CN108273051B - The rapidly and efficiently preparation method of pig A type C.perfringens inactivated vaccine - Google Patents
The rapidly and efficiently preparation method of pig A type C.perfringens inactivated vaccine Download PDFInfo
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Abstract
The invention belongs to veterinary biologics technical fields, are related to the rapidly and efficiently preparation method of a boar A type C.perfringens inactivated vaccine.The following steps are included: the pathological material of disease of the lethal sick pig of picking A type C.perfringens, scrapes intestinal contents, refrigeration or freezing；By culture medium, high pressure sterilization passes on 3 times until pathological material of disease is poured into pressure cooker, Anaerobic culturel after temperature reduces in pressure cooker；It takes the bacterium solution passed on three times to access the membrane digestion soup culture of meat liver stomach, is centrifuged after culture, takes supernatant；Inactivator is added to supernatant, shake well inactivates detoxification, obtains inactivated bacterial liquid；Inactivated bacterial liquid is mixed with adjuvant, obtains inactivated vaccine.Method and process provided by the invention is simple, at low cost, pollution is small, and the inactivated vaccine prepared is highly-safe, has no adverse reaction after injection, and immune efficacy is high, duration of immunity is long.
Description
Technical field
The invention belongs to veterinary biologics technical fields, and in particular to a boar A type C.perfringens inactivated vaccine
Rapidly and efficiently preparation method.
Background technique
C.perfringens is also known as clostridieum welchii, can produce a variety of exotoxins and enzyme, can decompose muscle and connective tissue
In sugar, generate bulk gas, lead to the serious wind-puff of tissue, then influence blood supply, cause tissue large area downright bad.According to
The synthesized primary toxins secreted of C.perfringens, are divided into five kinds of toxin types of A, B, C, D, E；Wherein A type C.perfringens
Mainly cause the lesion of gastrointestinal tract, is people and animals' gas gangrene, rabbit clostridium property diarrhea, horse necrotic enteritis and people's food poisoning
Main pathogenic bacteria, and cause the main pathogenic bacteria of deer hemorrhagic enteritis.It is most important in a variety of exotoxins that the type bacterium generates
It is alpha toxin.Pig A type C.perfringens is the main reason for causing piglet, growing and fattening pigs, farrowing sow and boar " sudden death ",
Characterized by enterotoxemia and necrotic enteritis caused by the gastrointestinal tract diffusivity bleeding, morbidity is anxious, and the course of disease is short, without any early period
Sign and die by visitation of God.
Currently, C.perfringens inactivated vaccine also has more research, Chinese patent application CN102698259A is public
A kind of chicken necrotizing enterocolitis (A type) inactivated vaccine preparation method is opened, by CVCC37 plants of seed liquors of A type C.perfringens by training
The 1%-2% for supporting base total amount is inoculated in using pancreas casein peptone and yeast extract powder as the liquid synthetic media of main raw material(s)
In, 37 DEG C of anaerobic fermentation cultures/or stationary culture 6 hours, culture terminate after through 3000r/min centrifugation 30min removal thallus, point
System is not concentrated by ultrafiltration with the millipore of 8Ku molecular cut off to be concentrated by ultrafiltration, is inactivated through 0.6% formalin de-
After poison is complete, 20% volume aluminium hydroxide of final concentration is added and is configured to vaccine.Chinese patent application CN104623651A discloses one
The preparation method of the A type C.perfringens inactivated vaccine of kind rabbit clostridium welchii disease, this method are with A type C.perfringens
Original strain finally obtains bacterium solution after firsts and seconds culture, which is mixed with white-oil adjuvant and obtains A type production gas
Capsular clostridium inactivated vaccine；Wherein, inactivator used is the formaldehyde that final mass score is 0.3%, and adjuvant is white-oil adjuvant.It is bent
(the safety of [1] Qu Haibo, Liu Hongbin, Bai Tongchen .A type C.perfringens toxoid vaccine and full bacterium inactivated vaccine such as hypo
Property and immune efficacy comparative studies [J] China veterinary drug magazine, 2010,44 (02): 15-16.) have studied the six batches of rabbit A types and produce gas
Capsular clostridium toxoid vaccine and its full bacterium inactivated vaccine, and research is compared to its safety and immune efficacy；Knot
Fruit shows that toxoid inactivated vaccine has no toxic side effect, and local stimulation is small, and safety is better than full inactivated vaccine, immune efficacy result
The malicious protective rate of attacking of the two no significant difference, two kinds of vaccines is not less than 4/5, and duration of immunity is at 6 months or more；Wherein inactivator
Using 0.4% formalin, adjuvant uses aluminium hydroxide gel.
Currently, the livestock that the research of A type C.perfringens inactivated vaccine is directed to is mostly deer, rabbit, ox, for pig A type pod
The research of film clostridium inactivated vaccine is less, and simply replacement can not obtain that safety is good, stability is high, good immune effect
Pig vaccine, and for the inactivation reagent of C.perfringens using the formaldehyde for having carcinogenic effect, adjuvant is more in the prior art
Using the aluminium hydroxide gel containing heavy metal.These vaccines still have some needs in use and further solve the problems, such as: (1)
Domestic animal reaction of inoculation is heavier；(2) injection dosage is big, and using there is inconvenient (3) preparation process cumbersome, the time is longer more.Therefore, it is necessary to
Preparation process and screening and culturing medium and adjuvant are improved, quickly to prepare efficient vaccine, to improve vaccine immunogenicity sum aggregate
Body immune response is horizontal.
Summary of the invention
The technical problem to be solved by the present invention is in view of the foregoing drawbacks, provide pig A type C.perfringens inactivated vaccine
Rapidly and efficiently preparation method.This method is simple and feasible, easy to operate, at low cost, pollution is small, the inactivated vaccine safety prepared
Property it is high, have no adverse reaction after injection, immune efficacy is high, duration of immunity is long.
To achieve the goals above, the present invention adopts the following technical scheme:
The rapidly and efficiently preparation method of one boar A type C.perfringens inactivated vaccine, comprising the following steps:
(1) acquisition of pathogen: the pathological material of disease of the lethal sick pig of picking A type C.perfringens scrapes intestinal contents, refrigeration
Or freezing；
(2) Spawn incubation: by culture medium in pressure cooker high pressure sterilization, until temperature reduce after, pathological material of disease is poured into high pressure
In pot, Anaerobic culturel is passed on 3 times, and wherein culture medium is the synthetic media of plain chocolate and glucose；
(3) prepared by bacterium solution: taking the bacterium solution passed on three times in step (2) to access the membrane digestion soup culture of meat liver stomach, culture terminates
After be centrifuged, take supernatant；
(4) bacterium solution inactivates: inactivator is added to the bacterium solution of step (3), shake well inactivates detoxification, obtains inactivated bacterial liquid,
Middle inactivator is binary ethylenimine and beta-propiolactone；
(5) vaccine preparation: inactivated bacterial liquid is mixed with adjuvant, obtains inactivated vaccine；Wherein, the adjuvant is multiple for immunostimulation
Close object.
The selection of cause of disease is related to the biological efficacy of vaccine, and it is cause of disease that the application, which directly scrapes intestinal contents, can be with
The problems such as vigor decline for avoiding preservation of bacteria strain from being likely to occur, toxicity decline, inhibit and kill other intestines under high-temperature and high-pressure conditions
Road pathogen；A type C.perfringens mass propagation produces acid and produces gas, ensure that the purity and concentration of A type C.perfringens.
Culture medium is the artificial nutriment of bacterium, and different according to bacterium require by artificial means to prepare various nutriment one
Rising becomes the basis maintained with bacterial growth, prepares applicable culture medium, nutriment needed for providing bacterium and growth conditions,
The rule for understanding its growth and breeding, can be improved the quality and quantity of vaccine.The application is synthesized using plain chocolate and glucose
Culture medium passes on 3 times, obtains pure culture；Strain after purification is digested into amplification cultivation in soup in pancreatin again, using meat liver stomach film
Soup is digested to cultivate, and the virulence for the A type clostridium which turns out is high compared to the virulence of ordinary culture medium culture, vaccine quality
It is good.
Inactivator is the chemical reagent or drug for inactivating microorganism, is a very important skill in biological products
Art, for improving the safety of biological products and preventing pathogen from spreading.Common inactivator is formaldehyde, but formaldehyde is a kind of
The carcinogen of intense irritation both may act on the amino-containing nucleotide base (such as A, G, U) of virus, and can operational virus
Glutelin.When acting on virus coat protein, easily make protein crosslink or virion aggregation, can no longer function to glutelin
Interior nucleic acid.In this way, the antigenicity of pathogen protein can be seriously damaged, and there may be pathogen survival；Use formalin-inactivated
Time is long, and inactivate effect vulnerable to temperature, pH, concentration, with the presence or absence of organic matter, the type of pathogen and nitrogen content etc. because
Element influences, and should arouse attention under study for action；Remaining free formaldehyde, if after injecting body with vaccine, the system that can generate swashs property reaction.
Using binary ethylenimine and beta-propiolactone as inactivator in the application, binary ethylenimine mainly acts on nucleic acid, protein immunization
Antigen is kept preferably, and small toxicity, but the virus stability inactivated is poor, and the validity period of vaccine is short；Beta-propiolactone directly with disease
Malicious nucleic acid effect, without acting on glutelin, is able to maintain immunogenicity, has strong inactivating efficacy, and inactivation time is short, pole
Facile hydrolysis, it is not necessary to consider residual of the finished product in vaccine, although there is many advantages, beta-propiolactone is a kind of potential carcinogenic
The factor, and the holding time it is too long cause self-polymerization influence inactivating efficacy.Binary ethylenimine and beta-propiolactone are collectively as going out
Agent living can act synergistically, and superiority and inferiority is complementary, not only shorten inactivation time, improve inactivation effect, further improve the peace of vaccine
Quan Xing maintains the immunogenicity of virus.
Adjuvant is the substance that antigen-specific immune response can be enhanced.The faint object of antigenicity can be remarkably reinforced in adjuvant
Matter induces body to generate specific immune response, and can use the smallest antigen and least inoculation times, generates enough
Immune response.Common adjuvant is aluminium hydroxide gel, but vaccine injection amount made of aluminium hydroxide gel is big, when immunity continues
Between it is short, domestic animal reaction weight, subcutaneous injection often has swelling or an agglomeration；The application, as adjuvant, is immunized using immunostimulating complex
Stimulation compound (ISCOM) is made of saponin(e, grease, cholesterol and antigen, passes through the hydrophobic effect between preceding 3 components
It is combined together.Hydrophobic or hydrophilic antigen can be incorporated on its compound, and compared with other adjuvants, ISCOM is safer,
It is capable of forming long acting biologically active reaction, the dual function offered with adjuvant and antigen.
Further, in above-mentioned steps (1), refrigerated storage temperature is 2-5 DEG C, and cryogenic temperature is -20 DEG C.
Further, in above-mentioned steps (2), pressure cooker sterilising temp is 110 DEG C, sterilization time 20min；Until temperature drops
To after 80 DEG C, pathological material of disease is poured into pressure cooker, 42-45 DEG C of high temperature anaerobic culture 4-6h is passed on.The application selection adds at 80 °
This bold improvement of pathological material of disease had not only killed unrelated pathogen but also had shortened the time of germ isolation and purification culture, greatly shortened
Vaccine production time.
Further, in above-mentioned steps (3), the bacterium solution passed on three times in step (2) is taken to access 37 DEG C of soup of meat liver stomach membrane digestion
6-10h is cultivated, 10-15min is centrifuged in 4000-6000r/min after culture, takes supernatant.
Further, meat liver stomach membrane digestion soup ingredient include: hog gastric mucosa, beef, beef liver, hydrochloric acid, sodium hydroxide, go from
Sub- water and ethyl alcohol.
According to one embodiment of the application, meat liver stomach membrane digestion soup ingredient include: hog gastric mucosa 150g, beef 100g,
Beef liver 100g, hydrochloric acid 10mL, sodium hydroxide 6g, deionized water 1000mL and ethyl alcohol 100mL.
Further, the preparation of meat liver stomach membrane digestion soup adds the following steps are included: beef, beef liver, hog gastric mucosa are blended
Enter distilled water, hydrochloric acid and ethyl alcohol, after mixing cold soaking 20-24h, is slowly heated to temperature 56 ± 1 DEG C of holdings 22-24h, every 1h
Stirring is primary, removes oil slick rouge, digestive juice is taken to boil, and with 2mol/L sodium hydroxide solution tune pH value, flannelette filtering takes supernatant
116 DEG C of sterilizing 30min are spare.
Further, in above-mentioned steps (4), it is molten that the binary ethylenimine that mass fraction is 0.2% is added to the bacterium solution of step (3)
Liquid makes its final mass score 0.02-0.05%, and mixed liquor is inactivated 20-24h in 20-30 DEG C of concussion, is added in β-the third afterwards
Ester makes its final concentration of 1g/L, inactivates 2h in 37 DEG C of concussions, the sterile hypo solution of 1mol/L is added, makes its final concentration
It is consistent with binary ethylenimine, inactivation 1h is terminated, inactivated bacterial liquid is obtained.
Further, in above-mentioned steps (5), the preparation of immunostimulating complex the following steps are included:
1) preparation of lipid mixture solution: lecithin and cholesterol are added in 20%Mega-10, make the two final concentration
It is all 10mg/mL；
2) preparation of saponin(e solution: QS-21 being dissolved in the PBS solution that pH is 7.2, and adjustment concentration is 10-15mg/
mL。
3) prepared by immunostimulating complex: being by volume 1:(3-5 by lipid mixture solution and saponin(e solution) sufficiently
5-7h is cracked under mixed room temperature；Ice-bath ultrasonic process, dialysis save backup after crossing 0.22 μm of filter membrane degerming in 4 DEG C.
Further, in above-mentioned steps (5), the volume ratio of inactivated bacterial liquid and adjuvant is 1:(2-4).
According to one embodiment of the application, the rapidly and efficiently side of preparation of a boar A type C.perfringens inactivated vaccine
Method, comprising the following steps:
(1) acquisition of pathogen: the pathological material of disease of the lethal sick pig of picking A type C.perfringens, scraping intestinal contents, 2 DEG C
Refrigeration；
(2) Spawn incubation: by culture medium, high pressure sterilization, sterilising temp are 110 DEG C in pressure cooker, and sterilization time is
20min；Until temperature is reduced to 80 DEG C, pathological material of disease is poured into pressure cooker, 44 DEG C of high temperature anaerobic culture 4h are passed on, passage 3
Secondary, wherein culture medium is the synthetic media of plain chocolate and glucose；
(3) prepared by bacterium solution: the bacterium solution passed on three times in step (2) being taken to access 37 DEG C of culture 8h of meat liver stomach membrane digestion soup, training
It is centrifuged 10min in 6000r/min after supporting, takes supernatant；Wherein, meat liver stomach membrane digestion soup preparation the following steps are included:
Beef 100g, beef liver 100g, hog gastric mucosa 150g are blended, distilled water 1000mL, hydrochloric acid 10mL and ethyl alcohol 100mL is added, is mixed
Cold soaking 20h after closing uniformly is slowly heated to 56 DEG C of temperature and keeps for 24 hours, and every 1h stirring is primary, removes oil slick rouge, digestive juice is taken to boil
Boiling, with 2mol/L sodium hydroxide solution 75mL tune pH value, flannelette filtering takes 116 DEG C of sterilizing 30min of supernatant spare.
(4) bacterium solution inactivates: the binary ethylenimine solution that mass fraction is 0.2%, which is added, to the bacterium solution of step (3) makes it most
Whole mass fraction is 0.04%, and mixed liquor is inactivated 20h in 30 DEG C of concussions, and the rear beta-propiolactone that is added makes its final concentration of 1g/L,
2h is inactivated in 37 DEG C of concussions, the sterile hypo solution of 1mol/L is added, keeps its final concentration consistent with binary ethylenimine, eventually
1h is only inactivated, inactivated bacterial liquid is obtained.
(5) vaccine preparation: by inactivated bacterial liquid, 1:3 is mixed by volume with adjuvant, obtains inactivated vaccine；Wherein, the adjuvant
For immunostimulating complex；Wherein, immunostimulating complex preparation the following steps are included:
1) preparation of lipid mixture solution: lecithin and cholesterol are added in 20%Mega-10, make the two final concentration
It is all 10mg/mL；
2) preparation of saponin(e solution: QS-21 being dissolved in the PBS solution that pH is 7.2, and adjustment concentration is 12mg/mL.
3) prepared by immunostimulating complex: lipid mixture solution and saponin(e solution are sufficiently mixed by volume for 1:5
7h is cracked at room temperature；Ice-bath ultrasonic process, dialysis save backup after crossing 0.22 μm of filter membrane degerming in 4 DEG C.
It is a further object to provide a kind of pig A types being prepared according to any of the above-described preparation method to produce gas pod
Film clostridium inactivated vaccine.
Beneficial effects of the present invention:
(1) directly scraping intestinal contents are cause of disease, inhibit and kill other enteric pathogenic bacterias under high-temperature and high-pressure conditions；A
Type C.perfringens mass propagation produces acid and produces gas, ensure that the purity and concentration of A type C.perfringens.80 degree plus pathological material of disease this
One bold improvement had not only killed unrelated pathogen but also had shortened the time of germ isolation and purification culture, and substantially reduced vaccine
Production Time.
(2) using milk as culture medium Rapid Fermentation separating-purifying strain, meat liver stomach membrane digestion soup is as amplification culture medium, training
The virulence of the A type clostridium raised is high compared to the virulence of ordinary culture medium culture, and vaccine is high-quality.
(3) effect that inactivation is largely improved using alkylating agent and beta-propiolactone synergistic effect, when shortening inactivation
Between, improve the safety of vaccine.
(4) vaccine includes the mycoprotein of inactivation, endotoxin and exotoxin, and immunogenicity is good, and protective rate is high.
(5) immune efficacy of vaccine is high, and the immunity duration is long.
(6) allergic reaction is small, does not influence spirit, appetite, injection site is without swelling, no necrosis.
(7) simple process, time are short, low in cost.
Specific embodiment
The present invention is further elaborated with reference to embodiments.These embodiments be only for illustrative purposes,
And do not limit the scope of the invention and essence.Based on the embodiment of the present invention, those of ordinary skill in the art are not making wound
Every other embodiment obtained under the premise of the property made labour, belongs to protection scope of the present invention.
Embodiment 1
The rapidly and efficiently preparation method of one boar A type C.perfringens inactivated vaccine:
(1) acquisition of pathogen: the pathological material of disease of the lethal sick pig of picking A type C.perfringens, scraping intestinal contents, 2 DEG C
Refrigeration；
(2) Spawn incubation: by the synthetic media of plain chocolate and glucose, high pressure sterilization, sterilising temp are in pressure cooker
110 DEG C, sterilization time 20min；Until temperature is reduced to 80 DEG C, pathological material of disease is poured into pressure cooker, 44 DEG C of high temperature anaerobic cultures
4h is passed on, and is passed on 3 times；
(3) prepared by bacterium solution: the bacterium solution passed on three times in step (2) being taken to access 37 DEG C of culture 8h of meat liver stomach membrane digestion soup, training
It is centrifuged 10min in 6000r/min after supporting, takes supernatant；Wherein, meat liver stomach membrane digestion soup preparation the following steps are included:
Beef 100g, beef liver 100g, hog gastric mucosa 150g are blended, distilled water 1000mL, hydrochloric acid 10mL and ethyl alcohol 100mL is added, is mixed
Cold soaking 20h after closing uniformly is slowly heated to 56 DEG C of temperature and keeps for 24 hours, and every 1h stirring is primary, removes oil slick rouge, digestive juice is taken to boil
Boiling, with 2mol/L sodium hydroxide solution 75mL tune pH value, flannelette filtering takes 116 DEG C of sterilizing 30min of supernatant spare.
(4) bacterium solution inactivates: the binary ethylenimine solution that mass fraction is 0.2%, which is added, to the bacterium solution of step (3) makes it most
Whole mass fraction is 0.04%, and mixed liquor is inactivated 20h in 30 DEG C of concussions, and the rear beta-propiolactone that is added makes its final concentration of 1g/L,
2h is inactivated in 37 DEG C of concussions, the sterile hypo solution of 1mol/L is added, keeps its final concentration consistent with binary ethylenimine, eventually
1h is only inactivated, inactivated bacterial liquid is obtained.
(5) vaccine preparation: by inactivated bacterial liquid, 1:3 is mixed by volume with adjuvant, obtains inactivated vaccine；Wherein, the adjuvant
For immunostimulating complex, the preparation method comprises the following steps: the 1) preparation of lipid mixture solution: lecithin and cholesterol are added to 20%
In Mega-10, making the two final concentration is all 10mg/mL；2) QS-21 the preparation of saponin(e solution: is dissolved in the PBS that pH is 7.2
In solution, adjustment concentration is 12mg/mL.3) prepared by immunostimulating complex: lipid mixture solution and saponin(e solution are pressed body
Product cracks 7h than being sufficiently mixed for 1:5 at room temperature；Ice-bath ultrasonic process, dialysis are standby in 4 DEG C of preservations after 0.22 μm of filter membrane degerming excessively
With.
Embodiment 2
The rapidly and efficiently preparation method of one boar A type C.perfringens inactivated vaccine:
(1) acquisition of pathogen: the pathological material of disease of the lethal sick pig of picking A type C.perfringens, scraping intestinal contents, 5 DEG C
Refrigeration；
(2) Spawn incubation: by the synthetic media of plain chocolate and glucose, high pressure sterilization, sterilising temp are in pressure cooker
110 DEG C, sterilization time 20min；Until temperature is reduced to 80 DEG C, pathological material of disease is poured into pressure cooker, 45 DEG C of high temperature anaerobic cultures
4h is passed on, and is passed on 3 times；
(3) prepared by bacterium solution: the bacterium solution passed on three times in step (2) being taken to access 37 DEG C of culture 10h of meat liver stomach membrane digestion soup, training
It is centrifuged 15min in 6000r/min after supporting, takes supernatant；Wherein, meat liver stomach membrane digestion soup preparation the following steps are included:
Beef 100g, beef liver 100g, hog gastric mucosa 150g are blended, distilled water 1000mL, hydrochloric acid 10mL and ethyl alcohol 100mL is added, is mixed
Cold soaking for 24 hours, is slowly heated to 57 DEG C of temperature and keeps for 24 hours after closing uniformly, and every 1h stirring is primary, removes oil slick rouge, digestive juice is taken to boil
Boiling, with 2mol/L sodium hydroxide solution 75mL tune pH value, flannelette filtering takes 116 DEG C of sterilizing 30min of supernatant spare.
(4) bacterium solution inactivates: the binary ethylenimine solution that mass fraction is 0.2%, which is added, to the bacterium solution of step (3) makes it most
Whole mass fraction is 0.05%, and mixed liquor is inactivated 20h in 30 DEG C of concussions, and the rear beta-propiolactone that is added makes its final concentration of 1g/L,
2h is inactivated in 37 DEG C of concussions, the sterile hypo solution of 1mol/L is added, keeps its final concentration consistent with binary ethylenimine, eventually
1h is only inactivated, inactivated bacterial liquid is obtained.
(5) vaccine preparation: by inactivated bacterial liquid, 1:4 is mixed by volume with adjuvant, obtains inactivated vaccine；Wherein, the adjuvant
For immunostimulating complex, the preparation method comprises the following steps: the 1) preparation of lipid mixture solution: lecithin and cholesterol are added to 20%
In Mega-10, making the two final concentration is all 10mg/mL；2) QS-21 the preparation of saponin(e solution: is dissolved in the PBS that pH is 7.2
In solution, adjustment concentration is 15mg/mL.3) prepared by immunostimulating complex: lipid mixture solution and saponin(e solution are pressed body
Product cracks 7h than being sufficiently mixed for 1:5 at room temperature；Ice-bath ultrasonic process, dialysis are standby in 4 DEG C of preservations after 0.22 μm of filter membrane degerming excessively
With.
Embodiment 3
The rapidly and efficiently preparation method of one boar A type C.perfringens inactivated vaccine:
(1) acquisition of pathogen: the pathological material of disease of the lethal sick pig of picking A type C.perfringens, scraping intestinal contents, 4 DEG C
Refrigeration；
(2) Spawn incubation: by the synthetic media of plain chocolate and glucose, high pressure sterilization, sterilising temp are in pressure cooker
110 DEG C, sterilization time 20min；Until temperature is reduced to 80 DEG C, pathological material of disease is poured into pressure cooker, 42 DEG C of high temperature anaerobic cultures
6h is passed on, and is passed on 3 times；
(3) prepared by bacterium solution: the bacterium solution passed on three times in step (2) being taken to access 37 DEG C of culture 6h of meat liver stomach membrane digestion soup, training
It is centrifuged 10min in 4000r/min after supporting, takes supernatant；Wherein, meat liver stomach membrane digestion soup preparation the following steps are included:
Beef 100g, beef liver 100g, hog gastric mucosa 150g are blended, distilled water 1000mL, hydrochloric acid 10mL and ethyl alcohol 100mL is added, is mixed
Cold soaking 20h after closing uniformly is slowly heated to 55 DEG C of holding 22h of temperature, and every 1h stirring is primary, removes oil slick rouge, digestive juice is taken to boil
Boiling, with 2mol/L sodium hydroxide solution 75mL tune pH value, flannelette filtering takes 116 DEG C of sterilizing 30min of supernatant spare.
(4) bacterium solution inactivates: the binary ethylenimine solution that mass fraction is 0.2%, which is added, to the bacterium solution of step (3) makes it most
Whole mass fraction is 0.02%, and mixed liquor is inactivated for 24 hours in 20 DEG C of concussions, and the rear beta-propiolactone that is added makes its final concentration of 1g/L,
2h is inactivated in 37 DEG C of concussions, the sterile hypo solution of 1mol/L is added, keeps its final concentration consistent with binary ethylenimine, eventually
1h is only inactivated, inactivated bacterial liquid is obtained.
(5) vaccine preparation: by inactivated bacterial liquid, 1:2 is mixed by volume with adjuvant, obtains inactivated vaccine；Wherein, the adjuvant
For immunostimulating complex, the preparation method comprises the following steps: the 1) preparation of lipid mixture solution: lecithin and cholesterol are added to 20%
In Mega-10, making the two final concentration is all 10mg/mL；2) QS-21 the preparation of saponin(e solution: is dissolved in the PBS that pH is 7.2
In solution, adjustment concentration is 10mg/mL.3) prepared by immunostimulating complex: lipid mixture solution and saponin(e solution are pressed body
Product cracks 5h than being sufficiently mixed for 1:3 at room temperature；Ice-bath ultrasonic process, dialysis are standby in 4 DEG C of preservations after 0.22 μm of filter membrane degerming excessively
With.
Embodiment 4
The rapidly and efficiently preparation method of one boar A type C.perfringens inactivated vaccine:
(1) acquisition of pathogen: the pathological material of disease of the lethal sick pig of picking A type C.perfringens, scraping intestinal contents, 4 DEG C
Refrigeration；
(2) Spawn incubation: by the synthetic media of plain chocolate and glucose, high pressure sterilization, sterilising temp are in pressure cooker
110 DEG C, sterilization time 20min；Until temperature is reduced to 80 DEG C, pathological material of disease is poured into pressure cooker, 43 DEG C of high temperature anaerobic cultures
5h is passed on, and is passed on 3 times；
(3) prepared by bacterium solution: the bacterium solution passed on three times in step (2) being taken to access 37 DEG C of culture 8h of meat liver stomach membrane digestion soup, training
It is centrifuged 12min in 5000r/min after supporting, takes supernatant；Wherein, meat liver stomach membrane digestion soup preparation the following steps are included:
Beef 100g, beef liver 100g, hog gastric mucosa 150g are blended, distilled water 1000mL, hydrochloric acid 10mL and ethyl alcohol 100mL is added, is mixed
Cold soaking 22h after closing uniformly is slowly heated to 56 DEG C of holding 22h of temperature, and every 1h stirring is primary, removes oil slick rouge, digestive juice is taken to boil
Boiling, with 2mol/L sodium hydroxide solution 75mL tune pH value, flannelette filtering takes 116 DEG C of sterilizing 30min of supernatant spare.
(4) bacterium solution inactivates: the binary ethylenimine solution that mass fraction is 0.2%, which is added, to the bacterium solution of step (3) makes it most
Whole mass fraction is 0.04%, and mixed liquor is inactivated 23h in 25 DEG C of concussions, and the rear beta-propiolactone that is added makes its final concentration of 1g/L,
2h is inactivated in 37 DEG C of concussions, the sterile hypo solution of 1mol/L is added, keeps its final concentration consistent with binary ethylenimine, eventually
1h is only inactivated, inactivated bacterial liquid is obtained.
(5) vaccine preparation: by inactivated bacterial liquid, 1:3 is mixed by volume with adjuvant, obtains inactivated vaccine；Wherein, the adjuvant
For immunostimulating complex, the preparation method comprises the following steps: the 1) preparation of lipid mixture solution: lecithin and cholesterol are added to 20%
In Mega-10, making the two final concentration is all 10mg/mL；2) QS-21 the preparation of saponin(e solution: is dissolved in the PBS that pH is 7.2
In solution, adjustment concentration is 12mg/mL.3) prepared by immunostimulating complex: lipid mixture solution and saponin(e solution are pressed body
Product cracks 6h than being sufficiently mixed for 1:3 at room temperature；Ice-bath ultrasonic process, dialysis are standby in 4 DEG C of preservations after 0.22 μm of filter membrane degerming excessively
With.
Comparative example 1
The bacterium solution inactivation process of 1 step of embodiment (4) is changed to: formalin is added to the bacterium solution of step (3), makes formaldehyde
Final mass score be 0.4%, 37 DEG C inactivation detoxification 3 days, obtain inactivated bacterial liquid；
Embodiment 5
Inactivated bacterial liquid, the untreated pig A type that inactivated bacterial liquid that 1 step of embodiment (4) obtains, comparative example 1 are obtained produce
Gas capsular clostridium virus, the mixed liquor without viral 1 inactivator of culture medium and embodiment, the culture medium without virus and comparison
The mixed liquor of 1 inactivator of example is inoculated into the ST cell of health, and does blank control (not being inoculated with), and blind passage to the third generation is observed
Cytopathy.Cell state is observed, as a result, it has been found that, inactivated bacterial liquid, the comparative example 1 that inoculation 1 step of embodiment (4) obtains obtain
Inactivated bacterial liquid, the mixed liquor without viral 1 inactivator of culture medium and embodiment, the culture medium without virus and comparative example 1 are gone out
The cell quantity of the mixed liquor of agent living is consistent with blank control group, meets normal cell growth state, for lesion occurs；And it is inoculated with
The cell whole lesion of untreated pig A type clostridium perfringens disease poison.
Embodiment 6
The sterility test of pig A type C.perfringens inactivated vaccine
Steriling test is tested by method as defined in " Chinese veterinary pharmacopoeia ", equal asepsis growth.
Embodiment 7
The security inspection of pig A type C.perfringens inactivated vaccine
(1) the minimum safety testing for using the single dose vaccine inoculation of age in days pig
Susceptible pig 20 of 30 ages in days health is taken, is randomly divided into 4 groups, 5/group.Preceding 3 groups of difference neck intramuscular injection embodiment
The vaccine (the 1st batch, the 2nd batch and the 3rd batch) of 3 batches of the 1 method preparation provided, 2ml/ is only；4th group be not inoculated with as pair
According to.It raises, observes 10 under the same terms, it is daily to observe whether dog spirit, diet, excrement are normal, and injection site and whole body have
It has no adverse reaction.
The result shows that the pig state of mind, diet, excrement are normal after single dose of intramuscular injection, injection site and
Whole body has no adverse reaction, and all pigs are strong to live.
(2) safety testing of single dose repeated inoculation vaccine
Susceptible pig 20 of 30 ages in days health is taken, is randomly divided into 4 groups, 5/group.Preceding 3 groups of difference intramuscular inoculations are according to embodiment
The vaccine of 3 batches of 1 method preparation, only, booster immunization is primary after 14 days by 2ml/, and 2ml/ is only；4th group be not inoculated with as pair
According to.It raises, observes 10 under the same terms, it is daily to observe whether pig spirit, diet, excrement are normal, and injection site and whole body have
It has no adverse reaction.
The result shows that the pig state of mind, diet, excrement are normal, injection site after intramuscular routes repeated inoculation vaccine
And whole body has no adverse reaction, all pigs are strong to live.
The safety testing of (3) overdose vaccine inoculations
Susceptible pig 20 of 30 ages in days health is taken, is randomly divided into 4 groups, 5/group.Preceding 3 groups of difference intramuscular inoculations are according to embodiment
3 batch vaccines of 1 preparation, 6ml/ is only；4th group is not inoculated with as control.It raises, observes 10 under the same terms, it is daily to see
Examine whether pig spirit, diet, excrement are normal, and injection site and whole body have no adverse reaction.
The result shows that the pig state of mind, diet, excrement are normal, injection part after intramuscular routes overdose vaccine inoculation
Position and whole body have no adverse reaction, and all pigs are strong to live.
(4) safety testing of the vaccine to farrowing sow
Vaccine is prepared according to 1 method of embodiment, is taken farrowing sow 10, is randomly divided into 2 groups, 5/group.1st group in antenatal
Intramuscular inoculation vaccine on the 20th, 8ml/ is only；2nd group is not inoculated with as control.It raises, observes 10 under the same terms, it is daily to observe
Whether pig spirit, diet, excrement normal, and injection site and whole body have no adverse reaction, and follow-up observation its farrow situation.
The result shows that farrowing sow in antenatal 20 age through muscle overdose inoculation after, the sow state of mind, diet, excrement
Normal, injection site and whole body have no adverse reaction, and all pigs are strong to live, and it is normal to farrow.
Conclusion
Safety test, the single dose repeated inoculation of the inoculation of a single dose have been carried out with 3 batches of pig A type clostridieum welchii inactivated vaccines
Safety testing, the inoculation of overdose safety test, be as a result safe.Show the vaccine for immunity inoculation pig
It is safe.Above-mentioned test (1)-(4) have also been carried out to embodiment 2, embodiment 3, embodiment 4, the results showed that, which is used for
Immunity inoculation pig is safe.
Embodiment 8
The potency test of pig A type C.perfringens inactivated vaccine
3 batches of pig A type clostridieum welchii inactivated vaccines of Example preparation take health susceptible pig 80, are randomly divided into 16 groups,
5/group.1,2,3 groups of pigs distinguish 3 batches of vaccines 2ml/ that intramuscular inoculations are prepared according to embodiment 1, and 4,5,6 groups of pigs distinguish muscle
3 batches of vaccines 2ml/ prepared according to embodiment 2 are inoculated with, 7,8,9 groups of pigs distinguish 3 batches that intramuscular inoculations are prepared according to embodiment 3
Only, 10,11,12 groups of pigs distinguish 3 batches of vaccines 2ml/ that intramuscular inoculations are prepared according to embodiment 4, control group 1,2,3 to vaccine 2ml/
3 crowdes of vaccine 2ml/ made from the preparation method that group pig difference intramuscular inoculation is provided according to Chinese patent CN101708332A only, sky
White group without immune.It is attacked respectively at 14 days, 30 days, 90 days, 120 days, 180 days, the culture solution 15ml living of oral clostridium on the 270th
Poison is observed 10, and incidence is recorded.The results are shown in Table 1.
The potency test of 1 pig A type C.perfringens inactivated vaccine of table
Remarks: protective rate=key number/test number living
To sum up, the vaccine safety of the application preparation is good, and local irritation is small, and the adverse reactions such as no lump, granuloma go out
It is existing；And immune efficacy is high, duration of immunity was long, up to 9 months or more；Efficacy test is carried out in duration of immunity, meets " Chinese veterinary drug
Allusion quotation " quality standard.
The above description is only a preferred embodiment of the present invention, is not intended to limit the scope of the invention, all utilizations
Equivalent structure or equivalent flow shift made by description of the invention is applied directly or indirectly in other relevant technology necks
Domain is included within the scope of the present invention.
Claims (7)
1. the rapidly and efficiently preparation method of a boar A type C.perfringens inactivated vaccine, which is characterized in that step are as follows:
(1) acquisition of pathogen: the pathological material of disease of the lethal sick pig of picking A type C.perfringens scrapes intestinal contents, refrigeration or cold
Freeze；
(2) Spawn incubation: by culture medium in pressure cooker high pressure sterilization, until temperature reduce after, pathological material of disease is poured into pressure cooker,
Anaerobic culturel passes on 3 times, and wherein culture medium is the synthetic media of plain chocolate and glucose；
(3) bacterium solution prepare: take the bacterium solution passed on three times in step (2) access the membrane digestion soup culture of meat liver stomach, after culture from
The heart takes supernatant；
(4) bacterium solution inactivates: the binary ethylenimine solution that mass fraction is 0.2%, which is added, to the bacterium solution of step (3) makes its final matter
Amount score is 0.02-0.05%, mixed liquor is inactivated 20-24h in 20-30 DEG C of concussion, the rear beta-propiolactone that is added makes its final concentration
For 1g/L, 2h is inactivated in 37 DEG C of concussions, the sterile hypo solution of 1mol/L is added, makes its final concentration and binary ethylenimine
Unanimously, inactivation 1h is terminated, inactivated bacterial liquid is obtained；
(5) vaccine preparation: inactivated bacterial liquid is mixed with adjuvant, obtains inactivated vaccine；
Wherein, inactivated bacterial liquid and the volume ratio of adjuvant are 1:(2-4)；
The adjuvant is immunostimulating complex；The preparation of the immunostimulating complex the following steps are included:
S1: the preparation of lipid mixture solution: lecithin and cholesterol are added in 20%Mega-10, make the two final concentration all
For 10mg/mL；
S2: the preparation of saponin(e solution: QS-21 being dissolved in the PBS solution that pH is 7.2, and adjustment concentration is 10-15mg/mL；
S3: immunostimulating complex preparation: being 1:(3-5 by lipid mixture solution and saponin(e solution by volume) it is sufficiently mixed
5-7h is cracked at room temperature；Ice-bath ultrasonic process, dialysis save backup after crossing 0.22 μm of filter membrane degerming in 4 DEG C.
2. preparation method according to claim 1, which is characterized in that in the step (1), refrigerated storage temperature is 2-5 DEG C, cold
Freezing temperature is -20 DEG C.
3. preparation method according to claim 1, which is characterized in that in the step (2), pressure cooker sterilising temp is
110 DEG C, sterilization time 20min；Until pathological material of disease is poured into pressure cooker after temperature drops to 80 DEG C, 42-45 DEG C of high temperature anaerobic training
Feeding 4-6h is passed on.
4. preparation method according to claim 1, which is characterized in that in the step (3), take in step (2) and pass three times
The bacterium solution in generation accesses 37 DEG C of culture 6-10h of meat liver stomach membrane digestion soup, is centrifuged 10- in 4000-6000r/min after culture
15min takes supernatant.
5. the preparation method according to claim 4, which is characterized in that the meat liver stomach membrane digestion soup ingredient includes: pig stomach
Mucous membrane, beef, beef liver, hydrochloric acid, sodium hydroxide, deionized water, ethyl alcohol.
6. the preparation method according to claim 4, which is characterized in that the preparation of the meat liver stomach membrane digestion soup includes following
Step: beef, beef liver, hog gastric mucosa are blended, and distilled water, hydrochloric acid and ethyl alcohol are added, after mixing cold soaking 20-24h, slowly
56 ± 1 DEG C of holding 22-24h of temperature are heated to, every 1h stirring is primary, removes oil slick rouge, takes digestive juice to boil, with 2mol/L hydrogen-oxygen
Change sodium solution tune pH value, flannelette filtering takes 116 DEG C of sterilizing 30min of supernatant spare.
7. a kind of pig A type C.perfringens inactivated vaccine that -6 any preparation methods are prepared according to claim 1.
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