CN108892725A - A kind of preparation method of E type C.perfringens antitoxic serum - Google Patents
A kind of preparation method of E type C.perfringens antitoxic serum Download PDFInfo
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- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
- C07K16/1282—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Clostridium (G)
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Abstract
The present invention relates to a kind of preparation methods of E type C.perfringens antitoxic serum；It is first to prepare E type C.perfringens exotoxin；And according to volume ratio 1 after 0.3% formalin-inactivated E type C.perfringens exotoxin:2 addition white-oil adjuvants are sufficiently mixed；And preservative is added as the first antigen；Using the E type C.perfringens toxoid after inactivation as the second antigen；Using the first antigen and the second antigen according to immune programme immunity inoculation animal, after the 14d after immunoprophylaxis, by animal jugular vein, sterile blood sampling is carried out；Serum is collected, E type C.perfringens antitoxic serum is obtained；E type C.perfringens hyper-immune serum prepared by the present invention is safe and efficient, and the urgent immunity for illness domestic animal is inoculated with, and safety is imitated.
Description
Invention field
The present invention relates to a kind of preparation methods of E type C.perfringens antitoxic serum.
Background technique
C.perfringens is once called as clostridieum welchii, according to 4 kinds of main exotoxins (α, β, ε, ι) that it is generated, will produce gas pod
Film clostridium is divided into five toxin type types of A, B, C, D, E.Bohenel is pointed out:" it is since they form secretion that clostridieum welchii, which causes a disease,
Clinical symptoms would not occur in toxin and metabolite, no toxin generation ".E type perfringens shuttle as one of the bacterium member
Bacterium Major Secretory α and ι exotoxin.After the bacterium infects calf and lamb, dysentery is generated, often acute onset is dead, and dissect is visible
Gastrointestinal mucosa hyperemia, oedema drawn game focal hemorrhage.It has been also reported that being separated to E type from the turkey meat product of processing produces gas pod
Film clostridium shows that the residual of the bacterium or pollution have food safety risk in the product.In addition, in the excrement of surface health and diarrhea ox
Just about 30% sample suspection contains C.perfringens in sample, there is 4% sample in the fecal specimens of surface health ox
Detect E type C.perfringens；There is 13% sample detection to arrive E type perfringens shuttle in the fecal specimens of diarrhea ox
Bacterium.
" thinking that C.perfringens causes a disease is the toxin secreted due to them and generation with outstanding (2001) etc. for Bohenel and bavin
Thank to product, clinical condition would not occur in no toxin production ".Meanwhile E type C.perfringens is pathogenic and anxious with morbidity, dead
Die the features such as fast.For the pathogenic characteristic of E type C.perfringens, to control E type C.perfringens and give aquaculture bring
Harm, vaccine immunization is main control measure, but in immune seeded process, and often there is immuning failure can
Energy；And it is increasing along with drug resistance of the C.perfringens to antibiotic.
Summary of the invention
In order to further ensure that livestock culturing industry develops, the present invention provides a kind of E type C.perfringens antitoxin blood
Clear preparation method.
A kind of preparation method of E type C.perfringens antitoxic serum, steps are as follows：
1, E type C.perfringens liquid is inoculated in blood agar culture-medium, 37 DEG C of Anaerobic culturel 48h；Then 5-10 are taken
For bacterium colony in thioglycollate medium, 39 DEG C of Anaerobic culturel 18h obtain seed liquor；Seed liquor is added to the production of sterilizing
It in malicious culture medium, is centrifuged after 43 DEG C of Anaerobic culturel 6h, discards precipitating, take supernatant liquid filtering, obtain the outer poison of E type C.perfringens
Element；
2, the E type C.perfringens exotoxin for taking preparation, uses the formalin-inactivated E of 0.3% (mass volume ratio g/mL)
Type C.perfringens exotoxin (formaldehyde that 0.3g is added in the E type C.perfringens exotoxin of i.e. every 1mL)；
3, into the E type C.perfringens exotoxin after inactivation according to volume ratio 1:2 addition white-oil adjuvants are sufficiently mixed；
And (the E type C.perfringens exotoxin addition of i.e. every 1mL of 0.01% (mass volume ratio g/mL) preservative thimerosal is added
The preservative of 0.01g), white-oil adjuvant E type C.perfringens toxoid vaccine is obtained as the first antigen；Step 2) is inactivated
E type C.perfringens toxoid afterwards is as the second antigen；
4, the 14d using the first antigen and the second antigen according to immune programme immunity inoculation animal, after immunoprophylaxis
Afterwards, by animal jugular vein, sterile blood sampling is carried out；Serum is collected, E type C.perfringens antitoxic serum is obtained.
The immune programme is as shown in table 1.
1 immune programme of table
The E type C.perfringens is deposit number NCTC8084, is preserved in United Kingdom National Type Culture Collection institute
(NCTC) E type C.perfringens (National Collection of Type Cultures 8084).
Various nutrient media components are：
Blood agar culture-medium：The glucose sugar of 3.8g blood agar basis and 1g is taken to be dissolved in 95mL deionized water, 121 DEG C of height
Pressure sterilizing 15min, when then culture medium is cooled to 50 DEG C or so, is added sterile defibrination Sheep Blood 5mL, is uniformly mixed
Afterwards, it pours into sterile plate；
Toxin producing medium：2g yeast extract, 2g casein, 1.2g L-arginine and 1g dextrin is taken to be dissolved in
In 100mL0.025mol/L phosphate buffer (PBS), then adjusting pH is 7.5,121 DEG C of high pressure sterilization 15min.
The 0.025mol/LPBS) preparation method is：8g sodium chloride (NaCl), 0.2g potassium chloride (KCl), 3.58g 12
Hypophosphite monohydrate disodium hydrogen (Na2HPO4.12H20), 0.27g potassium dihydrogen phosphate (KH2PO4) be dissolved in the deionized water of 1L, 121 DEG C
High pressure sterilization 15min.
Beneficial effect：
E type C.perfringens hyper-immune serum prepared by the present invention is safe and efficient, and the urgent immunity for illness domestic animal connects
Kind, safety is imitated.
Specific embodiment
Embodiment 1
The preparation of E type C.perfringens antitoxic serum：
In sterile super-clean bench, using sterile pipettor, 30 μ L E type C.perfringens bacterium solutions are taken, it is uniform to apply
It is distributed on blood agar culture-medium plate；In 37 DEG C of insulating boxs, Anaerobic culturel 48h；Then take 5-10 bacterium colony in liquid sulfur ethyl alcohol
Hydrochlorate culture medium, 39 DEG C of Anaerobic culturel 18h, the enrichment liquid of culture is seed liquor.Take culture after E8084 seed liquor according to
7：100 volume ratio is inoculated in sterile toxin producing medium, and 43 DEG C, (gas constitutes volume ratio to anaerobism：88%N2, 5%CO2With
7%H2) shaken cultivation 6h.4 DEG C of 10000rpm of culture solution are centrifuged 30min, take supernatant, bacteria filter filtration sterilization obtains outer poison
Plain solution.Formaldehyde will be added in exotoxin solution, and makes in exotoxin solution formaldehyde final concentration of 0.3% (g/mL) (outside every ml
0.3g formaldehyde is added in toxin soiutions)；
37 DEG C are placed in, is sufficiently vibrated, revolving speed 140rpm, inactivation for 24 hours, can inactivate completely.By the class after inactivation completely
White-oil adjuvant is added in toxin soiutions as the first antigen：The volume ratio 1 of toxoid solution and white-oil adjuvant after inactivation completely:
2；And thimerosal is added and is emulsified, make final concentration of 0.01% (g/mL) ((the E type of i.e. every 1mL of thimerosal in mixed solution
The preservative of C.perfringens exotoxin addition 0.01g)), after fully emulsified, white-oil adjuvant toxoid vaccine (first is made
Antigen).It is the second antigen by the toxoid solution after inactivation completely；Use the first antigen and the second antigen, difference immunity inoculation 3
Sheep (every sheep weight is about 30kg)；
The mode of immunity inoculation is as shown in table 1；After immunoprophylaxis 14d, by sheep jugular vein, sterile blood sampling is carried out.
37 DEG C of standing 1h of blood, 4 DEG C overnight, collects serum, obtains E type C.perfringens antitoxic serum.
1 sheep reinforced immunological program of table
Antitoxic serum safety detection：5 Kunming small white mouses are taken, every mouse weight is 20g or so；
Every 500 μ L of intravenous injection E type C.perfringens antitoxic serum prepared by the present invention is observed 10.As a result
The part of display injection is abnormal without swelling, extravasated blood or ulcer etc., and overall health of patients is normal, and no discomfort stress not wait adverse reactions.
(referring to 2001 editions《People's Republic of China's veterinary biologics quality standard》).
Urgent Protection：Kunming small white mouse (18-20g) 20 is taken, is divided into 4 groups, every group 5.4 groups are injected intraperitoneally respectively
1.0, (exotoxin protein content is the E type C.perfringens exotoxin serum prepared by the present invention of 0.5,0.25 and 0.1mL
2.26mg/mL), and the lethal time and overall health of patients of Kunming small white mouse are recorded, as shown in table 2.It is final to determine injection
Mice dying, lethal time about 5-6h can be caused when 0.25mLE type C.perfringens exotoxin serum.
Table 2 attacks the lethal time and overall health of patients of Kunming small white mouse after poison
Note：* it is depressed to represent spirit, move freely reduction；It is depressed that * represents spiritual height, motionless；* * represents death
Embodiment 2
Examination is protected using the urgent immunity that E type C.perfringens antitoxic serum prepared by the present invention carries out small white mouse
It tests.8 small white mouses (18-20g) are taken, are divided into test group and control group, every group 4.0.25mL is injected intraperitoneally in two groups of small white mouses
E type C.perfringens exotoxin, after attacking malicious 0.5h, test group intraperitoneal mouse inject 0.15mL serum, control group little Bai
The PBS of same dose is injected intraperitoneally in mouse, and 4 small white mouses of final test group all survive, all dead after control group small white mouse 6h.
Referring to (2001 editions《People's Republic of China's veterinary biologics quality standard》) carry out safety examination and sterile
Property examine.Further, the antibody level of serum is measured by serum neutralization titer, as the result is shown the E type perfringens shuttle of 0.1mL
Bacterium antitoxic serum can neutralize 250 E type C.perfringens exotoxin minimum lethal doses (7.5 μ L).It has carried out simultaneously tight
Anxious protection test.
Conclusion：E type C.perfringens hyper-immune serum prepared by the present invention is safe and efficient, for the urgent of illness domestic animal
Immunity inoculation, safety are imitated.
Various nutrient media components are as follows：
Blood agar culture-medium：The glucose sugar of 3.8g blood agar basis and 1g is taken to be dissolved in 95mL deionized water, 121 DEG C of height
Pressure sterilizing 15min, when then culture medium is cooled to 50 DEG C or so, is added sterile defibrination Sheep Blood 5mL, is uniformly mixed
Afterwards, it pours into sterile plate.
Toxin producing medium：2g yeast extract, 2g casein, 1.2g L-arginine and 1g dextrin is taken to be dissolved in
In 100mL0.025mol/L phosphate buffer (PBS), then adjusting pH is 7.5,121 DEG C of high pressure sterilization 15min.
The 0.025mol/L PBS preparation method is：8g sodium chloride (NaCl), 0.2g potassium chloride (KCl), 3.58g 12
Hypophosphite monohydrate disodium hydrogen (Na2HPO4.12H20), 0.27g potassium dihydrogen phosphate (KH2PO4) be dissolved in the deionized water of 1L, 121 DEG C
High pressure sterilization 15min.
Claims (4)
1. a kind of preparation method of E type C.perfringens antitoxic serum, it is characterised in that：Steps are as follows：
1) E type C.perfringens liquid is inoculated in blood agar culture-medium, 37 DEG C of Anaerobic culturel 48h；Then 5-10 bacterium colony is taken
In thioglycollate medium, 39 DEG C of Anaerobic culturel 18h obtain seed liquor；Seed liquor is added to the production poison training of sterilizing
It supports in base, is centrifuged after 43 DEG C of Anaerobic culturel 6h, discards precipitating, take supernatant liquid filtering, obtain E type C.perfringens exotoxin；
2) the E type C.perfringens exotoxin of preparation is taken, the first of 0.3g is added in the E type C.perfringens exotoxin of every 1mL
Aldehyde inactivation；
3) into the E type C.perfringens exotoxin after inactivation according to volume ratio 1:2 addition white-oil adjuvants are sufficiently mixed；Often
The preservative of 0.01g is added in the E type C.perfringens exotoxin of 1mL, obtains white-oil adjuvant E type C.perfringens toxoid
Vaccine is as the first antigen；Using the E type C.perfringens toxoid after step 2) inactivation as the second antigen；
4) using the first antigen and the second antigen according to immune programme immunity inoculation animal, after the 14d after immunoprophylaxis, lead to
Animal jugular vein is crossed, sterile blood sampling is carried out；Serum is collected, E type C.perfringens antitoxic serum is obtained；
The immune programme is as shown in table 1：
1 immune programme of table
2. a kind of preparation method of E type C.perfringens antitoxic serum as described in claim 1, it is characterised in that：Institute
The E type C.perfringens stated is deposit number NCTC8084, be preserved in United Kingdom National Type Culture Collection E type perfringens
Clostridium.
3. a kind of preparation method of E type C.perfringens antitoxic serum as described in claim 1, it is characterised in that：Institute
The preservative stated is thimerosal.
4. a kind of preparation method of E type C.perfringens antitoxic serum as described in claim 1, it is characterised in that：Respectively
Planting nutrient media components is：
Blood agar culture-medium：The glucose sugar of 3.8g blood agar basis and 1g is taken to be dissolved in 95mL deionized water, 121 DEG C of high pressures are gone out
When then culture medium is cooled to 50 DEG C or so, sterile defibrination Sheep Blood 5mL is added in bacterium 15min, after mixing,
It pours into sterile plate；
Toxin producing medium：2g yeast extract, 2g casein, 1.2g L-arginine and 1g dextrin is taken to be dissolved in 100mL
In 0.025mol/L phosphate buffer PBS, then adjusting pH is 7.5,121 DEG C of high pressure sterilization 15min；
The 0.025mol/LPBS preparation method is：8g sodium chloride, 0.2g potassium chloride, 3.58g disodium hydrogen phosphate dodecahydrate,
0.27g potassium dihydrogen phosphate is dissolved in the deionized water of 1L, 121 DEG C of high pressure sterilization 15min.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117467001A (en) * | 2023-12-28 | 2024-01-30 | 金宇保灵生物药品有限公司 | Preparation method and application of clostridium perfringens antitoxin A serum |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1440810A (en) * | 2003-03-13 | 2003-09-10 | 山东农业大学 | Prepn of clostridium welchil toxoid vaccine and antitoxin serum |
| US20110129479A1 (en) * | 2009-12-02 | 2011-06-02 | Tobin Monte B | Immunogen selection directed in immunoglobulin packages in plasma and colostrum and method of making and using same |
| CN103432579A (en) * | 2013-09-12 | 2013-12-11 | 山东农业大学 | Preparation method of lamb-dysentery clostridium perfringens type B toxoid vaccine |
| CN104623652A (en) * | 2015-03-06 | 2015-05-20 | 山东农业大学 | Method for preparing two types of bivalent antigen vaccines of A, C type clostridium perfringens toxoid and thallus of necrotic enteritis |
| CN104829712A (en) * | 2015-04-23 | 2015-08-12 | 山东农业大学 | C and D type C.perfringens antitoxin serum and preparation method thereof |
| CN107875377A (en) * | 2017-11-06 | 2018-04-06 | 山东农业大学 | A kind of preparation method of E types C.perfringens toxoid vaccine |
| CN109954135A (en) * | 2017-12-25 | 2019-07-02 | 金宇保灵生物药品有限公司 | Ox A type C.perfringens inactivates toxoid vaccine and preparation method thereof |
-
2018
- 2018-07-25 CN CN201810824023.2A patent/CN108892725A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1440810A (en) * | 2003-03-13 | 2003-09-10 | 山东农业大学 | Prepn of clostridium welchil toxoid vaccine and antitoxin serum |
| US20110129479A1 (en) * | 2009-12-02 | 2011-06-02 | Tobin Monte B | Immunogen selection directed in immunoglobulin packages in plasma and colostrum and method of making and using same |
| CN103432579A (en) * | 2013-09-12 | 2013-12-11 | 山东农业大学 | Preparation method of lamb-dysentery clostridium perfringens type B toxoid vaccine |
| CN104623652A (en) * | 2015-03-06 | 2015-05-20 | 山东农业大学 | Method for preparing two types of bivalent antigen vaccines of A, C type clostridium perfringens toxoid and thallus of necrotic enteritis |
| CN104829712A (en) * | 2015-04-23 | 2015-08-12 | 山东农业大学 | C and D type C.perfringens antitoxin serum and preparation method thereof |
| CN107875377A (en) * | 2017-11-06 | 2018-04-06 | 山东农业大学 | A kind of preparation method of E types C.perfringens toxoid vaccine |
| CN109954135A (en) * | 2017-12-25 | 2019-07-02 | 金宇保灵生物药品有限公司 | Ox A type C.perfringens inactivates toxoid vaccine and preparation method thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117467001A (en) * | 2023-12-28 | 2024-01-30 | 金宇保灵生物药品有限公司 | Preparation method and application of clostridium perfringens antitoxin A serum |
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Application publication date: 20181127 |