CN102120028B - Method for extracting lipopolysaccharides from avian pasteurella multocida and preparing lipopolysaccharide vaccine - Google Patents
Method for extracting lipopolysaccharides from avian pasteurella multocida and preparing lipopolysaccharide vaccine Download PDFInfo
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- CN102120028B CN102120028B CN 201110055100 CN201110055100A CN102120028B CN 102120028 B CN102120028 B CN 102120028B CN 201110055100 CN201110055100 CN 201110055100 CN 201110055100 A CN201110055100 A CN 201110055100A CN 102120028 B CN102120028 B CN 102120028B
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Abstract
The invention provides a method for extracting lipopolysaccharides from avian pasteurella multocida and preparing a lipopolysaccharide vaccine. The method comprises the following eight steps of: 1, preparing culture solution of avian pasteurella multocida; 2, collecting the avian pasteurella multocida from the culture solution of avian pasteurella multocida; 3, crushing the avian pasteurella multocida by using ultrasonic wave; 4, crudely extracting solution of lipopolysaccharides from the avian pasteurella multocida crushed by the ultrasonic wave; 5, extracting concentrated solution of lipopolysaccharides from the crudely extracted solution of lipopolysaccharides; 6, performing enzymolysis on deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in the concentrated solution of lipopolysaccharides by using DNA and RNA enzymes; 7, preparing the purified lipopolysaccharides; and 8, preparing the lipopolysaccharide vaccine of avian pasteurella multocida from the purified lipopolysaccharides. According to animal immunization experiments and animal virus attacking experiments, after the lipopolysaccharide vaccine of avian pasteurella multocida, prepared by the method, is used for immunizing chickens for three times, the immunized chickens can be effectively prevented from suffering from avian pasteurella multocida disease.
Description
Technical field
The invention belongs to the animal vaccine preparing technical field, especially a kind of method of extracting lipopolysaccharide and making the lipopolysaccharide vaccine from the fowl pasteurella multocida.
Background technology
The fowl pasteurella multocida disease is the poultry contagious disease caused by the fowl pasteurella multocida, being the world distributes, in China, be also common multiple infectious disease, nearly all poultry can infect this disease, and it infects the popular sound development that is having a strong impact on domestic fowl farming.
At present China for the prevention of fowl pasteurella multocida disease and treatment, main what adopt is Drug therapy, as antibiotic etc., once but easily recurrence after drug withdrawal, and long-term prescription easily induces the fowl pasteurella multocida to produce drug resistance, also may have side effects, such as meeting causes the decline of laying hen laying rate.Therefore, in order more effectively to prevent the popular of fowl pasteurella multocida disease, need to carry out immunity with effective vaccine.
The conventional vaccine of fowl pasteurella multocida disease mainly contains two kinds of attenuated live vaccines and inactivated vaccines; but there is certain shortcoming in attenuated live vaccines; such as immune duration is shorter, the protection effect is on the low side, side effect is bigger than normal; can cause immuning failure during misapplication, even can cause the death of some poultry.
And inactivated vaccine may cause the loss of some antigenic substance in inactivation process, certain immune effect can only be arranged the strain infection of homologous serotype, and protect effect and immune duration to it would be better that attenuated live vaccines is better.
Lipopolysaccharide is the endotoxic material base of fowl pasteurella multocida, when the fowl pasteurella multocida is burst apart, discharges, and is that very strong heating is former.People generally believe that lipopolysaccharide plays an important role in the pathogenic course of fowl pasteurella multocida at present.The lipopolysaccharide that research shows the fowl pasteurella multocida is playing assosting effect in the sticking of neutrophil cell.The lipopolysaccharide be separated to the fowl Pasteurella Multocida Strains that is B:2 from serotype can cause that animal produces the symptom of hueppe's disease.Except have pathogenic, therefore the lipopolysaccharide of fowl pasteurella multocida can also bring out humoral immunoresponse(HI), is considered to a kind of protective antigen.
Up to now, the method for extracting lipopolysaccharide and make fowl pasteurella multocida lipopolysaccharide vaccine from the fowl pasteurella multocida yet there are no relevant report.
Summary of the invention
For addressing the above problem, the invention provides a kind of method of extracting lipopolysaccharide and making the lipopolysaccharide vaccine from the fowl pasteurella multocida, the method is first extracted lipopolysaccharide from the fowl pasteurella multocida, then the lipopolysaccharide solution of extraction and Freund's complete adjuvant are mixed and made into to fowl pasteurella multocida disease lipopolysaccharide vaccine according to the ratio of 1 ︰ 1.5, show that through the zoopery checking fowl pasteurella multocida disease lipopolysaccharide vaccine a process for preparing can provide the opposing fowl ability that pasteurella multocida is attacked for the chicken by immune effectively, protective rate is 66.7%, can effectively prevent poultry to produce the fowl pasteurella multocida disease.
For achieving the above object, the present invention adopts following technical scheme:
A kind of method of extracting lipopolysaccharide and making the lipopolysaccharide vaccine from the fowl pasteurella multocida, 1. the method through preparing fowl pasteurella multocida culture fluid, 2. collect fowl pasteurella multocida thalline from fowl pasteurella multocida culture fluid, 3. ultrasound wave carries out fragmentation to fowl pasteurella multocida thalline, 4. slightly carry lipopolysaccharide solution from the fowl pasteurella multocida thalline of ultrasonic disruption, 5. extract the lipopolysaccharide concentrated solution from the lipopolysaccharide solution of slightly carrying, 6. carry out DNA and the RNA in enzymolysis lipopolysaccharide concentrated solution with DNA enzyme and RNA enzyme, 7. prepare the lipopolysaccharide of purification and 8. the lipopolysaccharide of purification is made to teleoptile pasteurella multocida lipopolysaccharide vaccine totally eight steps, described in above-mentioned eight steps 2. in relate to the centrifugal rotational speed of centrifuge, describedly relate to hyperacoustic power in 3., radiated time, intermittently number of seconds and radiation number of times, described relating in 5. changed the waterside number, describedly relate to enzymolysis time in 6., it is described that to relate to by concentration in 7. be the pH value that 6 mol/L NaOH carry out regulator solution, the described volume ratio that relates to lipopolysaccharide solution and Freund's complete adjuvant in 8. is the normal experiment technological means, any one in these normal experiment technological means perhaps exists as independent experiment condition, but these normal experiment technological means integrated applications are essential in " method of extracting lipopolysaccharide and making the lipopolysaccharide vaccine from the fowl pasteurella multocida ".
Described eight steps of method that the present invention extracts lipopolysaccharide and makes the lipopolysaccharide vaccine from the fowl pasteurella multocida are described below:
1. prepare fowl pasteurella multocida culture fluid
Use the inoculating loop scraping
one ringthe fowl pasteurella multocida of slant preservation, will scrape
one ringthe inoculating loop of fowl pasteurella multocida with the streak inoculation of " Z " font on culture plate, culture plate is positioned in 37 ℃ of incubators and cultivates 24 hours, a single colony inoculation of growing on the culture plate of picking cultivation after 24 hours is in the fluid medium of 5 milliliters, this fluid medium of 5 milliliters being placed on to the velocity fluctuation with 160 rev/mins in the shaking table of 37 ℃ cultivates 24 hours, again 5 milliliters of liquid culture medium of shaken cultivation all poured in the fluid medium of 200 milliliters and cultivated and within 24 hours, prepare fowl pasteurella multocida culture fluid with the velocity fluctuation of 160 rev/mins in the shaking table of 37 ℃,
2. collect fowl pasteurella multocida thalline from fowl pasteurella multocida culture fluid
The above-mentioned fowl pasteurella multocida culture fluid of 1. preparing is joined in the centrifuge tube of 250 milliliters, to add these 250 milliliters of centrifuge tubes of fowl pasteurella multocida culture fluid to be placed on centrifuge and under the rotating speed of 5000 rev/mins centrifugal 15 minutes, then the supernatant in 250 milliliters of centrifuge tubes after centrifugal 15 minutes is discarded, the precipitate of staying in these 250 milliliters of centrifuge tubes is exactly collected fowl pasteurella multocida thalline;
3. ultrasound wave carries out fragmentation to fowl pasteurella multocida thalline
At the above-mentioned distilled water that adds 10 milliliters in 2. in the collected fowl pasteurella multocida thalline of 250 milliliters of centrifuge tubes, then these 250 milliliters of centrifuge tubes are placed in to the ultrasonic disruption instrument, the power that the ultrasonic disruption instrument is set is 500 watts, every Ultrasonic Radiation stops radiation after 20 seconds 20 seconds be a broken cyclic process, all broken cyclic processes continue 30 minutes altogether, and what after 30 minutes, in these 250 milliliters of centrifuge tubes, obtain is exactly the fowl pasteurella multocida thalline through ultrasonic disruption;
4. slightly carry lipopolysaccharide solution from the fowl pasteurella multocida thalline of ultrasonic disruption
Slightly carry lipopolysaccharide and be divided into three steps from fowl pasteurella multocida thalline, three steps are described below:
The first step, extract above-mentioned 10 milliliters of fowl pasteurella multocida thalline that obtain through ultrasonic disruption in 3. and join in 50 milliliters of centrifuge tubes, and then add the phenol that 10 milliliters of its concentration are 90% in these 50 milliliters of centrifuge tubes, putting upside down these 50 milliliters of centrifuge tubes makes 10 milliliters of fowl pasteurella multocida thalline and 10 milliliters of phenol fully mix for several times, 50 milliliters of centrifuge tubes of this that then will fully mix are placed in the water-bath of 70 ℃ and heat and use the Glass rod rapid stirring 30 minutes, take out these 50 milliliters of centrifuge tubes after 30 minutes and at room temperature place 20 minutes from water-bath, by these 50 milliliters of centrifuge tubes, be placed on centrifuge again and under the rotating speed of 4000 rev/mins centrifugal 30 minutes, after centrifugal end, the upper solution of these 50 milliliters of centrifuge tubes is drawn and is joined in another triangular flask of 50 milliliters, middle level solution in these 50 milliliters of centrifuge tubes discards not, last milliliter stand-by to adding in the lower floor's solution in these 50 milliliters of centrifuge tubes distilled water to supply volume to 10 again,
Second step, add the phenol that 10 milliliters of its concentration are 90% in the lower floor's solution+distilled water in these 50 milliliters of centrifuge tubes stand-by in the first step, putting upside down these 50 milliliters of centrifuge tubes makes it fully mix for several times, after fully mixing, these 50 milliliters of centrifuge tubes are placed in the water-bath of 70 ℃ and heat and use the Glass rod rapid stirring 30 minutes, take out these 50 milliliters of centrifuge tubes after 30 minutes and at room temperature place 20 minutes from water-bath, by these 50 milliliters of centrifuge tubes, be placed on centrifuge again and under the rotating speed of 4000 rev/mins centrifugal 30 minutes, the upper solution of these 50 milliliters of centrifuge tubes is drawn and joined in another 50 milliliters the triangular flask in the first step after centrifugal end and store, middle level solution in these 50 milliliters of centrifuge tubes discards not, last to adding distilled water to supply volume to 10 a milliliter preparation in the lower floor's solution in these 50 milliliters of centrifuge tubes, reuse again,
The 3rd step, repeat second step for several times until, by another triangular flask of 50 milliliters storage full scale, the described upper solution in another 50 milliliters of triangular flasks that storage is full 50 milliliters is exactly the lipopolysaccharide solution of slightly carrying from the fowl pasteurella multocida thalline of ultrasonic disruption;
5. extract the lipopolysaccharide concentrated solution from the lipopolysaccharide solution of slightly carrying
By the 50 ml lipopolysaccharide solution of slightly carrying, all join in bag filter, bag filter is placed in tap water flowing water to continual rinsing 48 hours, again bag filter is put in distilled water and soaked, immersion in every 6 hours is changed first water and is changed altogether distilled water 8 times, then bag filter being put into to the beaker that polyethylene glycol 6000 is housed is concentrated, until 50 ml lipopolysaccharide solution in bag filter are concentrated to 7.5 milliliters of concentrated solutions, described in bag filter, 7.5 milliliters of concentrated solutions are the lipopolysaccharide concentrated solution;
6. carry out DNA and the RNA in enzymolysis lipopolysaccharide concentrated solution with DNA enzyme and RNA enzyme
Above-mentioned described 7.5 ml lipopolysaccharide concentrated solutions in are 5. poured in the centrifuge tube of 10 milliliters, then to the DNA enzyme and the RNA enzyme that add respectively 375 micrograms in these 10 milliliters of centrifuge tubes; These 10 milliliters of centrifuge tubes are placed in 37 ℃ of water-baths and place 5 hours, again these 10 milliliters of centrifuge tubes are placed in 100 ℃ of water-baths and place 10 minutes, place after 10 minutes and take out these 10 milliliters of centrifuge tubes and be cooled to room temperature from 100 ℃ of water-baths, be placed on centrifuge by these 10 milliliters of centrifuge tubes at ambient temperature and under the rotating speed of 3000 rev/mins centrifugal 30 minutes, draw and pour in another centrifuge tube of 100 milliliters standby by the supernatant in these 10 milliliters of centrifuge tubes after centrifugal 30 minutes;
7. the lipopolysaccharide for preparing purification
To adding volume in another above-mentioned 6. standby 100 milliliters of centrifuge tubes, it is the dehydrated alcohol of 6 times of its above-mentioned 6. described supernatant liquid with the NaOH adjusting pH value to 9.0 of 6 mol/L, again this another 100 milliliters of centrifuge tubes are placed 12 hours 4 ℃ the time, then on centrifuge and under the rotating speed of 6000 rev/mins centrifugal 15 minutes, after centrifugal 15 minutes, the liquid in this another 100 milliliters of centrifuge tubes is outwelled, is stayed the lipopolysaccharide that precipitate in this another 100 milliliters of centrifuge tubes is prepared purification;
8. the lipopolysaccharide of purification is made to teleoptile pasteurella multocida lipopolysaccharide vaccine
In above-mentioned 7. another 100 milliliters of centrifuge tubes, in the lipopolysaccharide of prepared purification, add the ultra-pure water of 1 milliliter to be dissolved and obtain the lipopolysaccharide solution of high concentration, measure the concentration of described lipopolysaccharide solution with anthrone method, till slowly adding ultra-pure water to the concentration of described lipopolysaccharide solution to reach 0.5 mg/ml;
In the described lipopolysaccharide solution that is 0.5 mg/ml to above-mentioned concentration, pour in 50 ml beakers, to adding volume in this 50 ml beaker, be the Freund's complete adjuvant of 1.5 times of described lipopolysaccharide liquor capacities again, put into a magnetic bar simultaneously in this 50 ml beaker and stir 1 hour under the magnetic stirring apparatus effect, stirring the solution in this 50 ml beaker after 1 hour is exactly the fowl pasteurella multocida lipopolysaccharide vaccine of making.
Include 1.5% tryptone, 0.5% soy peptone, 0.5% sodium chloride, 2% agar and a small amount of water on described culture plate.
Described fluid medium includes 1.5% tryptone, 0.5% soy peptone, 0.5% sodium chloride and a small amount of water.
Owing to adopting technical scheme as above, the present invention has following superiority:
1, the present invention is when carrying out fragmentation with ultrasound wave to fowl pasteurella multocida thalline, the power of ultrasonic disruption instrument used is 500 watts, every Ultrasonic Radiation stops radiation after 20 seconds 20 seconds be a broken cyclic process, in this broken cyclic process, radiation after 20 seconds due to the formed void effect of ultrasound wave, can make fowl pasteurella multocida thalline break, the ultrasonic disruption instrument that can make in 20 seconds that stops radiation being repaired.Reach the pasteurella multocida of the fowl through the ultrasonic disruption thalline that can obtain maximum under the prerequisite of not damaging the ultrasonic disruption instrument after all broken cyclic processes in 30 minutes.
When 2, the present invention slightly carries lipopolysaccharide solution from the fowl pasteurella multocida thalline of ultrasonic disruption, phenol concentration used is 90%, when the phenol that is 90% with concentration of the fowl pasteurella multocida through ultrasonic disruption mixes and is heated to 70 ℃ and during by the Glass rod vigorous stirring, phenol and lipopolysaccharide solution have formed single-phase, have formed phenol phase and water after cooling; The lipopolysaccharide major part is dissolved in water, and most of protein be dissolved in phenol mutually in, insoluble substance is deposited in biphase intersection, through this method, can obtain the fowl pasteurella multocida lipopolysaccharide solution of slightly carrying of maximum.
When 3, the present invention extracts the lipopolysaccharide concentrated solution from the lipopolysaccharide solution of slightly carrying, by the fowl pasteurella multocida lipopolysaccharide solution of slightly carrying, all join in bag filter, bag filter is placed in tap water flowing water to continual rinsing 48 hours, again bag filter is put in distilled water and soaked, immersion in every 6 hours is changed first water and is changed altogether distilled water 8 times, then bag filter is put into to the beaker that polyethylene glycol 6000 is housed and concentrated, this method can obtain the fowl pasteurella multocida lipopolysaccharide solution of high concentration.
When 4, the present invention comes DNA in enzymolysis lipopolysaccharide concentrated solution and RNA with DNA enzyme and RNA enzyme, can make by the effect of DNA enzyme and RNA enzyme DNA and RNA in fowl pasteurella multocida lipopolysaccharide concentrated solution be able to enzymolysis, to remove DNA and the RNA be contained in the lipopolysaccharide concentrated solution, obtain more highly purified fowl pasteurella multocida lipopolysaccharide solution.
When 5, the present invention prepares the lipopolysaccharide of purification, to adding volume in another above-mentioned 6. standby 100 milliliters of centrifuge tubes, it is the dehydrated alcohol of 6 times of its above-mentioned 6. described supernatant liquid with the NaOH adjusting pH value to 9.0 of 6 mol/L, again this another 100 milliliters of centrifuge tubes are placed 12 hours 4 ℃ the time, can make in this way fowl pasteurella multocida lipopolysaccharide precipitate fully, obtain the lipopolysaccharide of the purification of maximum.
6, the present invention is when just the lipopolysaccharide of purification is made teleoptile pasteurella multocida lipopolysaccharide vaccine, the lipopolysaccharide solution of extraction and Freund's complete adjuvant are mixed and made into to fowl pasteurella multocida disease lipopolysaccharide vaccine according to the ratio of 1 ︰ 1.5, Freund's complete adjuvant is containing the cell wall constituent of mycobacterium tuberculosis, a kind of water in oil emulsion, can strengthen the antibody response to antigen, can very effectively induce the antibody that produces high titre, the Freund's complete adjuvant activity comes from the lipopolysaccharide vaccine of preparation immunogenic sustained release and stimulates the local immunity reaction, the fowl pasteurella multocida lipopolysaccharide vaccine of preparation can strengthen immune effect in this way.
The specific embodiment
The present invention is a kind of method of extracting lipopolysaccharide and making the lipopolysaccharide vaccine from the fowl pasteurella multocida, 1. the method through preparing fowl pasteurella multocida culture fluid, 2. collect fowl pasteurella multocida thalline from fowl pasteurella multocida culture fluid, 3. the fowl pasteurella multocida thalline of ultrasonic disruption, 4. slightly carry lipopolysaccharide solution from the fowl pasteurella multocida thalline of ultrasonic disruption, 5. extract the lipopolysaccharide concentrated solution from the lipopolysaccharide solution of slightly carrying, 6. carry out DNA and the RNA in enzymolysis lipopolysaccharide concentrated solution with DNA enzyme and RNA enzyme, 7. prepare the lipopolysaccharide of purification and 8. the lipopolysaccharide of purification is made to teleoptile pasteurella multocida lipopolysaccharide vaccine totally eight steps.
Related fowl pasteurella multocida CVCC474(in the inventive method
avian Pasteurella multocidacVCC474) store and provide by China Veterinery Drug Inspection Office.
Described in above-mentioned eight steps 2. in relate to the centrifugal rotational speed of centrifuge, describedly relate to hyperacoustic power in 3., radiated time, intermittently number of seconds and radiation number of times, described relating in 5. changed the waterside number, describedly relate to enzymolysis time in 6., it is described that to relate to by concentration in 7. be the pH value that 6 mol/L NaOH carry out regulator solution, the described volume ratio that relates to lipopolysaccharide solution and Freund's complete adjuvant in 8. is the normal experiment technological means, any one in these normal experiment technological means perhaps exists as independent experiment condition, but these normal experiment technological means integrated applications are essential among method of the present invention.
Eight steps of the present invention are described below:
1. prepare fowl pasteurella multocida culture fluid
Use the inoculating loop scraping
one ringthe fowl pasteurella multocida of slant preservation, will scrape
one ringthe inoculating loop of fowl pasteurella multocida with the streak inoculation of " Z " font on culture plate, culture plate is positioned in 37 ℃ of incubators and cultivates 24 hours, a single colony inoculation of growing on the culture plate of picking cultivation after 24 hours is in the fluid medium of 5 milliliters, this fluid medium of 5 milliliters being placed on to the velocity fluctuation with 160 rev/mins in the shaking table of 37 ℃ cultivates 24 hours, again 5 milliliters of liquid culture medium of shaken cultivation all poured in the fluid medium of 200 milliliters and cultivated and within 24 hours, prepare fowl pasteurella multocida culture fluid with the velocity fluctuation of 160 rev/mins in the shaking table of 37 ℃.
It should be noted that:
Include 1.5% tryptone, 0.5% soy peptone, 0.5% sodium chloride, 2% agar and a small amount of water on described culture plate.
Described fluid medium includes 1.5% tryptone, 0.5% soy peptone, 0.5% sodium chloride and a small amount of water.Above-mentioned 1. in the fluid medium of twice use identical.
2. collect fowl pasteurella multocida thalline from fowl pasteurella multocida culture fluid
The above-mentioned fowl pasteurella multocida culture fluid of 1. preparing is joined in the centrifuge tube of 250 milliliters, to add these 250 milliliters of centrifuge tubes of fowl pasteurella multocida culture fluid to be placed on centrifuge and under the rotating speed of 5000 rev/mins centrifugal 15 minutes, then the supernatant in 250 milliliters of centrifuge tubes after centrifugal 15 minutes is discarded, the precipitate of staying in these 250 milliliters of centrifuge tubes is exactly collected fowl pasteurella multocida thalline, for next step ultrasonic disruption is prepared.
3. ultrasound wave carries out fragmentation to fowl pasteurella multocida thalline
At the above-mentioned distilled water that adds 10 milliliters in 2. in the collected fowl pasteurella multocida thalline of 250 milliliters of centrifuge tubes, the distilled water of 10 milliliters can suspend fowl pasteurella multocida thalline to get up in these 250 milliliters of centrifuge tubes, then these 250 milliliters of centrifuge tubes are placed in to the ultrasonic disruption instrument, the power that the ultrasonic disruption instrument is set is 500 watts, every Ultrasonic Radiation stops radiation after 20 seconds 20 seconds be a broken cyclic process, all broken cyclic processes continue 30 minutes altogether, what after 30 minutes, in these 250 milliliters of centrifuge tubes, obtain is exactly the fowl pasteurella multocida thalline through ultrasonic disruption, the purpose of this step is, by ultrasonic disruption, fowl pasteurella multocida thalline is carried out to cracking, make as much as possible the discharging of lipopolysaccharide wherein.
4. slightly carry lipopolysaccharide solution from the fowl pasteurella multocida thalline of ultrasonic disruption
Slightly carry lipopolysaccharide and be divided into three steps from fowl pasteurella multocida thalline, three steps are described below:
The first step, extract above-mentioned 10 milliliters of fowl pasteurella multocida thalline that obtain through ultrasonic disruption in 3. and join in 50 milliliters of centrifuge tubes, and then add the phenol that 10 milliliters of its concentration are 90% in these 50 milliliters of centrifuge tubes, putting upside down these 50 milliliters of centrifuge tubes several (at least 3 times) fully mixes 10 milliliters of fowl pasteurella multocida thalline and 10 milliliters of phenol, 50 milliliters of centrifuge tubes of this that then will fully mix are placed in the water-bath of 70 ℃ and heat and use the Glass rod rapid stirring 30 minutes, take out these 50 milliliters of centrifuge tubes after 30 minutes and at room temperature place 20 minutes from water-bath, by these 50 milliliters of centrifuge tubes, be placed on centrifuge again and under the rotating speed of 4000 rev/mins centrifugal 30 minutes, after centrifugal end, the upper solution of these 50 milliliters of centrifuge tubes is drawn and is joined in another triangular flask of 50 milliliters, middle level solution in these 50 milliliters of centrifuge tubes discards not, last milliliter stand-by to adding in the lower floor's solution in these 50 milliliters of centrifuge tubes distilled water to supply volume to 10 again.
Second step, add the phenol that 10 milliliters of its concentration are 90% in the lower floor's solution+distilled water in these 50 milliliters of centrifuge tubes stand-by in the first step, putting upside down these 50 milliliters of centrifuge tubes several (at least 3 times) fully mixes it, after fully mixing, these 50 milliliters of centrifuge tubes are placed in the water-bath of 70 ℃ and heat and use the Glass rod rapid stirring 30 minutes, take out these 50 milliliters of centrifuge tubes after 30 minutes and at room temperature place 20 minutes from water-bath, by these 50 milliliters of centrifuge tubes, be placed on centrifuge again and under the rotating speed of 4000 rev/mins centrifugal 30 minutes, the upper solution of these 50 milliliters of centrifuge tubes is drawn and joined in another 50 milliliters the triangular flask in the first step after centrifugal end and store, middle level solution in these 50 milliliters of centrifuge tubes discards not, last to adding distilled water to supply volume to 10 a milliliter preparation in the lower floor's solution in these 50 milliliters of centrifuge tubes, reuse again.
The 3rd step, repeat second step for several times until, by another triangular flask of 50 milliliters storage full scale, the described upper solution in another 50 milliliters of triangular flasks that storage is full 50 milliliters is exactly the lipopolysaccharide solution of slightly carrying from the fowl pasteurella multocida thalline of ultrasonic disruption.
The 3rd step of this step will constantly repeat until, by another triangular flask of 50 milliliters storage full scale, can slightly carry more lipopolysaccharide solution, also for next step extraction lipopolysaccharide concentrated solution, to prepare.
5. extract the lipopolysaccharide concentrated solution from the lipopolysaccharide solution of slightly carrying
By the 50 ml lipopolysaccharide solution of slightly carrying, all join in bag filter, bag filter is placed in tap water flowing water to continual rinsing 48 hours, again bag filter is put in distilled water and soaked, immersion in every 6 hours is changed first water and is changed altogether distilled water 8 times, then bag filter being put into to the beaker that polyethylene glycol 6000 is housed is concentrated, until 50 ml lipopolysaccharide solution in bag filter are concentrated to 7.5 milliliters of concentrated solutions, described in bag filter, 7.5 milliliters of concentrated solutions are the lipopolysaccharide concentrated solution, and this step can improve the concentrated solution concentration of lipopolysaccharide by concentration process.Contain minim DNA and RNA in described lipopolysaccharide concentrated solution.
6. carry out DNA and the RNA in enzymolysis lipopolysaccharide concentrated solution with DNA enzyme and RNA enzyme
Above-mentioned described 7.5 ml lipopolysaccharide concentrated solutions in are 5. poured in the centrifuge tube of 10 milliliters, again to the DNA enzyme and the RNA enzyme that add respectively 375 micrograms in these 10 milliliters of centrifuge tubes, can make to add respectively minim DNA and the RNA that the DNA enzyme of 375 micrograms and the concentration of RNA enzyme in described 7.5 ml lipopolysaccharide concentrated solutions contain in being controlled at 50 ug/ml left and right and making described lipopolysaccharide concentrated solution to be able to enzymolysis.These 10 milliliters of centrifuge tubes are placed in 37 ℃ of water-baths and place 5 hours, again these 10 milliliters of centrifuge tubes are placed in 100 ℃ of water-baths and place 10 minutes, place after 10 minutes and take out these 10 milliliters of centrifuge tubes and be cooled to room temperature from 100 ℃ of water-baths, be placed on centrifuge by these 10 milliliters of centrifuge tubes at ambient temperature and under the rotating speed of 3000 rev/mins centrifugal 30 minutes, draw and pour in another centrifuge tube of 100 milliliters standby by the supernatant in these 10 milliliters of centrifuge tubes after centrifugal 30 minutes.
7. the lipopolysaccharide for preparing purification
To adding volume in another above-mentioned 6. standby 100 milliliters of centrifuge tubes, it is the dehydrated alcohol of 6 times of its above-mentioned 6. described supernatant liquid with the NaOH adjusting pH value to 9.0 of 6 mol/L, again this another 100 milliliters of centrifuge tubes are placed 12 hours 4 ℃ the time, then on centrifuge and under the rotating speed of 6000 rev/mins centrifugal 15 minutes, after centrifugal 15 minutes, the liquid in this another 100 milliliters of centrifuge tubes is outwelled, is stayed the lipopolysaccharide that precipitate in this another 100 milliliters of centrifuge tubes is prepared purification.
8. the lipopolysaccharide of purification is made to teleoptile pasteurella multocida lipopolysaccharide vaccine
In above-mentioned 7. another 100 milliliters of centrifuge tubes, in the lipopolysaccharide of prepared purification, add the ultra-pure water of 1 milliliter to be dissolved and obtain the lipopolysaccharide solution of high concentration, measure the concentration of described lipopolysaccharide solution with anthrone method, till slowly adding ultra-pure water to the concentration of described lipopolysaccharide solution to reach 0.5 mg/ml, now the described lipopolysaccharide solution in another 100 milliliters of centrifuge tubes is approximately 10 milliliters of left and right.
In the described lipopolysaccharide solution that is 0.5 mg/ml to above-mentioned concentration, pour in 50 ml beakers, to adding volume in this 50 ml beaker, be the Freund's complete adjuvant of 1.5 times of described lipopolysaccharide liquor capacities again, put into a magnetic bar simultaneously in this 50 ml beaker and stir 1 hour under the magnetic stirring apparatus effect, stirring the solution in this 50 ml beaker after 1 hour is exactly the fowl pasteurella multocida lipopolysaccharide vaccine of making, described fowl pasteurella multocida lipopolysaccharide vaccine is about 25 milliliters, lipopolysaccharide content in 25 milliliters of described fowl pasteurella multocida lipopolysaccharide vaccines is about 0.2 mg/ml.Due to the experiment environment, condition, means and mode of operation difference, the described fowl pasteurella multocida lipopolysaccharide vaccine of making or >=25 milliliters, or<25 milliliters, described 25 milliliters are not determined value, only for reference.
The inoculating loop used in eight steps of the present invention, culture plate, incubator, shaking table, centrifuge, ultrasonic disruption instrument, water-bath, bag filter, beaker, triangular flask, magnetic bar, Glass rod etc. are the common experimental apparatus of biology laboratory and material, all can buy in bio-engineering corporation.
The tryptone used in eight steps of the present invention, soy peptone, distilled water, phenol, polyethylene glycol 6000, DNA enzyme, RNA enzyme, dehydrated alcohol, NaOH, ultra-pure water, anthrone, sulphuric acid, Freund's complete adjuvant etc. are the biology laboratory general reagent, all can buy in chemical reagents corporation or bio-engineering corporation.
Tryptone used in the present invention, soy peptone, phenol, polyethylene glycol 6000, sodium hydroxide, dehydrated alcohol, the DNA enzyme, the RNA enzyme, Freund's complete adjuvant, anthrone, sulphuric acid, bag filters etc. all can buy in chemical reagents corporation or bio-engineering corporation.
The relevant immunoprophylaxis effect of extracting lipopolysaccharide and make fowl pasteurella multocida lipopolysaccharide vaccine from the fowl pasteurella multocida is with reference to as follows:
The qualifications of animal immune experiment and the experiment of animal counteracting toxic substances is: the lipopolysaccharide content in fowl pasteurella multocida lipopolysaccharide vaccine is 0.2 mg/ml.
1, animal immune experiment:
It is chicken that animal immune is tested experiment thing used, get not raised through immune chicken of 30 1 ages in days, while Deng 30 chickens, growing to 4 week age, they are divided into to two groups at random, every group of 15 chickens, the fowl pasteurella multocida disease lipopolysaccharide vaccine that in wherein one group prepared by all equal immune the present invention of 15 chickens, this group is customized for to the lipopolysaccharide immune group, the chicken of this immune group all immune three times, when the time of immunity is 4 week age for the first time, when the time of immunity is 6 week age for the second time, when the time of immunity is 8 week age for the third time, the dosage of each immunity is 1 milliliter/every chicken, immunization ways is subcutaneous injection, 15 chickens of other one group do not carry out any immunity, just with the chicken of lipopolysaccharide immune group, under identical condition, raised, this group is customized for to nonimmune group.
2, animal counteracting toxic substances experiment:
Can the purpose of this experiment be to prepare fowl pasteurella multocida disease lipopolysaccharide vaccine and provide the opposing ability that the fowl pasteurella multocida is attacked to chicken in order to detect the present invention, and concrete operation method is as follows:
With fowl pasteurella multocida CVCC474 bacterial strain, all chickens in above-mentioned lipopolysaccharide vaccine immunity group and nonimmune group are all carried out to the intramuscular injection counteracting toxic substances, dosage during counteracting toxic substances is 1 * 10
4cVCC474/ chicken of individual fowl pasteurella multocida; all chickens in lipopolysaccharide immune group and nonimmune group are continued to raise 2 weeks after counteracting toxic substances; record the number of the chicken still survived in two groups after 2 weeks; and calculate the protective rate of two groups; the higher explanation immunoprophylaxis of protective rate effect is better, and the computational methods of the protective rate of two groups are as follows:
(the quantity ÷ 15 of the chicken that counteracting toxic substances was still survived after 2 weeks) * 100%
Result is as shown in table 1; chicken in counteracting toxic substances lipopolysaccharide immune group after 2 weeks still has 10 survivals; the protective rate that fowl pasteurella multocida disease lipopolysaccharide vaccine is described is 66.7%, and the chicken of nonimmune group is all dead after 2 weeks at counteracting toxic substances, and the protective rate that illustrates nonimmune group is 0%.
The above results shows: the fowl pasteurella multocida disease lipopolysaccharide vaccine that utilizes the present invention to prepare carries out making effectively to be prevented the fowl pasteurella multocida disease by immune chicken after three immunity to chicken.
table 1 counteracting toxic substances is still quantity and the protection of the chicken of survival after two weeks
| Group | The quantity of the chicken that counteracting toxic substances was still survived after two weeks | Protective rate (%) |
| The lipopolysaccharide immune group | 10 | 66.7% |
| Nonimmune group | 0 | 0% |
When the qualifications of animal immune experiment and the experiment of animal counteracting toxic substances changes to some extent, its immunizing dose also will be followed to some extent and change.
Although method of the present invention is that chamber makes fowl pasteurella multocida lipopolysaccharide vaccine by experiment; but the method according to this invention equally can be for producing fowl pasteurella multocida lipopolysaccharide vaccine in batches, and batch production fowl pasteurella multocida lipopolysaccharide vaccine and the using method (immunizing dose) provided according to this description belong to protection scope of the present invention equally.
Claims (1)
1. a method of extracting lipopolysaccharide and making the lipopolysaccharide vaccine from the fowl pasteurella multocida, 1. the method through preparing fowl pasteurella multocida culture fluid, 2. collect fowl pasteurella multocida thalline from fowl pasteurella multocida culture fluid, 3. ultrasound wave carries out fragmentation to fowl pasteurella multocida thalline, 4. slightly carry lipopolysaccharide solution from the fowl pasteurella multocida thalline of ultrasonic disruption, 5. extract the lipopolysaccharide concentrated solution from the lipopolysaccharide solution of slightly carrying, 6. carry out DNA and the RNA in enzymolysis lipopolysaccharide concentrated solution with DNA enzyme and RNA enzyme, 7. prepare the lipopolysaccharide of purification and 8. the lipopolysaccharide of purification is made to teleoptile pasteurella multocida lipopolysaccharide vaccine totally eight steps, it is characterized in that: described eight steps are described below:
1. prepare fowl pasteurella multocida culture fluid
Use the inoculating loop scraping
one ringthe fowl pasteurella multocida of slant preservation, will scrape
one ringthe inoculating loop of fowl pasteurella multocida with the streak inoculation of " Z " font on culture plate, culture plate is positioned in 37 ℃ of incubators and cultivates 24 hours, a single colony inoculation of growing on the culture plate of picking cultivation after 24 hours is in the fluid medium of 5 milliliters, this fluid medium of 5 milliliters being placed on to the velocity fluctuation with 160 rev/mins in the shaking table of 37 ℃ cultivates 24 hours, again 5 milliliters of liquid culture medium of shaken cultivation all poured in the fluid medium of 200 milliliters and cultivated and within 24 hours, prepare fowl pasteurella multocida culture fluid with the velocity fluctuation of 160 rev/mins in the shaking table of 37 ℃,
2. collect fowl pasteurella multocida thalline from fowl pasteurella multocida culture fluid
The above-mentioned fowl pasteurella multocida culture fluid of 1. preparing is joined in the centrifuge tube of 250 milliliters, to add these 250 milliliters of centrifuge tubes of fowl pasteurella multocida culture fluid to be placed on centrifuge and under the rotating speed of 5000 rev/mins centrifugal 15 minutes, then the supernatant in 250 milliliters of centrifuge tubes after centrifugal 15 minutes is discarded, the precipitate of staying in these 250 milliliters of centrifuge tubes is exactly collected fowl pasteurella multocida thalline;
3. ultrasound wave carries out fragmentation to fowl pasteurella multocida thalline
At the above-mentioned distilled water that adds 10 milliliters in 2. in the collected fowl pasteurella multocida thalline of 250 milliliters of centrifuge tubes, then these 250 milliliters of centrifuge tubes are placed in to the ultrasonic disruption instrument, the power that the ultrasonic disruption instrument is set is 500 watts, every Ultrasonic Radiation stops radiation after 20 seconds 20 seconds be a broken cyclic process, all broken cyclic processes continue 30 minutes altogether, and what after 30 minutes, in these 250 milliliters of centrifuge tubes, obtain is exactly the fowl pasteurella multocida thalline through ultrasonic disruption;
4. slightly carry lipopolysaccharide solution from the fowl pasteurella multocida thalline of ultrasonic disruption
Slightly carry lipopolysaccharide and be divided into three steps from fowl pasteurella multocida thalline, three steps are described below:
The first step, extract above-mentioned 10 milliliters of fowl pasteurella multocida thalline that obtain through ultrasonic disruption in 3. and join in 50 milliliters of centrifuge tubes, and then add the phenol that 10 milliliters of its concentration are 90% in these 50 milliliters of centrifuge tubes, putting upside down these 50 milliliters of centrifuge tubes makes 10 milliliters of fowl pasteurella multocida thalline and 10 milliliters of phenol fully mix for several times, 50 milliliters of centrifuge tubes of this that then will fully mix are placed in the water-bath of 70 ℃ and heat and use the Glass rod rapid stirring 30 minutes, take out these 50 milliliters of centrifuge tubes after 30 minutes and at room temperature place 20 minutes from water-bath, by these 50 milliliters of centrifuge tubes, be placed on centrifuge again and under the rotating speed of 4000 rev/mins centrifugal 30 minutes, after centrifugal end, the upper solution of these 50 milliliters of centrifuge tubes is drawn and is joined in another triangular flask of 50 milliliters, middle level solution in these 50 milliliters of centrifuge tubes discards not, last milliliter stand-by to adding in the lower floor's solution in these 50 milliliters of centrifuge tubes distilled water to supply volume to 10 again,
Second step, add the phenol that 10 milliliters of its concentration are 90% in the lower floor's solution+distilled water in these 50 milliliters of centrifuge tubes stand-by in the first step, putting upside down these 50 milliliters of centrifuge tubes makes it fully mix for several times, after fully mixing, these 50 milliliters of centrifuge tubes are placed in the water-bath of 70 ℃ and heat and use the Glass rod rapid stirring 30 minutes, take out these 50 milliliters of centrifuge tubes after 30 minutes and at room temperature place 20 minutes from water-bath, by these 50 milliliters of centrifuge tubes, be placed on centrifuge again and under the rotating speed of 4000 rev/mins centrifugal 30 minutes, the upper solution of these 50 milliliters of centrifuge tubes is drawn and joined in another 50 milliliters the triangular flask in the first step after centrifugal end and store, middle level solution in these 50 milliliters of centrifuge tubes discards not, last to adding distilled water to supply volume to 10 a milliliter preparation in the lower floor's solution in these 50 milliliters of centrifuge tubes, reuse again,
The 3rd step, repeat second step for several times until, by another triangular flask of 50 milliliters storage full scale, the described upper solution in another 50 milliliters of triangular flasks that storage is full 50 milliliters is exactly the lipopolysaccharide solution of slightly carrying from the fowl pasteurella multocida thalline of ultrasonic disruption;
5. extract the lipopolysaccharide concentrated solution from the lipopolysaccharide solution of slightly carrying
By the 50 ml lipopolysaccharide solution of slightly carrying, all join in bag filter, bag filter is placed in tap water flowing water to continual rinsing 48 hours, again bag filter is put in distilled water and soaked, immersion in every 6 hours is changed first water and is changed altogether distilled water 8 times, then bag filter being put into to the beaker that polyethylene glycol 6000 is housed is concentrated, until 50 ml lipopolysaccharide solution in bag filter are concentrated to 7.5 milliliters of concentrated solutions, described in bag filter, 7.5 milliliters of concentrated solutions are the lipopolysaccharide concentrated solution;
6. carry out DNA and the RNA in enzymolysis lipopolysaccharide concentrated solution with DNA enzyme and RNA enzyme
Above-mentioned described 7.5 ml lipopolysaccharide concentrated solutions in are 5. poured in the centrifuge tube of 10 milliliters, then to the DNA enzyme and the RNA enzyme that add respectively 375 micrograms in these 10 milliliters of centrifuge tubes; These 10 milliliters of centrifuge tubes are placed in 37 ℃ of water-baths and place 5 hours, again these 10 milliliters of centrifuge tubes are placed in 100 ℃ of water-baths and place 10 minutes, place after 10 minutes and take out these 10 milliliters of centrifuge tubes and be cooled to room temperature from 100 ℃ of water-baths, be placed on centrifuge by these 10 milliliters of centrifuge tubes at ambient temperature and under the rotating speed of 3000 rev/mins centrifugal 30 minutes, draw and pour in another centrifuge tube of 100 milliliters standby by the supernatant in these 10 milliliters of centrifuge tubes after centrifugal 30 minutes;
7. the lipopolysaccharide for preparing purification
To adding volume in another above-mentioned 6. standby 100 milliliters of centrifuge tubes, it is the dehydrated alcohol of 6 times of its above-mentioned 6. described supernatant liquid with the NaOH adjusting pH value to 9.0 of 6 mol/L, again this another 100 milliliters of centrifuge tubes are placed 12 hours 4 ℃ the time, then on centrifuge and under the rotating speed of 6000 rev/mins centrifugal 15 minutes, after centrifugal 15 minutes, the liquid in this another 100 milliliters of centrifuge tubes is outwelled, is stayed the lipopolysaccharide that precipitate in this another 100 milliliters of centrifuge tubes is prepared purification;
8. the lipopolysaccharide of purification is made to teleoptile pasteurella multocida lipopolysaccharide vaccine
In above-mentioned 7. another 100 milliliters of centrifuge tubes, in the lipopolysaccharide of prepared purification, add the ultra-pure water of 1 milliliter to be dissolved and obtain the lipopolysaccharide solution of high concentration, measure the concentration of described lipopolysaccharide solution with anthrone method, till slowly adding ultra-pure water to the concentration of described lipopolysaccharide solution to reach 0.5 mg/ml;
In the described lipopolysaccharide solution that is 0.5 mg/ml to above-mentioned concentration, pour in 50 ml beakers, to adding volume in this 50 ml beaker, be the Freund's complete adjuvant of 1.5 times of described lipopolysaccharide liquor capacities again, put into a magnetic bar simultaneously in this 50 ml beaker and stir 1 hour under the magnetic stirring apparatus effect, stirring the solution in this 50 ml beaker after 1 hour is exactly the fowl pasteurella multocida lipopolysaccharide vaccine of making.
2, the method for extracting lipopolysaccharide and making the lipopolysaccharide vaccine from the fowl pasteurella multocida as claimed in claim 1 is characterized in that: include 1.5% tryptone, 0.5% soy peptone, 0.5% sodium chloride, 2% agar and a small amount of water on described culture plate.
3, the method for extracting lipopolysaccharide and making the lipopolysaccharide vaccine from the fowl pasteurella multocida as claimed in claim 1 is characterized in that: described fluid medium includes 1.5% tryptone, 0.5% soy peptone, 0.5% sodium chloride and a small amount of water.
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