CN107875377A - A kind of preparation method of E types C.perfringens toxoid vaccine - Google Patents
A kind of preparation method of E types C.perfringens toxoid vaccine Download PDFInfo
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
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Abstract
The invention discloses a kind of preparation method of E types C.perfringens toxoid vaccine, comprise the following steps：(1) E type C.perfringens strains are inoculated on sterile blood plating medium, Anaerobic culturel, carry out the recovery of bacterial strain；(2) the E type C.perfringens strains after recovery are inoculated in liquid sulfur glycollate broth, increasing bacterium is carried out under anaerobic condition, seed liquor is made；(3) seed liquor is inoculated in toxin producing medium, Anaerobic culturel, exotoxin liquid is prepared；(4) the exotoxin liquid of preparation is added thimerosal, that is, E type C.perfringens toxoid vaccines is prepared through inactivation, emulsification treatment.Present invention exploitation is prepared for the E type C.perfringens toxoid vaccines that immunity is strong, has no toxic side effect.The vaccine can effectively prevent the diseases such as calf caused by thus bacterium, lamb dysentery.
Description
Technical field
The present invention relates to veterinary biologicses technical field, and in particular to a kind of E types C.perfringens toxoid vaccine
Preparation method.
Background technology
C.perfringens, clostridieum welchii is once called as, according to its caused 4 kinds of main exotoxins (α, β, ε, ι), by aerogenesis pod
Film clostridium is divided into A, B, C, five toxin type types of D, E.Bohenel is pointed out：" it is due to that they form secretion that clostridieum welchii, which causes a disease,
Toxin and metabolite, no toxin, which produces, clinical symptoms would not occurs ".E type perfringens shuttles as one of the bacterium member
Bacterium Major Secretory α and ι exotoxin.Although the relevant E types C.perfringens disease of few classical symptoms in animal productiong, still
There is report to point out that E type C.perfringens accounts for the 5% of all C.perfringens separation strains, while 5% E type perfringens
Clostridium can be relevant with 50% ox lethal hemolytic enteritis.And E type C.perfringens primary attack lambs, calf etc. are anti-
Hay animal.Early 20th century, in Britain, it was recently reported that the generation of the ι toxin enterotoxemia of calf and lamb, had been reported that at that time
A number of ox hemolytic, necrotic enteritis are the generation of dysentery, and have also detected that E type bacterium from the sheep of morbidity and ox
Strain and ι toxin.In Brazil, E types C.perfringens accounting in the bacterial strain of separation is bigger, is connect in the ox of health and diarrhoea
Nearly 30% sample carries ι toxin, wherein the ox for having 9% can die suddenly.E types bacterium can also infect multiple species simultaneously,
From the goat with enterotoxemia clinical symptoms, deer, in wild boar, E type C.perfringens bacterial strains are also isolated to.Therefore, it is
Potential hazard of the prevention E types C.perfringens to calf, lamb, it is necessary to carried out to the vaccine of E type C.perfringens
Research.
The chemical composition for the main exotoxins that C.perfringens is formed is protein, has preferable immunogenicity.Cause
This, according to the pathogenesis of C.perfringens, prepares different classes of clostridium perfringens toxoid, class is made after being inactivated
Toxin vaccine, prevented and treated for livestock and poultry, infallible immune and therapeutic effect can be reached.At present be related to A types-
Report prepared by D type C.perfringens toxoid vaccine, but on the preparation of E type C.perfringens toxoid vaccines, so far
The present has not yet to see report.
The content of the invention
For above-mentioned prior art, it is an object of the invention to provide a kind of system of E types C.perfringens toxoid vaccine
Preparation Method.
To achieve the above object, the present invention adopts the following technical scheme that：
A kind of preparation method of E types C.perfringens toxoid vaccine, comprises the following steps：
(1) E type C.perfringens strains are inoculated on sterile blood plating medium, Anaerobic culturel, carry out bacterial strain
Recovery；
(2) the E type C.perfringens strains after recovery are inoculated in liquid sulfur glycollate broth, anaerobism
Under the conditions of carry out increasing bacterium, be made seed liquor；
(3) seed liquor is inoculated in toxin producing medium, Anaerobic culturel, filtration sterilization, exotoxin liquid is prepared；
(4) the exotoxin liquid of preparation is added thimerosal, that is, E type perfringens is prepared through inactivation, emulsification treatment
Clostridium toxoid vaccine.
Preferably, in step (1), the E types C.perfringens strain accepted standard strain E types NCTC8084
(National Collection of Type Cultures, Britain's Culture Collection).The bacterial strain can be by existing
There are the conven-tional channels in technology to directly obtain.The bacterial strain is through test of many times, compared with other E type C.perfringens bacterial strains, production
Malicious best results.
Preferably, in step (1), the sterile blood plating medium is prepared by the following method：
By 3.8g blood agars basis, 1g glucose, it is dissolved in 100mL deionized water, 121 DEG C, 15min autoclavings,
50 DEG C are cooled to, 5mL is added and takes off fiber Sheep Blood, rock uniformly, be poured into the plate of sterilizing, produce.
Preferably, in step (1), the temperature of Anaerobic culturel is 37 DEG C, and the time of Anaerobic culturel is 36h.
Preferably, in step (2), 41 DEG C of Anaerobic culturel 18h carry out increasing bacterium.Its increasing bacterium of different types of C.perfringens
There is notable difference in the condition of culture, for E type C.perfringens, temperature and time of the present invention to its Zengjing Granule
Investigation is optimized in condition, as a result finds, temperature is too high or the too low growth for being all unfavorable for E type C.perfringens of temperature,
Incubation time is too short, and enriching effect can be made not notable；Incubation time is long, can influence the production exotoxin in bacterial strain later stage.Work as culture
Temperature is 41 DEG C, and when incubation time is 18h, its enriching effect is optimal.
Preferably, in step (3), the toxin producing medium is prepared by the following method：
2g peptones, 1g dextrin, 2g yeast extracts, 1.2g L-arginines are dissolved in 100mL PBSs, with dense salt
It is 7.5 that acid, which adjusts pH, autoclaving, is produced.
Preferably, in step (3), the condition of Anaerobic culturel is：43 DEG C of anaerobism shaken cultivation 6h.
It is further preferred that during Anaerobic culturel, the gas of anaerobic condition, which is formed by volume fraction, is：88%N2, 5%CO2、
7%H2。
Anaerobic culturel is carried out in toxin producing medium to prepare exotoxin liquid, is to prepare E type C.perfringens toxoids
The committed step of vaccine, because under the conditions of identical adjuvant, immune effect of vaccine is often proportional with content of toxins, content of toxins
Higher, vaccine effect is better.
Because the condition that different types of C.perfringens excretes poison is different, be difficult to each other use for reference or
With reference to, for the preparation of E type C.perfringens exotoxin liquid, the present invention from the nutritional ingredient of toxin producing medium, pH conditions, detest
Gas composition of the temperature of oxygen culture, time and anaerobic condition etc. is explored, and is as a result found, production of the invention poison culture
Base especially meets the demand that E type C.perfringens excretes poison to nutritional ingredient, peptone, yeast of the present invention with commercialization
The finished products such as extract substitute the raw material such as the uncontrollable beef of initial quality, beef liver as raw material, pass through screening and culturing medium
Be formulated and optimize the condition of culture of toxin producing, the Toxin producing C of culture medium is greatly improved, and toxigenicity can stablize,
It is quality controllable, prepare it is easy to use, cheap.PH conditions are also to influence the key factor of Output of toxin, poison when pH is 7.5
Plain yield highest, pH can reduce the yield of toxin below or above 7.5.More it is essential that the temperature of Anaerobic culturel and when
Between condition, although in bacterial growth Suitable ranges, improve cultivation temperature and be advantageous to the growth of thalline, thalli growth and
Bacterium production poison adheres to the different stages separately, and the production of highest toxin can be not necessarily obtained most beneficial for the cultivation temperature of thalli growth
Amount, gropes to find, 43 DEG C of anaerobism shaken cultivation 6h can obtain highest Output of toxin through test of many times.In addition, anaerobic environment
Gas composition grown for E types C.perfringens and excrete poison and can all have an impact, if the gas componant changes
Become such as mixed gas composition ratio to change, may result in E type C.perfringens and do not grow, while will not also secrete poison
Element.
Preferably, in step (4), inactivate the method that uses for：Formaldehyde is added in outside toxin solution, makes its final concentration of
0.3%, 37 DEG C are placed in, fully vibration, rotating speed 140rmp, inactivate 24h.
Because the exotoxin secreted by different types of C.perfringens has difference, external toxin solution is caused to be gone out
Treatment conditions living have differences, and external toxin solution carries out the effect of the inactivation treatment mainly concentration with formaldehyde, temperature, inactivation
Time etc. is relevant, using the formaldehyde and high-temperature inactivation of high concentration, although inactivation time can be reduced, the loss to antigen also compared with
Greatly；Inactivation time is too short, can make that inactivation is insufficient, and inactivation time is long, cost is on the one hand added, on the other hand to antigen
There is loss.Consider the efficiency of inactivation and antigenic influence, the present invention have been carried out preferably, as a result sending out to inactivation condition
It is existing, optimal inactivating efficacy can be obtained using 0.3% formaldehyde, 37 DEG C, inactivation 24h.
Preferably, in step (4), into the exotoxin liquid after inactivation add white-oil adjuvant emulsified, exotoxin liquid with
The volume ratio that white-oil adjuvant adds is 1:2.
Preferably, the composition of white-oil adjuvant is：After 12 parts of white oils and 1 part of Span-80 mixing, add account for white oil and
The aluminum stearate of Span-80 mixed liquors gross mass 1.5%；Positive carbon number is the quality percentage shared by C16-C20 alkane in white oil
Number is 10-20%.Found through test of many times, using above-mentioned composition white-oil adjuvant prepare vaccine, its antibody titer highest and
Side effect is minimum.
Beneficial effects of the present invention：
Exploitation is prepared for the E type C.perfringens toxoid vaccines that immunity is strong, has no toxic side effect to the present invention first.Should
Vaccine can effectively prevent the diseases such as calf caused by thus bacterium, lamb dysentery.
Embodiment
It is noted that described further below is all exemplary, it is intended to provides further instruction to the application.It is unless another
Indicate, all technologies used herein and scientific terminology are with usual with the application person of an ordinary skill in the technical field
The identical meanings of understanding.
In order that the technical scheme of the application can clearly be understood by obtaining those skilled in the art, below with reference to tool
The embodiment of body describes the technical scheme of the application in detail.
Test material used is the conventional test material in this area in the embodiment of the present invention, can pass through commercial channel
It is commercially available.Wherein, blood agar basis, THIOGLYCOLLIC ACID salt broth (FT), yeast extract etc. are conventional commercially available
Product.
Embodiment 1：Calf, lamb dysentery E type C.perfringens toxoid vaccines preparation
Comprise the following steps that：
(1) exotoxin is produced：Pipettor takes E type C.perfringens strain NCTC8084 (National Collection
Of Type Cultures8084) preserve liquid be coated on sterile blood plating medium (sterile blood plating medium：3.8g blood agar
Basis, 1g glucose, is dissolved in 100mL deionized water, 121 DEG C, 15min autoclavings, is cooled to 50 DEG C or so, adds
5mL takes off fiber Sheep Blood, rocks the plate for uniformly, being poured into sterilizing), 37 DEG C of Anaerobic culturel 36h.Taken with sterile oese
5-10 colony inoculation increases bacterium in sterile THIOGLYCOLLIC ACID salt broth (FT), 41 DEG C of Anaerobic culturel 18h.After taking culture
E8084 seed liquors according to 7：100 volume ratio is inoculated in homemade sterile toxin producing medium (toxin producing medium：2g albumen
Peptone, 1g dextrin, 2g yeast extracts, 1.2g L-arginines are dissolved in 100mL PBSs, and it is 7.5 to adjust pH with concentrated hydrochloric acid, high
Pressure sterilizing), (gas is formed 43 DEG C of anaerobism：88%N2, 5%CO2, 7%H2) shaken cultivation 6h.4 DEG C of 10000rpm of nutrient solution, from
Heart 30min, supernatant is taken, bacteria filter filtration sterilization, obtains exotoxin.
(2) exotoxin inactivates：The exotoxin produced is added into formaldehyde, and makes final concentration of 0.3%, and it is 7.5 to adjust pH, is put
In 37 DEG C, fully vibration, rotating speed 140rpm, 24h is inactivated, you can inactivation is complete.
(3) prepared by toxoid vaccine：By the toxoid after inactivating well with white-oil adjuvant with 1:2 volume ratio is emulsified,
And add thimerosal, make its final concentration of 0.01%, after fully emulsified, toxoid vaccine is made.
Embodiment 2：
(1) safety detection
3 1.5kg or so rabbit is taken, is subcutaneously injected 5mL toxoid vaccine (prepared by embodiment 1), Continuous Observation 10d,
As a result injection site is shown without swelling, situations such as granuloma.
(2) vaccine potency is examined
With body weight 22g or so Kunming mouse 6, it is divided into two groups, it is (real that 0.2mL toxoid vaccines are subcutaneously injected in injection respectively
Example 1 is applied to prepare) and 0.2mL physiological saline, it is each to inject minimal lethal dose E type clostridium perfringens toxoids after 28 days
0.3mL, observe 5, control group small white mouse is all dead；Immune group small white mouse 3 is not only dead, continues observation 5 days, no dysentery etc.
Disease symptom, health survival.Prove the vaccine of the present invention to the protective rate of experimental animal up to 100%.
Comparative example 1：
Pipettor takes E type C.perfringens strains NCTC8084 (National Collection of Type
Cultures8084) preserve liquid and be coated on sterile blood plating medium (sterile blood plating medium：3.8g blood agars basis, 1g
Glucose, it is dissolved in 100mL deionized water, 121 DEG C, 15min autoclavings, is cooled to 50 DEG C or so, adds 5mL and take off fiber
Sheep Blood, rock the plate for uniformly, being poured into sterilizing), 37 DEG C of Anaerobic culturel 36h.5-10 bacterium is taken with sterile oese
Fall to be inoculated in sterile THIOGLYCOLLIC ACID salt broth (FT), 37 DEG C of Anaerobic culturel 12h increase bacterium.Take the E8084 after culture
Seed liquor is according to 7：100 volume ratio is inoculated in homemade sterile toxin producing medium (toxin producing medium：2g peptones, 1g pastes
Essence, 2g yeast extracts, 1.2g L-arginines are dissolved in 100mL PBSs, and it is 7.4 to adjust pH with concentrated hydrochloric acid, autoclaving),
(gas is formed 40 DEG C of anaerobism：88%N2, 5%CO2, 7%H2) shaken cultivation 5h.4 DEG C of 10000rpm of nutrient solution, 30min is centrifuged,
Supernatant is taken, bacteria filter filtration sterilization, obtains exotoxin.
Comparative example 2：
Pipettor takes E type C.perfringens strains NCTC8084 (National Collection of Type
Cultures8084) preserve liquid and be coated on sterile blood plating medium (sterile blood plating medium：3.8g blood agar is basic, 1g
Glucose, it is dissolved in 100mL deionized water, 121 DEG C, 15min autoclavings, is cooled to 50 DEG C or so, adds 5mL and take off fiber
Sheep Blood, rock the plate for uniformly, being poured into sterilizing), 37 DEG C of Anaerobic culturel 46h.5-10 bacterium is taken with sterile oese
Fall to be inoculated in sterile THIOGLYCOLLIC ACID salt broth (FT), 38 DEG C of Anaerobic culturel 14h increase bacterium.Take the E8084 after culture
Seed liquor is according to 7：100 volume ratio is inoculated in homemade sterile toxin producing medium (toxin producing medium：2g peptones, 1g pastes
Essence, 2g yeast extracts, 1.2g L-arginines are dissolved in 100mL PBSs, and it is 7.4 to adjust pH with concentrated hydrochloric acid, autoclaving),
(gas is formed 38 DEG C of anaerobism：85%N2, 9%CO2, 6%H2) shaken cultivation 5h.4 DEG C of 10000rpm of nutrient solution, 30min is centrifuged,
Supernatant is taken, bacteria filter filtration sterilization, obtains exotoxin.
Comparative example 3：
Pipettor takes E type C.perfringens strains NCTC8084 (National Collection of Type
Cultures8084) preserve liquid and be coated on sterile blood plating medium (sterile blood plating medium：3.8g blood agars basis, 1g
Glucose, it is dissolved in 100mL deionized water, 121 DEG C, 15min autoclavings, is cooled to 50 DEG C or so, adds 5mL and take off fiber
Sheep Blood, rock the plate for uniformly, being poured into sterilizing), 37 DEG C of Anaerobic culturel 36h.5-10 bacterium is taken with sterile oese
Fall to be inoculated in sterile THIOGLYCOLLIC ACID salt broth (FT), 39 DEG C of Anaerobic culturel 12h increase bacterium.Take the E8084 after culture
Seed liquor is according to 7：100 volume ratio is inoculated in homemade sterile toxin producing medium (toxin producing medium：2g peptones, 1g pastes
Essence, 2g yeast extracts, 1.2g L-arginines are dissolved in 100mL PBSs, and it is 7.4 to adjust pH with concentrated hydrochloric acid, autoclaving),
(gas is formed 45 DEG C of anaerobism：88%N2, 5%CO2, 7%H2) shaken cultivation 4h.4 DEG C of 10000rpm of nutrient solution, 30min is centrifuged,
Supernatant is taken, bacteria filter filtration sterilization, obtains exotoxin.
The exotoxin that respectively prepared by Example 1, comparative example 1-3, tail vein injection body weight is the small of 16-18g after dilution
White mouse, 2 are injected per titre, every injection 0.2ml, observes the mouse death condition in 72h after injection.Record can make mouse
2/2 dead minimum toxin amount occurs.As a result find, ectotoxic minimum toxin amount prepared by comparative example 1-3 is respectively to implement
2.4 times, 5.6 times and 3.8 times of example 1.Illustrate under different condition of culture, the production poison amount significant difference of E type C.perfringens,
Cultivated using the method for the present invention, the production poison amount highest of E type C.perfringens.
The preferred embodiment of the application is the foregoing is only, is not limited to the application, for the skill of this area
For art personnel, the application can have various modifications and variations.It is all within spirit herein and principle, made any repair
Change, equivalent substitution, improvement etc., should be included within the protection domain of the application.
Claims (9)
1. a kind of preparation method of E types C.perfringens toxoid vaccine, it is characterised in that comprise the following steps：
(1) E type C.perfringens strains are inoculated on sterile blood plating medium, Anaerobic culturel, carry out the recovery of bacterial strain；
(2) the E type C.perfringens strains after recovery are inoculated in liquid sulfur glycollate broth, anaerobic condition
Under carry out increasing bacterium, be made seed liquor；
(3) seed liquor is inoculated in toxin producing medium, Anaerobic culturel, filtration sterilization, exotoxin liquid is prepared；
(4) the exotoxin liquid of preparation is added thimerosal, that is, E type C.perfringens is prepared through inactivation, emulsification treatment
Toxoid vaccine.
2. preparation method according to claim 1, it is characterised in that in step (1), the sterile blood plating medium by
Following method is prepared：
By 3.8g blood agars basis, 1g glucose, it is dissolved in 100mL deionized water, 121 DEG C, 15min autoclavings, cools down
To 50 DEG C, add 5mL and take off fiber Sheep Blood, rock uniformly, be poured into the plate of sterilizing, produce.
3. preparation method according to claim 1, it is characterised in that in step (1), the temperature of Anaerobic culturel is 37 DEG C,
The time of Anaerobic culturel is 36h.
4. preparation method according to claim 1, it is characterised in that in step (2), 41 DEG C of Anaerobic culturel 18h are increased
Bacterium.
5. preparation method according to claim 1, it is characterised in that in step (3), the toxin producing medium is by such as lower section
Method is prepared：
2g peptones, 1g dextrin, 2g yeast extracts, 1.2g L-arginines are dissolved in 100mL PBSs, adjusted with concentrated hydrochloric acid
PH is 7.5, autoclaving, is produced.
6. preparation method according to claim 1, it is characterised in that in step (3), the condition of Anaerobic culturel is：43℃
Anaerobism shaken cultivation 6h.
7. preparation method according to claim 1, it is characterised in that during Anaerobic culturel, the gas of anaerobic condition, which is formed, to be pressed
Volume fraction is：88%N2, 5%CO2, 7%H2。
8. preparation method according to claim 1, it is characterised in that in step (4), inactivate the method that uses for：Outwards
Formaldehyde is added in toxin solution, make its final concentration of 0.3%, be placed in 37 DEG C, fully vibration, rotating speed 140rmp, inactivate 24h.
9. preparation method according to claim 1, it is characterised in that in step (4), add into the exotoxin liquid after inactivation
Enter white-oil adjuvant to be emulsified, exotoxin liquid is 1 with the volume ratio that white-oil adjuvant adds:2.
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| CN108892725A (en) * | 2018-04-18 | 2018-11-27 | 山东农业大学 | A kind of preparation method of E type C.perfringens antitoxic serum |
| CN110314227A (en) * | 2019-06-20 | 2019-10-11 | 青海省畜牧兽医科学院 | A kind of preparation method of the overworked macaque vaccine of sheep |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108892725A (en) * | 2018-04-18 | 2018-11-27 | 山东农业大学 | A kind of preparation method of E type C.perfringens antitoxic serum |
| CN110314227A (en) * | 2019-06-20 | 2019-10-11 | 青海省畜牧兽医科学院 | A kind of preparation method of the overworked macaque vaccine of sheep |
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