
6 cattle of D group are used escherichia coli XJMEY9 to carry out counteracting toxic substances, the counteracting toxic substances bacterium is cultivated and uses 37 ℃ of LB fluid mediums to cultivate 20h, counteracting toxic substances dosage is 1000cfu/ breast district, every cattle selects breast district, same position to carry out counteracting toxic substances, the counteracting toxic substances method is for being used the lactogenesis pin that 5mL bacterium liquid is injected in newborn pond, and massage 1-2min, observe the part such as that counteracting toxic substances breast district occurred in 5 days is red, swollen, hot, pain or follow the clinic mastitis symptom such as the General Symptoms cattle that is defined as falling ill after counteracting toxic substances 5 days;
6 cattle of E group are used the K5 escherichia coli that are numbered that separate from the mammitis of cow pathological material of disease to carry out counteracting toxic substances, and the cultivation of counteracting toxic substances bacterium, counteracting toxic substances dosage, the district's selection of counteracting toxic substances breast, counteracting toxic substances method and morbidity judgement are consistent with the D group.
(2) counteracting toxic substances result
The counteracting toxic substances result shows that the sickness rate of immune cattle in each grouping is lower than 33.3%, and the sickness rate of matched group is greater than 66.7%, according to the computational methods of immune protective rate (%)=(matched group Incidence-immune group Incidence)/matched group Incidence * 100%, the protective rate of this vaccine to staphylococcus aureus: A group 66.7%; B group 100%; C group 100%, average protective rate is 88.9%; This vaccine is to colibacillary protective rate: D group 100%; E group 100%, average protective rate is 100%.
The clinical challenge test protective rate of table 1 dairy cow mammitis staphylococcus aureus-Escherichia coli bigeminy polyvalent vaccine
2, dairy cow mammitis staphylococcus aureus-Escherichia coli bigeminy polyvalent vaccine field example
60 of the chosen distance term conceived holstein cows of 30-60 days, each 30 of immune group and matched groups, immune group is carried out immunity for the first time in first 40～60 days in childbirth, divides and within puerperium 30-40 days, carries out immunity for the second time; Immune musculi colli vaccinate 3ml for the first time, immune 2ml for the second time.Matched group does not carry out any immunity.After secondary immunity, carry out the tracking determination and analysis of 6 months, add up in 6 months by staphylococcus aureus and or the clinic mastitis incidence that causes of colon bacillus, secondary immunity after latent mammitis 3rd month the time (latent mammitis is judged: the average milk yield in 7 days 3rd month time the after sickness rate somatic cell from milk number/mL >=500,000) and secondary immunity.
Table 2 dairy cow mammitis staphylococcus aureus-Escherichia coli bigeminy polyvalent vaccine field example
3, dairy cow mammitis staphylococcus aureus-Escherichia coli bigeminy polyvalent vaccine field example
100 of the chosen distance term conceived holstein cows of 30-60 days, each 50 of immune group and matched groups; Immune group is carried out immunity for the first time in first 40～60 days in childbirth, divides and within puerperium 30-40 days, carries out immunity for the second time
;immune musculi colli vaccinate 3ml for the first time, immune 2ml for the second time.Matched group does not carry out any immunity.After secondary immunity, carry out the tracking determination and analysis of 6 months, add up in 6 months by staphylococcus aureus and or the clinic mastitis incidence that causes of colon bacillus, secondary immunity after latent mammitis 3rd month the time (latent mammitis is judged: the average milk yield in 7 days 3rd month time the after sickness rate somatic cell from milk number/mL >=500,000) and secondary immunity.
Table 3 dairy cow mammitis staphylococcus aureus-Escherichia coli bigeminy polyvalent vaccine field example
Above three groups of data show: dairy cow mammitis staphylococcus aureus-Escherichia coli bigeminy polyvalent vaccine is aspect the prevention mammitis of cow, and obviously, effect is remarkable in effect.
Claims (10)
1. dairy cow mammitis staphylococcus aureus-Escherichia coli bigeminy polyvalent vaccine, it is characterized in that: described vaccine by volume portion rate comprises 4 parts of antigens, described antigen comprises a, any 2 kinds or combination of more than two kinds in the golden yellow subspecies Staphylococcus of staphylococcus aureus aureus subsp.aureusCGMCC NO.4595,4596,4597,4598 mycelial polysaccharides, proteantigen; B, any 2 kinds or combination of more than two kinds in CGMCC NO.4595,4596,4597,4598 toxin antigens; C, the tropina antigen of colon bacillus Escherichia coliCGMCC NO.4594; D, the toxin antigen of CGMCC NO.4594; With the e of 6 parts, the oily adjuvant of Montanide ISA70M VG immunity forms.
2. dairy cow mammitis staphylococcus aureus-Escherichia coli bigeminy polyvalent vaccine according to claim 1, it is characterized in that in described 4 parts of antigens, in CGMCC NO.4595,4596,4597,4598 mycelial polysaccharides, proteantigen, any 2 kinds or combination of more than two kinds are containing 50%; CGMCC NO.4594 tropina antigen is containing 12.5%; In CGMCC NO.4595,4596,4597,4598 toxin antigens, any 2 kinds or combination of more than two kinds are containing 12.5%; CGMCC NO.4594 toxin antigen is containing 25%.
3. the preparation method of a dairy cow mammitis staphylococcus aureus-Escherichia coli bigeminy polyvalent vaccine according to claim 1 is characterized in that the method has following steps:
(1) preparation of CGMCC NO.4595,4596,4597,4598 strains and CGMCC NO.4594 strain;
(2) preparation of the tropina antigen of CGMCC NO.4595,4596,4597,4598 mycelial polysaccharides, proteantigen and CGMCC NO.4594;
(3) preparation of the toxin antigen of CGMCC NO.4595,4596,4597,4598 toxin antigen and CGMCC NO.4594;
(4) by the toxin antigen of the tropina antigen of any 2 kinds or combination of more than two kinds in the CGMCC NO.4595,4596,4597 of any 2 kinds or combination of more than two kinds in the CGMCC NO.4595,4596,4597 of above-mentioned acquisition, 4598 mycelial polysaccharides, proteantigen and acquisition, 4598 toxin antigen and CGMCC NO.4594 and CGMCC NO.4594 and the oily adjuvant of Montanide ISA70M VG immunity by above-mentioned volume parts than mixing, make vaccine.
4. the preparation method of dairy cow mammitis staphylococcus aureus-Escherichia coli bigeminy polyvalent vaccine according to claim 3 is characterized in that can prepare according to following steps by any one in described CGMCC NO.4595,4596,4597,4598 strains:
(1) a small amount of strain of picking under aseptic condition, rule containing on 7% Sheep Blood flat board, places it in incubator and cultivate, and temperature is controlled under 37 ℃ and cultivates 24-28h;
(2) the single bacterium colony of picking from the strain of cultivating, be inoculated in improvement taxi driver brother rival subculture base test tube, is placed in the jolting incubator and cultivates 20h, and jolting incubator temperature remains on 37 ℃, and jolting speed keeps 170rpm;
(3) from test tube, get in Colombia's fluid medium triangular flask that culture is inoculated into improvement, inoculum concentration, for cultivating 1% of dosage, places it in the jolting incubator and cultivates 20h, and the jolting incubator keeps 37 ℃, and the jolting revolution keeps 170rpm;
(4) the culture microscopy without miscellaneous bacteria after, be placed in 4 ℃ of refrigerators standby.
5. the preparation method of dairy cow mammitis staphylococcus aureus-Escherichia coli bigeminy polyvalent vaccine according to claim 3 is characterized in that prepared according to following steps by described CGMCC NO.4594 strain:
(1) a small amount of strain of picking under aseptic condition is rule on LB solid culture flat board, places it in incubator and cultivates, and temperature is controlled under 37 ℃ and cultivates 24h;
(2) under aseptic condition, the single bacterium colony of picking from the strain of cultivating, be inoculated in LB fluid medium test tube, is placed in the jolting incubator and cultivates 20h, and jolting incubator temperature remains on 37 ℃, and the jolting revolution keeps 170rpm;
(3) get culture from test tube and be inoculated in LB fluid medium triangular flask, inoculum concentration, for cultivating 1% of dosage, places it in the jolting incubator and cultivates 20h, and the jolting incubator keeps 37 ℃, and jolting cultivation speed keeps 170rpm;
(4) to the culture microscopy without miscellaneous bacteria after, be placed in 4 ℃ of refrigerators standby.
6. the preparation method of dairy cow mammitis staphylococcus aureus-Escherichia coli bigeminy polyvalent vaccine according to claim 3 is characterized in that in described CGMCC NO.4595,4596,4597,4598 can prepare according to following steps by any one mycelial polysaccharides, proteantigen:
(1) from the strain prepared, use painting bacterium rod bacterium liquid to be seeded in uniformly on Colombia's solid culture flat board of improvement, bacterium liquid is placed in incubator and cultivates after absorbing, and the incubator temperature remains on 37 ℃, cultivates 24h;
(2) scraping lawn on the culture plate from cultivating, be adjusted into weight in wet base 30g/L with phosphate buffered saline, with the histiocyte pressure-even pulp crusher, processes bacteria suspension, collects the supernatant of bacteria suspension;
(3) supernatant of acquisition is cooled to 4 ℃, the centrifugal 15min of 8000 X g, regather supernatant, add the DNase of 25mg and the RNase of 75mg to every 1000ml in supernatant, place it in incubator and hatch 2h under 37 ℃ of conditions, be cooled to 4 ℃ and add again 10mMPMSF2ml, each 4ml of 10mM TLCK and TPCK;
(4) subsequently supernatant is measured to mycelial polysaccharides, proteantigen concentration by the Braedford method, with phosphate buffered saline be adjusted into 15g/L(±-0.3g/L), then the membrane filtration that is successively 0.8um and 0.45um with aperture, put-20 ℃ standby.
7. the preparation method of dairy cow mammitis staphylococcus aureus-Escherichia coli bigeminy polyvalent vaccine according to claim 3 is characterized in that prepared according to following steps by described CGMCC NO.4594 tropina antigen:
(1) from the strain of preparation, getting culture is seeded in the fermentation tank that the LB fluid medium is housed in 1% ratio, and the amount of LB fluid medium is no more than 40% of fermentation tank volume, in temperature, be that 37 ℃, pressure are 0.05MPa afterwards, turnover volume of air per minute, than under 1:1 and 170rpm stirring condition, is cultivated 16h;
(2) culture obtained from cultivating is 4 ℃ in temperature, and the centrifugal 30min of 8000 X g collects the bacterial sediment thing, with phosphate buffered saline, is adjusted into weight in wet base 30g/L, with the histiocyte pressure-even pulp crusher, processes bacteria suspension, collects the supernatant of bacteria suspension;
(3) supernatant of acquisition is cooled to 4 ℃, the centrifugal 15min of 8000 X g, regather supernatant, add the DNase of 25mg and the RNase of 75mg to every 1000ml in supernatant, place it in incubator and hatch 2h under 37 ℃ of conditions, be cooled to 4 ℃ and add again 10mMPMSF2ml, each 4ml of 10mM TLCK and TPCK;
(4) subsequently supernatant is measured to mycelial polysaccharides, proteantigen concentration by the Braedford method, with phosphate buffered saline be adjusted into 15g/L(±-0.3g/L), then the membrane filtration that is successively 0.8um and 0.45um with aperture, put-20 ℃ standby.
8. the preparation method of dairy cow mammitis staphylococcus aureus-Escherichia coli bigeminy polyvalent vaccine according to claim 3 is characterized in that can prepare according to following steps by any one in described CGMCC NO.4595,4596,4597,4598 verticillium toxin antigens:
(1) from strain, getting bacterium liquid is seeded in the fermentation tank of Colombia's fluid medium that improvement is housed in 1% ratio, cultivate base unit weight and be no more than 40% of fermentation tank volume, in temperature, be that 37 ℃, pressure are 0.05MPa afterwards, turnover volume of air per minute, than under 1:1 and 170rpm stirring condition, is cultivated 20h;
(2) by culture, in temperature, be 4 ℃, the centrifugal 30min of 8000 X g, collect supernatant;
(3) add formaldehyde in supernatant, make the ultimate density of formaldehyde be controlled at 0.15%, temperature deactivation 48h or be deactivation 24h under 25 ℃ of conditions in temperature under 4 ℃ of conditions, in inactivation process, fully stir 2-3 time every day;
(4) 20 times of the ultrafiltration and concentration membrance concentration that deactivation liquid is 10KDa by the molecular retention amount, put-20 ℃ standby.
9. the preparation method of dairy cow mammitis staphylococcus aureus-Escherichia coli bigeminy polyvalent vaccine according to claim 3 is characterized in that prepared according to following steps by described CGMCC NO.4594 toxin antigen:
(1) from the strain of preparation, getting bacterium liquid is seeded in the fermentation tank that the LB fluid medium is housed in 1% ratio, cultivate base unit weight and be no more than 40% of fermentation tank volume, in temperature, be that 37 ℃, pressure are 0.05MPa afterwards, turnover volume of air per minute, than under 1:1 and 170rpm stirring condition, is cultivated 24h;
(2) by culture, in temperature, be 4 ℃, the centrifugal 30min of 8000 X g, collect supernatant;
(3) add formaldehyde in supernatant, make the ultimate density of formaldehyde be controlled at 0.15%, temperature deactivation 48h or be deactivation 24h under 25 ℃ of conditions in temperature under 4 ℃ of conditions, in inactivation process, fully stir 2-3 time every day;
(4) 20 times of the ultrafiltration and concentration membrance concentration that deactivation liquid is 10KDa by the molecular retention amount, put-20 ℃ standby.
10. according to the preparation method of claim 4,6,8 described dairy cow mammitis staphylococcus aureus-Escherichia coli bigeminy polyvalent vaccines, it is characterized in that described improvement taxi driver brother rival subculture base, by Colombia's culture medium, add 1.5% sodium chloride, after sterilizing, be chilled to 55-60 ℃, add the milk surum of 10% calf serum and 10% to mix after cooling and make.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110130527 CN102205117B (en) | 2011-05-19 | 2011-05-19 | Dairy cow mammitis staphylococcus aureus-Escherichia coli bigeminy polyvalent vaccine and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110130527 CN102205117B (en) | 2011-05-19 | 2011-05-19 | Dairy cow mammitis staphylococcus aureus-Escherichia coli bigeminy polyvalent vaccine and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102205117A CN102205117A (en) | 2011-10-05 |
| CN102205117B true CN102205117B (en) | 2013-12-18 |
Family
ID=44694359
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110130527 Expired - Fee Related CN102205117B (en) | 2011-05-19 | 2011-05-19 | Dairy cow mammitis staphylococcus aureus-Escherichia coli bigeminy polyvalent vaccine and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102205117B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102716475A (en) * | 2012-06-29 | 2012-10-10 | 黑龙江省科学院微生物研究所 | Preparation method of cow mammitis vaccines |
| CN110387341A (en) * | 2019-07-17 | 2019-10-29 | 安徽赛德齐瑞生物科技有限公司 | A kind of staphylococcus culture fluid and its cultural method |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101991847A (en) * | 2010-11-16 | 2011-03-30 | 赤峰博恩药业有限公司 | Triple inactivated vaccine against dairy cattle mastitis and preparation method thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030036639A1 (en) * | 2001-01-03 | 2003-02-20 | Emery Daryll A. | Immunizing compositions and methods of use |
| CN100536917C (en) * | 2005-07-13 | 2009-09-09 | 崔玉东 | Cow mastitis concatenate inactivated vaccine |
-
2011
- 2011-05-19 CN CN 201110130527 patent/CN102205117B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101991847A (en) * | 2010-11-16 | 2011-03-30 | 赤峰博恩药业有限公司 | Triple inactivated vaccine against dairy cattle mastitis and preparation method thereof |
Non-Patent Citations (6)
| Title |
|---|
| 《Temperature and milk compositon responses of the non-infused mammary gland to infusion of E.colii LPS AND S.aureus vaccine and to intravenous E.coli LPS in the goat》;Jarman RF;《Ventralbl Veterinarmed A》;19871231;第34卷(第8期);561-73 * |
| 《Vaccine against6 bovine mastitis in the New Zealand context :what is the best way forward?》;Denis M;《N Z VET J》;20110216;第57卷(第3期);132-40 * |
| 《奶牛乳房炎疫苗防治的研究进展》;高宇;《河北科技师范学院学报》;20110330;第25卷(第1期);74-77 * |
| Denis M.《Vaccine against6 bovine mastitis in the New Zealand context :what is the best way forward?》.《N Z VET J》.2011,第57卷(第3期),132-40. |
| Jarman RF.《Temperature and milk compositon responses of the non-infused mammary gland to infusion of E.colii LPS AND S.aureus vaccine and to intravenous E.coli LPS in the goat》.《Ventralbl Veterinarmed A》.1987,第34卷(第8期),561-73. |
| 高宇.《奶牛乳房炎疫苗防治的研究进展》.《河北科技师范学院学报》.2011,第25卷(第1期),74-77. |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102205117A (en) | 2011-10-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102499982B (en) | Method for producing trivalent inactivated vaccine against Haemophilus parasuis infection | |
| CN104334187B (en) | Diplococcus meningitidis composition and its method | |
| Zhang et al. | Influences of season, parity, lactation, udder area, milk yield, and clinical symptoms on intramammary infection in dairy cows | |
| CN103182076B (en) | Swine mycoplasma pneumoniae inactivated vaccine and preparation method thereof | |
| CN102258776B (en) | Combined inactivated vaccine against mycoplasma hyopneumoniae (MHP) and mycoplasma hyorhinis and preparation method thereof | |
| CN104829712A (en) | C and D type C.perfringens antitoxin serum and preparation method thereof | |
| CN102949714B (en) | Swine Streptococcosis trivalent inactivated vaccine and preparation method thereof | |
| CN102205117B (en) | Dairy cow mammitis staphylococcus aureus-Escherichia coli bigeminy polyvalent vaccine and preparation method thereof | |
| CN102294026A (en) | Milk cow streptococcal mastitis inactivated vaccine and preparation method thereof | |
| CN108721616B (en) | A kind of avian pasteurella multocida capsular polysaccharide-protein conjugate vaccines and preparation method thereof | |
| CN105039233B (en) | A kind of B. abortus molecular marker vaccine strain and its application | |
| CN104163858B (en) | Pasteurella multocida acellular antigen, preparation method and applications thereof | |
| CN101991847A (en) | Triple inactivated vaccine against dairy cattle mastitis and preparation method thereof | |
| CN104998256A (en) | Preparation method of triple inactivated vaccine for pigs | |
| WO2016119078A1 (en) | Combined use of haemophilus parasuis lc strain and lz-20100109 strain | |
| CN110075289A (en) | A kind of haemophilus parasuis, Streptococcus suis and Actinobacillus pleuropneumoniae triple inactivated vaccine and its application | |
| CN109609418A (en) | One plant of erysipelothrix porci and its application | |
| CN106520623B (en) | A kind of serum 7-type haemophilus parasuis low virulent strain and its application | |
| CN103157101B (en) | Combined inactivate vaccine for haemophilus parasuis disease and streptococcus suis disease and preparation method for same | |
| CN105420141A (en) | Haemophilus parasuis strain | |
| CN101721695B (en) | Novel trivalent inactivated vaccine for swine streptococcosis | |
| CN104250623B (en) | One plant of mycoplasma hyorhinis bacterial strain, vaccine combination and its preparation method and application | |
| CN107875377A (en) | A kind of preparation method of E types C.perfringens toxoid vaccine | |
| CN109106946B (en) | Inactivated staphylococcus aureus vaccine for dairy cow mastitis and preparation method thereof | |
| CN105713855A (en) | Strains, application of strains, vaccine and preparation method of vaccine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C53 | Correction of patent of invention or patent application | ||
| CB03 | Change of inventor or designer information |
Inventor after: Wang Zhicai Inventor after: Wang Dengfeng Inventor after: Li Jianjun Inventor after: Yang Xueyun Inventor after: Xu Meng Inventor after: Wu Jianyong Inventor before: Wang Zhicai Inventor before: Wang Dengfeng |
|
| COR | Change of bibliographic data |
Free format text: CORRECT: INVENTOR; FROM: WANG ZHICAI WANG DENGFENG TO: WANG ZHICAI WANG DENGFENG LI JIANJUN YANG XUEYUN XU MENG WU JIANYONG |
|
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20131218 Termination date: 20210519 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |