CN104027796B - A kind of swine eperythrozoonosis vaccine and preparation method thereof - Google Patents
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Abstract
The invention discloses a kind of swine eperythrozoonosis vaccine and preparation method thereof.Comprise in the present invention: the preparation technology such as emulsifying of the cultivation of eperythrozoon suis, eperythrozoon suis deactivation, vaccine adjuvant obtains the eperythrozoon suis vaccine of deactivation; its physicochemical property such as stability, viscosity is detected; and by mice detect vaccine safety, carry out immunization experiment to piglets; carried out the immune detection of specific immunity and non-specific two aspects, and inactivated vaccine is to the detection of the protective effect of animal and vaccine clinical effect.Advantageous feature: can find out that this vaccine has safety and protectiveness from clinical and experimental data, also has processing technology simple, the feature such as easily to obtain, cost is low, can in a large number for veterinary clinic.The prevention of the present invention to eperythrozoon suis has important effect, has very important clinical meaning to minimizing swine eperythrozoonosis to the loss that pig industry is brought.
Description
Technical field
The invention belongs to veterinary vaccines technical field, particularly vaccine of preventing of a kind of swine eperythrozoonosis and preparation method thereof, is a kind of effective, the safe vaccine of veterinary applications.
Background technology
Swine eperythrozoonosis is caused by eperythrozoon suis (M.suis), has host specificity feature, often cause the Arbo infectious disease of the symptoms such as organism fever, haemolysis, jaundice.This disease can cause the weak and anemia of newborn piglet and ablactational baby pig, and Growth of Pigs is slow, the summary symptom such as sow breeding difficulty, conception rate are low, non-estrus, miscarriage and stillborn fetus.It not only causes the reduction of Animal product quality and output, also can cause the large quantities of death of pig.The pig immune function more seriously infecting eperythrozoonosis declines greatly, can other disease of secondary infection, such as wean wild boar porcine circovirus and eperythrozoon suis, eperythrozoon suis and skin acaricide mixed infection, atypical classical swine fever and swine eperythrozoonosis mixed infection etc., the treatment of these mixed infections to this disease adds difficulty, and to raising pigs, industrial belt carrys out serious economic loss.This sick infection has universality, and is inapparent infection, and bringing serious hidden danger and harm to China's pig industry, therefore, is very important to the prevention of M.suis disease.But, still lack at present and a kind ofly can be widely used in clinical safe, effective, that production cost is lower vaccine this disease is prevented.
Over entering year, also there is the research of eperythrozoon suis vaccine.The subunit vaccine of the research such as Lv Zhou, Zheng Xiuhong, has certain effect to mice, but can vaccine produce immanoprotection action in other animal bodies needs further research, and its clinical meaning is very few; The vaccine of the research such as HoelzleK can produce strong immune level, but can not shield.This inactivated vaccine combines the research to its immune effect and protective effect of clinical and laboratory two aspects, detects from specificity and non-specific 2 aspects etc. its immune effect.
Based on the problems referred to above, the invention provides a kind of can the inactivated vaccine preventing eperythrozoon suis and preparation method thereof, for swine eperythrozoonosis prevention provides theoretical basis.
Summary of the invention
The object of the invention is to solve existing market and there is no a kind of vaccine of safe and effective prevention eperythrozoon suis clinically, and a kind of eperythrozoon suis inactivated vaccine and preparation method thereof is provided.
A preparation method for swine eperythrozoonosis vaccine, comprises the following steps:
The erythrocyte infecting eperythrozoon suis is inoculated in cell culture medium, culture medium prescription: 60-65wt%PPLO Mycoplasma Broth Base liquid, 5-10wt%M199 basal liquid, the Serine that the glucose solution that the yeast leachate of the 0.25wt% of 9-10wt%, 2.5-3.5wt% concentration are 0.5g/mL, 1-2wt% concentration are 4.5mg/mL, 0.5-1wt% concentration are NAD, 10-15wt% deactivation porcine blood serum composition of 4mg/mL; Become single pathogen to cultivate with the ultrasound wave eperythrozoon suis that dissociates in incubation, detect its number and reach 2.5-3.0x10
9individual/mL; Then eperythrozoon suis deactivation cultivation obtained is for the preparation of inactivated vaccine.
Culture medium prescription is preferred: 60wt%PPLO Mycoplasma Broth Base liquid, 10wt%M199 basal liquid, and the concentration of the glucose solution that concentration is 0.25g/mL yeast leachate, 2.5wt% concentration is 0.5g/mL of 10wt%, Serine that 2wt% concentration is 4.5mg/mL, 0.5wt% is NAD, 15wt% deactivation porcine blood serum composition of 4mg/mL.
The preparation method of described swine eperythrozoonosis vaccine:
The erythrocyte infecting eperythrozoon suis is inoculated in cell culture medium, is placed in 37 DEG C, containing 5%-10%CO
2incubator cultivate, replaced medium after erythrocyte hemolysis after 72h, then continuous culture 30d, every 6d changes a culture fluid, fully reduces erythrocyte membrane composition, dissociates after eperythrozoon suis becomes single pathogen continue to cultivate propagation with ultrasound wave, again ultrasonicly to dissociate, detect its number.
Said method adopts formalin-inactivated eperythrozoon suis.Specifically concentration is adopted to be that the formaldehyde of 0.3-0.5% is at 37 DEG C of deactivation eperythrozoon suis 20-28h.Preferred employing concentration is that the formaldehyde of 0.4% is at 37 DEG C of deactivation eperythrozoon suis 24h.
The eperythrozoon suis of deactivation is mixed homogeneously as aqueous phase with oil phase by said method, obtains oil-in-water inactivated vaccine and be the inactivated vaccine prepared after emulsifying.Specifically by oil phase and aqueous phase by volume 1:1-2:1 mixing, the concentration of antigen is 4.28x10
8-4.48x10
8individual/mL.Preferably by oil phase and aqueous phase by volume 1:1 mixing, the concentration of antigen is 4.38x10
8individual/mL.
A kind of swine eperythrozoonosis vaccine is the vaccine prepared by said method.
In prior art in the In vitro culture of eperythrozoon suis, Nonakaetal has attempted the In vitro culture of M.suis, selects the Iger (family name) adding flesh former times to cultivate (rEM), and observes the rising of Infestation rat; Domestic Zhang Shoufa etc., Fang Chunlin etc., Zhang Xuefeng, Zheng Haihong etc. add the PRMI-1640 of the compositions such as Ox blood serum, with negative animal erythrocyte for carrier all reports that Infestation rat has rising to a certain degree; Li Xiaoyun Mycoplasma culture medium culturing M.suis, define the blood shadow of eperythrozoon suis, and value-added velocity ratio is slower after cultivating, this can affect the counting of eperythrozoon suis, and innovative point of the present invention is that the culture medium that eperythrozoon suis used prepared by vaccine is improved on the bases such as Li Xiaoyun, mainly decrease PPLO basal liquid concentration, add M199 basal liquid, instead of Ox blood serum etc. with porcine blood serum; Adopt ultrasonic method 30min-1h to make eperythrozoon suis become the individuality of single existence, make the increment of eperythrozoon suis obvious, be conducive to counting simultaneously.
Innovative point of the present invention is mainly the culture effect of eperythrozoon suis, the eperythrozoon suis propagation that Li Xiao cloud method is cultivated is slower, because the agglomerating existence of eperythrozoon suis, have impact on its propagation, the present invention with ultrasonic method eperythrozoon suis is separated into single after cultivate again and be conducive to the propagation of eperythrozoon suis, speed of its propagation can reach 30-40%.
Effect of the present invention is also, can cause good immunoreation, and can not cause other untoward reaction to the antigen of eperythrozoon suis, and processing technology process of the present invention and structure simple, cost is lower, safe and reliable, is suitable for promoting the use of on a large scale.
Detailed description of the invention
Be intended to further illustrate the present invention below in conjunction with case study on implementation, and unrestricted the present invention.
What the preparation of vaccine adopted is more effectively, deactivation mode easily, also more directly perceived, feasible to its detection.
The mode of room and clinical combination by experiment the immune effect of vaccine, protection effect can be tested, for later clinical application provides reliable foundation, improve the feasibility of research.
The preparation of embodiment 1, vaccine comprises the following steps:
1, the cultivation of eperythrozoon suis.The erythrocyte infecting eperythrozoon suis is inoculated in Tissue Culture Flask, is placed in 37 DEG C, containing 5%CO
2incubator cultivate, after about 72h, erythrocyte hemolysis becomes taeniasis blood shadow, then continuous culture 30 days, abundant minimizing erythrocyte membrane composition, become single pathogen with the ultrasound wave eperythrozoon suis blood shadow that dissociates, continue to cultivate with after red blood cell count(RBC) plate counting, breed to peak to 48h, proliferation number reaches 30-40%, reduce subsequently, the eperythrozoon suis being linked to be the propagation that 2-6 quantity does not wait can be seen in the visual field, then by the single antigen of its ultrasonic one-tenth, detect its number, number reaches 2.87x10
9individual/mL, prepares for vaccine.Culture medium prescription: 60-65%PPLO Mycoplasma Broth Base liquid, 5-10%M199 basal liquid, the Serine that the glucose solution that the 0.25% yeast leachate of 9-10% (yeast leach cream is water-soluble is prepared into yeast leachate), 2.5-3.5% concentration are 0.5g/mL, 1-2% concentration are 4.5mg/mL, 0.5-1% concentration are NAD, 10-15% deactivation porcine blood serum composition of 4mg/mL.
2, the preparation of vaccine aqueous phase.Select formaldehyde to be the inactivator of inactivation antigen, after utilizing different concentration to carry out deactivation, eperythrozoon suis is counted, find out best deactivation concentration; After determining inactivator concentration, determine the time of deactivation, the whole inactivation time determined is 24h, and the concentration of formaldehyde is 0.4%, and the temperature of deactivation is 37 DEG C.
3, the preparation of vaccine oil phase.Selection adjuvant is vaccine adjuvant, and each reagent adds according to optimum proportioning composition, the oil phase for preparing after mixing.The basically identical oil vacuole of the size that oil phase is formed under the microscope, in the different visual field, number and each oil vacuole difference are little, oil phase mix homogeneously.
4, the preparation of vaccine.Oil phase and aqueous phase 1:1 are mixed, the protein concentration of swine erythrocyte body is 8.703mg/mL, and the concentration of antigen is 4.38x10
8individual/mL; Make aqueous phase and oil phase mix homogeneously with magnetic stirring apparatus, obtain oil-in-water inactivated vaccine after emulsifying and be the inactivated vaccine prepared.Detect the aspects such as the model of the stability of vaccine, viscosity, the uniformity and oil-adjuvant vaccine.Stability: in vaccine instillation clear water after 10min, vaccine oil droplet does not occur dispersion phenomenon in water, vaccine is centrifugal 2500,3000, after the centrifugal 15min of 3500rpmin, all do not observe with or without lamination; Degradation: vaccine has not observed demulsifying phenomenon after being placed on 37 DEG C of constant incubators 10d, 20d, 30d; Viscosity test: 0.5mL required time released by vaccine from the pipette of 1mL is 5s, its viscosity is relatively applicable to being expelled in animal body.
The evaluation of embodiment 2, vaccine effect
1, safety evaluatio.
Prepare vaccine as described above, by vaccine injection 20 Kunming white mouses prepared, every intramuscular injection 0.2mL, observes diet and the mental status of mice, does not find that very large change, 21d mice are without death.
By vaccine injection 10 sows prepared, 10 suckling pigs, 10 nursery pig, 10 growing and fattening pigs, observing the mental status of pig, breathing, pulse normally, there is not death in 21d.
2, white mice Efficacy evaluation
By 30 Kunming white mice, be divided into 2 groups at random, often organize 15, the vaccine that intramuscular injection 0.2mL prepares, after 7d, booster immunization once, the normal saline of matched group injection Isodose.Observe the mice mental status of matched group and immune group, after 21d, put to death mice, claim the weight of the body weight of mice, spleen and thymus, measure the Thymus and spleen index of mice.The Thymus and spleen index of vaccine group mice is all high than matched group, shows that vaccine can improve the immunoreation to mice.The Thymus and spleen index result of mice is as follows:
| Group | n | Thymus index/(g/100g) | Index and spleen index/(g/100g) |
| Vaccine group | 15 | 0.2026-0.2284 | 0.4435-0.7037 |
| Matched group | 15 | 0.1489-0.1963 | 0.3499-0.4805 |
3, the Efficacy evaluation of piglet
10 piglets are divided into 2 groups at random, matched group and vaccine immunity group, the vaccine of the piggy injection 4mL of immune group, after the 1st immunity, and each booster immunization of 7d, 14d 1 time.Take blood to be divided into 2 parts, 1 part does not add anticoagulant for separating of serum at every turn, and 1 part does not add anticoagulant for separating of lymphocyte, utilizes indirect ELISA to detect the change of Serum Antibody level, detects cellular immune level with lymphocyte transformation experiment.
Lymphocyte result shows, matched group is according to the difference of immune time, and difference is not remarkable, and the OD value of vaccine group presents the trend risen gradually.
Lymphocyte transformation experimental result is as follows:
4, non-specific detection
ALT, CREA (creatinine) physical signs utilizing automatic clinical chemistry analyzer to detect in serum detects; Meanwhile, also have detected the index that the malonaldehyde (MDA) in serum, superoxide dismutase (SOD) etc. can react body nonspecific immunity.Result shows, and immune and non-immune piglet ALT, CREA difference is not remarkable, illustrates that the impact of vaccine on Liver and kidney is little.The immunity of malonaldehyde (MDA), superoxide dismutase (SOD) testing result and non-immune piglet AMDA, SOD difference are not remarkable, and this shows that the metabolism of vaccine on piglet is without impact.
ALT testing result
CREA testing result
MDA result
SOD result
5, vaccine protective experiment
20 clinical health piglets are former with injecting swine eperythrozoonosis after eperythrozoon suis vaccine of the present invention, and piglet clinical health, swine eperythrozoonosis does not occur; And the piggy injection swine eperythrozoonosis that matched group 20 clinical healths do not inject eperythrozoon suis vaccine of the present invention is former, there is 3 to occur swine eperythrozoonosis clinical symptoms, shows that vaccine has protection.
Result
The present invention is a kind of safe, vaccine eperythrozoon suis to protective effect.
Claims (6)
1. a preparation method for swine eperythrozoonosis vaccine, is characterized in that, comprises the following steps:
The erythrocyte infecting eperythrozoon suis is inoculated in cell culture medium, culture medium prescription: 60-65wt%PPLO Mycoplasma Broth Base liquid, 5-10wt%M199 basal liquid, the Serine that the glucose solution that the yeast leachate of the 0.25wt% of 9-10wt%, 2.5-3.5wt% concentration are 0.5g/mL, 1-2wt% concentration are 4.5mg/mL, 0.5-1wt% concentration are NAD, 10-15wt% deactivation porcine blood serum composition of 4mg/mL; Be placed in 37 DEG C, containing 5%-10%CO
2incubator cultivate, replaced medium after erythrocyte hemolysis after 72h, continuous culture 30d again, every 6d changes a culture fluid, abundant minimizing erythrocyte membrane composition, become after single pathogen to continue to cultivate propagation with the ultrasound wave eperythrozoon suis that dissociates, again ultrasonicly to dissociate, detect its number and reach 2.5-3.0x10
9individual/mL; Then will cultivate the eperythrozoon suis deactivation that obtains, using the eperythrozoon suis of deactivation as aqueous phase and oil phase by volume 1:1-2:1 mix homogeneously, the concentration of antigen is 4.28x10
8-4.48x10
8individual/mL, obtains oil-in-water inactivated vaccine and is the inactivated vaccine prepared after emulsifying.
2. the preparation method of swine eperythrozoonosis vaccine according to claim 1, it is characterized in that, culture medium prescription: 60wt%PPLO Mycoplasma Broth Base liquid, 10wt%M199 basal liquid, the concentration of the glucose solution that concentration is 0.25g/mL yeast leachate, 2.5wt% concentration is 0.5g/mL of 10wt%, Serine that 2wt% concentration is 4.5mg/mL, 0.5wt% is NAD, 15wt% deactivation porcine blood serum composition of 4mg/mL.
3. the preparation method of swine eperythrozoonosis vaccine according to claim 1, is characterized in that,
Adopt formalin-inactivated eperythrozoon suis.
4. the preparation method of swine eperythrozoonosis vaccine according to claim 3, is characterized in that,
Concentration is adopted to be that the formaldehyde of 0.3-0.5% is at 37 DEG C of deactivation eperythrozoon suis 20-28h.
5. the preparation method of swine eperythrozoonosis vaccine according to claim 4, is characterized in that,
Concentration is adopted to be that the formaldehyde of 0.4% is at 37 DEG C of deactivation eperythrozoon suis 24h.
6. the preparation method of swine eperythrozoonosis vaccine according to claim 1, is characterized in that, by oil phase and aqueous phase by volume 1:1 mixing, the concentration of antigen is 4.38x10
8individual/mL.
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