CN102168073A - Method for improving proliferation capability of mycoplasma gallisepticum in vaccine production - Google Patents
Method for improving proliferation capability of mycoplasma gallisepticum in vaccine production Download PDFInfo
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Abstract
The invention relates to a method for improving proliferation capability of mycoplasma gallisepticum in vaccine production, vaccines are prepared through the process steps of preparing seeds, preparing a culture medium of the mycoplasma gallisepticum, preparing vaccine-making bacterial liquid and preparing the inactivated vaccines, after improvement, the content of PPLO (pleuropneumonia-like organism) broth, porcine serum and glucose is increased, MEM (minimum essential medium) ingredients are removed, and the mixture ratio of the ingredients in the culture medium are more scientific and the nutritional value is higher after adjustment. The bacterial liquid concentration of a semi-finished product prepared by the method is as high as 1.0*1013CCU/ml-1.0*1014CCU/ml, and the bacterial liquid does not need to be concentrated and can be directly or indirectly used for preparing the inactivated vaccines after dilution, thereby not only simplifying the production process, but also reducing the production cost.
Description
Technical field
The present invention relates to a kind of method of improved raising chicken virus mycoplasma multiplication capacity, specifically, is a kind of chicken virus mycoplasma production of vaccine that is applicable to, is used to improve the cultural method of chicken virus mycoplasma multiplication capacity.Belong to the biological medicine technology field.
Background technology
The chicken virus mycoplasma disease be the chicken that causes by chicken virus mycoplasma (Mycoplasma gallisepticum, be called for short MG), turkey chronic respiratory tract disease (Chronicrespiratory disease, CRD).Mainly show as cough clinically, have a running nose, send rale during breathing, mouth breathing when serious.Scale strengthens along with raising chickens, feeding manner changes and stocking density improves, and this sick sickness rate is more and more higher.According to statistics, behind the mycoplasma gallisepticum infection chicken group, the weak young rate of chick increases about 10%, the laying rate of laying hen descends 10～20%, fryer lose weight 38%, marketing period prolongs, feed conversion rate reduces, chicken infected immunity is suppressed and causes the secondary of other disease or occur together, and can cause a large amount of medication expenses indirectly, is one of important diseases of harm poultry husbandry.At present the control of mycoplasma infection mainly is the foundation of pharmacological agent, vaccine prevention and healthy drove.CRD is effective though heal with medicine, and can not remove carrier state, and the disadvantage of drug residue is arranged again; Effectively vaccine immunity is the important means that prevention and control should disease, the very important field of vaccine immunity research becoming.
In the chicken virus mycoplasma production of vaccine, cultivate the main using modified FreyShi substratum of chicken virus mycoplasma or other mycoplasma culture mediums at present.Its seedling with bacterium liquid culture process is: qualified chicken virus mycoplasma production is inoculated in substratum with seed by 10% amount, and enlarged culturing progressively is until required amount; Every Dai Jun puts 36～37 ℃ and cultivates 24～48h, treats that the PH of substratum drops to about 7.0 results; Can only reach 1.0 * 109CCU/ml by chicken virus mycoplasma seedling that this traditional culture process obtained with the bacterial concentration of bacterium liquid (work in-process) is the highest.
Poor with the chicken virus mycoplasma multiplication capacity that above-mentioned traditional substratum and culture process are cultivated, cultivate titre low (it is low to cultivate bacterial concentration); when producing inactivated vaccine with bacterium liquid (work in-process), seedling can not directly use; can be prepared into the product vaccine after need carrying out necessarily concentrating to work in-process; and half-finished concentration operation is loaded down with trivial details, the concentration process loss is bigger; the final protection that influences the finished product vaccine is renderd a service, and the production of vaccine cost is improved.
Summary of the invention
Problem to be solved by this invention provides a kind of method that improves chicken virus mycoplasma multiplication capacity in the production of vaccine, in order to improve chicken virus mycoplasma multiplication capacity and bacterium drop degree.
Problem of the present invention solves with following technical proposals:
A kind of method that improves chicken virus mycoplasma multiplication capacity in the production of vaccine, described vaccine is made through seed preparation, the preparation of chicken virus mycoplasma substratum, the preparation of seedling bacterium liquid and inactivated vaccine preparation section, after the improvement, described chicken virus mycoplasma substratum is prepared by following technology:
PPLO meat soup 23.0～26.0g,
Glucose 8.0～10.0g,
Mass concentration is 1% thaliium acetate solution 10ml,
Mass concentration is 25% yeast leach liquor 100ml,
Mass concentration is phenol red solution 1.0～2.0ml of 1%,
With deionized water constant volume to 790～840ml,, after cooling, under aseptic condition, add following composition at 115 ℃ of sterilization 20min:
Mass concentration is 10% arginine solution (filtration sterilization) 10ml,
Porcine blood serum 150～200ml,
Penicillin 80～1,200,000 units,
Transfer pH value to 7.6～7.8 with sodium hydroxide solution.
The method of chicken virus mycoplasma multiplication capacity in the above-mentioned raising production of vaccine, described seedling are cultivated with bacterium liquid and are carried out as follows:
(1) chicken virus mycoplasma production is inoculated in the described mycoplasma culture medium by 10% volume with seed, cultivates 16～18h results at 36～37 ℃; Choose 10% of the long-pending amount of results bacteria liquid, with the volume substratum, go down to posterity again 1 time with quadrat method;
(2) the bacterium liquid after above-mentioned the going down to posterity is inoculated in mycoplasma culture medium of the present invention by 10% amount, cultivates 24～48h for 36～37 ℃, treat that the PH of substratum drops to about 7.0 results.According to this method enlarged culturing progressively, until required amount, this bacterium liquid promptly can be used for preparing vaccine as work in-process.
The present invention carries out viable count with above-mentioned work in-process bacterium liquid and measures, and the chicken virus mycoplasma bacterial concentration of acquisition can reach 1.0 * 1013～CCU/ml～1.0 * 1014CCU/ml.
The present invention is on the basis of traditional chicken virus mycoplasma cultural method, improved substratum, improved culture process, improved the chicken virus mycoplasma multiplication capacity and bacterium drop degree (has been brought up to 1.0 present * 1013CCU/ml～1.0 * 1014CCU/ml) by 1.0 * 108 original～9CCU/ml, the seedling of cultivating can directly or prepare the finished product vaccine with bacterium liquid after the dilution, exempted concentration technology, it is poor to have solved traditional method cultivation chicken virus mycoplasma multiplication capacity, bacterium drop degree is low, problems such as concentrated cost height, complex operation.
The present invention the experiment proved that following advantage is arranged:
1, the present invention cultivates chicken virus mycoplasma with the mycoplasma culture medium and the improved multiplication culture technology of improvement, has improved the multiplication capacity and the bacterium drop degree of thalline, and bacterium drop degree is up to 1.0 * 1013CCU/ml～1.0 * 1014CCU/ml.When cultivating seedling with bacterium liquid, in preceding two generations, adopt the short period of time mode of 16～18h to cultivate, incubation time is short, metabolism products such as the decline factor that produces or toxin are few, can reduce of the influence of the toxic action of meta-bolites to the growing microorganism vigor, improve the growing microorganism vigor, the mycoplasma culture of this high vigor is carried out enlarged culturing containing under the condition of sufficient nutrition, bacterial concentration is improved greatly.
2, the chicken virus mycoplasma bacterium liquid of cultivating with the inventive method need not to concentrate, and can directly or prepare the finished product vaccine after the dilution; This cultural method is applied to can simplify production technique, reduce production costs in the production of chicken virus mycoplasma vaccine.
3, use present method and prepare the chicken virus mycoplasma vaccine, behind the finished product vaccine immune chicken group, good immune effect, immunizing power is strong, can resist the attack of the strong poison of chicken virus mycoplasma.
The present invention removes and is used for chicken virus mycoplasma CR strain, also can be used for chicken virus mycoplasma R strain or other bacterial strains.
Embodiment
The invention will be further described below in conjunction with embodiment.
Embodiment 1
Use chicken virus mycoplasma (CR strain) and by mycoplasma culture medium of the present invention, mycoplasma culture medium, improvement FreyShi substratum chicken virus mycoplasma carried out multiplication culture respectively:
1, mycoplasma culture medium culture method of the present invention
A, preparation mycoplasma culture medium of the present invention:
PPLO meat soup 23g,
Glucose 8.0g,
1% thaliium acetate solution 10ml,
25% yeast leach liquor 100ml,
1% phenol red solution 1.5ml,
To 840ml,, after cooling, under aseptic condition, add following composition with the deionized water constant volume at 115 ℃ of sterilization 20min:
10% arginine solution (filtration sterilization) 10ml,
Porcine blood serum 150ml,
Penicillin 1,000,000 units,
Transfer pH value to 7.8 with sodium hydroxide solution (2M).
B, seedling are cultivated with bacterium liquid:
(1) chicken virus mycoplasma (CR strain) production is inoculated in the mycoplasma culture medium of the present invention (1800ml) by 10% with seed, cultivates the 18h results for 37 ℃; Choose 10% of results bacterium liquid, with volume substratum (1800ml), go down to posterity again 1 time with quadrat method;
(2) the bacterium liquid after above-mentioned the going down to posterity is inoculated in mycoplasma culture medium of the present invention (18000ml) by 10% amount, cultivates 30h for 37 ℃, the PH that measures substratum has dropped to 7.1, results bacterium liquid.Continue to go down to posterity, bacterium liquid is inoculated in mycoplasma culture medium of the present invention (180000ml) by 10% amount, cultivates 31h for 37 ℃, and the PH that measures substratum has dropped to 7.0, results bacterium liquid.
(3) get an amount of above-mentioned work in-process bacterium liquid and carry out viable count mensuration, concentration is 1.0 * 1014CCU/ml.
Comparative example:
2, mycoplasma culture medium culture method
A, preparation " mycoplasma culture medium "
PPLO meat soup 21.0g,
Glucose 5.0g,
10% arginine solution 10.0ml,
10 times of concentrated MEM nutrient solution 10.0ml,
1% thaliium acetate solution 10ml,
25% yeast leach liquor 100ml,
80,000 units/ml penicillin 10.0ml,
Pig (horse) serum 100ml,
1% phenol red solution 1.0ml,
Be settled to 1000ml with deionized water, adjust pH value to 7.8 with sodium hydroxide solution (2M), 0.22 micron Entkeimung is standby.
B, seedling are cultivated with bacterium liquid:
(1) chicken virus mycoplasma (CR strain) production is inoculated in the mycoplasma culture medium (1800ml) by 10% with seed, cultivates 44h for 37 ℃, the PH of substratum drops to 7.0 o'clock results.
(2) the bacterium liquid after above-mentioned the going down to posterity is inoculated in mycoplasma culture medium (18000ml) by 10% amount, cultivates 42h for 37 ℃, the PH that measures substratum has dropped to 7.1, results bacterium liquid.Continue to go down to posterity, bacterium liquid is inoculated in mycoplasma culture medium (180000ml) by 10% amount, cultivates 42h for 37 ℃, and the PH that measures substratum has dropped to 7.1, results bacterium liquid.
(3) get an amount of above-mentioned work in-process bacterium liquid and carry out viable count mensuration, concentration is 1.0 * 108CCU/ml.
3, improvement FreyShi culture medium culturing method
A, preparation improvement FreyShi substratum
(1) basal liquid
NaCl 5g
KCl 0.4g
MgSO4·7H2O 0.2g
Na2HPO4·12H2O 1.6g
K2HPO4 0.1g
Glucose 10g
Milk protein hydrolysate 5g
Be dissolved in the 1000ml deionized water 115 ℃ of sterilizations in 20 minutes.
(2) substratum
Basal liquid 100ml
Porcine blood serum 13ml
25% yeast leach liquor 10ml
Penicillin 100,000 units
1% phenol red 0.1ml
1/80 thaliium acetate 1ml
Transfer pH value to 7.7 with 1N NaOH.
B, seedling are cultivated with bacterium liquid:
(1) chicken virus mycoplasma (CR strain) production is inoculated in the improvement FreyShi substratum (1800ml) by 10% with seed, cultivates 36h for 37 ℃, the PH of substratum drops to 7.1 o'clock results.
(2) the bacterium liquid after above-mentioned the going down to posterity is inoculated in improvement FreyShi substratum (18000ml) by 10% amount, cultivates 38h for 37 ℃, the PH that measures substratum has dropped to 6.9, results bacterium liquid.Continue to go down to posterity, bacterium liquid is inoculated in improvement FreyShi substratum 4(180000ml by 10% amount), cultivate 37h for 37 ℃, the PH that measures substratum has dropped to 7.0, results bacterium liquid.
(3) get an amount of above-mentioned work in-process bacterium liquid and carry out viable count mensuration, concentration is 1.0 * 109CCU/ml.
Described chicken virus mycoplasma name is called Mycoplasma gallisepticum, abbreviates MG as, and bacterial strain is the CR strain, and preserving number is: CGMCC No.3900 was deposited in Chinese microbial preservation council common micro-organisms center on 06 25th, 2010.
Embodiment 2
Use chicken virus mycoplasma (R strain) and by mycoplasma culture medium of the present invention, mycoplasma culture medium, improvement FreyShi substratum chicken virus mycoplasma is carried out multiplication culture respectively.
1, mycoplasma culture medium culture method of the present invention
A, preparation mycoplasma culture medium of the present invention:
PPLO meat soup 24g,
Glucose 10.0g,
1% thaliium acetate solution 10ml,
25% yeast leach liquor 100ml,
1% phenol red solution 1.5ml,
To 790ml,, after cooling, under aseptic condition, add following composition with the deionized water constant volume at 115 ℃ of sterilization 20min:
10% arginine solution (filtration sterilization) 10ml
Porcine blood serum 200ml
Penicillin 1,000,000 units
Transfer pH value to 7.7 with sodium hydroxide solution (2M).
B, seedling are cultivated with bacterium liquid:
(1) chicken virus mycoplasma (R strain) production is inoculated in the mycoplasma culture medium of the present invention (1800ml) by 10% with seed,, cultivate the 18h results for 37 ℃; Choose results bacterium liquid 10% with volume substratum (1800ml), go down to posterity again 1 time with quadrat method;
(2) the bacterium liquid after above-mentioned the going down to posterity is inoculated in mycoplasma culture medium of the present invention (18000ml) by 10% amount, cultivates 32h for 37 ℃, the PH that measures substratum has dropped to 7.0, results bacterium liquid.Continue to go down to posterity, bacterium liquid is inoculated in mycoplasma culture medium of the present invention (180000ml) by 10% amount, cultivates 30h for 37 ℃, and the PH that measures substratum has dropped to 7.1, results bacterium liquid.
(3) get an amount of above-mentioned work in-process bacterium liquid and carry out viable count mensuration, concentration is 1.0 * 1013CCU/ml.
Comparative example:
2, mycoplasma culture medium culture method
A, press correlation method among the embodiment 1, the preparation mycoplasma culture medium;
B, seedling are cultivated with bacterium liquid:
(1) chicken virus mycoplasma (CR strain) production is inoculated in the mycoplasma culture medium (1800ml) by 10% with seed, cultivates 42h for 37 ℃, the PH of substratum drops to 7.0 o'clock results.
(2) the bacterium liquid after above-mentioned the going down to posterity is inoculated in mycoplasma culture medium (18000ml) by 10% amount, cultivates 39h for 37 ℃, the PH that measures substratum has dropped to 7.1, results bacterium liquid.Continue to go down to posterity, bacterium liquid is inoculated in mycoplasma culture medium (180000ml) by 10% amount, cultivates 40h for 37 ℃, and the PH that measures substratum has dropped to 7.1, results bacterium liquid.
(3) get an amount of above-mentioned work in-process bacterium liquid and carry out viable count mensuration, concentration is 1.0 * 109CCU/ml.
3, improvement FreyShi culture medium culturing method
A, press correlation method among the embodiment 1, preparation improvement FreyShi substratum is transferred pH value to 7.8;
B, seedling are cultivated with bacterium liquid:
(1) chicken virus mycoplasma (CR strain) production is inoculated in the improvement FreyShi substratum (1800ml) by 10% with seed, cultivates 37h for 37 ℃, the PH of substratum drops to 7.1 o'clock results.
(2) the bacterium liquid after above-mentioned the going down to posterity is inoculated in improvement FreyShi substratum (18000ml) by 10% amount, cultivates 36h for 37 ℃, the PH that measures substratum has dropped to 7.1, results bacterium liquid.Continue to go down to posterity, bacterium liquid is inoculated in improvement FreyShi substratum 4(180000ml by 10% amount), cultivate 37h for 37 ℃, the PH that measures substratum has dropped to 7.0, results bacterium liquid.
(3) get an amount of above-mentioned work in-process bacterium liquid and carry out viable count mensuration, concentration is 1.0 * 108CCU/ml.
Embodiment 3
Prepare the chicken virus mycoplasma inactivated vaccine with chicken virus mycoplasma (CR strain) the work in-process bacterium liquid of using mycoplasma culture medium cultivation of the present invention among the embodiment 1, and carry out the safety verification and the efficacy test of vaccine.
A, the preparation of chicken virus mycoplasma (CR strain) inactivated vaccine
(1) deactivation: in chicken virus mycoplasma bacterium liquid, add the formaldehyde solution deactivation, 34% formaldehyde solution: deactivation nutrient solution=2:1000(volume: volume), 37 ℃ of effect 20h, every 2h shakes up once.
(2) water preparation: get the sterilization tween-80 and join in 96 parts of deactivation nutrient solutions for 4 parts, in the deactivation container, shake up, make aqueous-phase material.
(3) oil phase preparation: get 94 parts of white oils for animals, Jia Siben-80 6 part, add 2 parts of 2% aluminum stearates behind the mixing, be heated to dissolving fully after, 115 ℃ of sterilizations 40 minutes, it is standby promptly to get the oil phase thing after the cooling.
(4) emulsification: get oil phase and join in the colloidal mill for 3 parts, in stirring at low speed, slowly add 1 part of water, stirred 12 minutes, promptly make chicken virus mycoplasma (CR strain) inactivated vaccine with 8000r/min.
B, safety verification: get 8 of 40 age in days SPF chickens, the leg muscle vaccinate, every 1ml observed 14 days continuously.The result shows, annotates the seedling chicken and does not have the fervescence phenomenon, and the diet and the mental status are no abnormal.The injection site does not have untoward reactions such as redness, inorganization liquefaction and granulation tissue hyperplasia, and the injection site absorbs good.
C, efficacy test: get 8 of 40 age in days SPF chickens, the subcutaneous or leg muscle vaccinate of nape portion, every 0.5ml, after 30 days, together with 6 of the identical contrast chickens of condition, attack the every 1ml of R strain culture 600ml(at the secret room internal spraying of 3m2 and contain 109 color variable color units), droplet is about 2 microns.Observed 14, and cutd open inspection and observe the air bag pathology, to its scoring, the average air bag injury protection of immune group chicken rate should be more than 60%, and vaccine is judged to qualified.Assay, the vaccine protection ratio of this production reaches 100%.
Claims (2)
1. method that improves chicken virus mycoplasma multiplication capacity in the production of vaccine, described vaccine is made through seed preparation, the preparation of chicken virus mycoplasma substratum, the preparation of seedling bacterium liquid and inactivated vaccine preparation section, it is characterized in that described chicken virus mycoplasma substratum is prepared by following technology:
PPLO meat soup 23.0～26.0g,
Glucose 8.0～10.0g,
Mass concentration is 1% thaliium acetate solution 10ml,
Mass concentration is 25% yeast leach liquor 100ml,
Mass concentration is phenol red solution 1.0～2.0ml of 1%,
With deionized water constant volume to 790～840ml,, after cooling, under aseptic condition, add following composition at 115 ℃ of sterilization 20min:
Mass concentration is 10% arginine solution (filtration sterilization) 10ml,
Porcine blood serum 150～200ml,
Penicillin 80～1,200,000 units,
Transfer pH value to 7.6～7.8 with sodium hydroxide solution.
2. the method for chicken virus mycoplasma multiplication capacity is characterized in that in the raising production of vaccine according to claim 1,
Described cultivation seedling is carried out as follows with bacterium liquid:
(1) chicken virus mycoplasma production is inoculated in the described mycoplasma culture medium by 10% with seed, cultivates 16～18h results at 36～37 ℃; Choose 10% of the long-pending amount of results bacteria liquid, with the volume substratum, go down to posterity again 1 time with quadrat method;
(2) the bacterium liquid after above-mentioned the going down to posterity is inoculated in mycoplasma culture medium of the present invention by 10% amount, cultivates 24～48h for 36～37 ℃, treat that the PH of substratum drops to about 7.0 results; According to this method enlarged culturing progressively, until required amount, this bacterium liquid promptly can be used for preparing vaccine as work in-process.
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