CN108424450A - Horse source tetanus antibody - Google Patents
Horse source tetanus antibody Download PDFInfo
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- CN108424450A CN108424450A CN201810319299.5A CN201810319299A CN108424450A CN 108424450 A CN108424450 A CN 108424450A CN 201810319299 A CN201810319299 A CN 201810319299A CN 108424450 A CN108424450 A CN 108424450A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- 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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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
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Abstract
The present invention relates to a kind of horse source tetanus antibodies.The present invention is selected without natural antibody and age, weight, the good horses of health status, is carried out the immune of horses using the lockjaw Asia toxin Fragment of various dose or with tetanus toxoid hybrid antigen, is obtained antibody.Beneficial effects of the present invention are：The present invention obtains horse source tetanus antibody using the good immunogenicity that tetanus toxin Asia toxin has, and can infinitely amplify production, system easy to control the quality, entirely without toxicity；The antibody of the present invention is suitable with the horse source tetanus antibody effect being immunized with tetanus toxoid.
Description
Technical field
The invention belongs to immunological technique fields, and in particular to lockjaw Asia toxin is used for that horses are immunized, and it is broken to generate Ma Yuan
Cold antibody.
Background technology
Lockjaw is a kind of serious infectious diseases, and case fatality rate is up to 20%-40%.Any wound has generation broken
The possibility of cold.Tetanus antibody is to prevent and treat tetanic fast and effective drug, lockjaw currently on the market in short term
Medicine has lockjaw horse antitoxin (TAT) and Tetanus Human Immunoglobulin (TIG).TIG is exempted from by hepatitis B vaccine
The high blood plasma of tetanus antibody potency, extracted specificity are acquired in the blood donor being immunized again through tetanus toxoid after epidemic disease
Immunoglobulin preparation.Skin test is not necessarily to when TIG is used, can direct injection, but since it belongs to blood product, there are infection third
The potential risk of the infectious diseases such as liver, AIDS, and limited by source, yield is relatively low, and cost is higher, often occurs " one in the market
Needle is hard to find " the phenomenon that.Developing country and undeveloped country be difficult oneself prepare and purchase TIG for it is tetanic prevention and
Treatment.So TAT treatment tetanus infections are easier to realize in this two classes country, and in a very long time in future
Inside still it will continue in clinical application.
TAT is that blood plasma obtained by horse is immunized by tetanus toxoid, purified after gastric enzyme digests made of liquid antitoxin
Immunoglobulin preparation contains specific Ab fragments, has the function of neutralizing tetanus toxin.It is also deposited in the preparation of TAT at present
In some problems：(1) in horses lockjaw booster immunization epidemic disease, due to tetanus toxoid complicated component, it is not likely to produce Gao Te
Heterogenetic antibody；(2) still there is certain toxicity in toxoid, take a blood sample after the injection tetanus toxoid antigen of frequent high dose,
Make horses liver cell that degeneration necrosis gradually occur, blood sampling horses are immunized under normal circumstances and start in the tenth Cheng Yihou, liver enlargement
Denaturation, becomes " soybean residue " sample, and last liver edge rupture leads to horses death.Therefore, the quality of immunizing antigen is improved, is dropped
The Quality advance of hypotoxicity, reduction and TAT for production cost all has significance.
Tetanus toxoid (TT) is the clostridium tetani of the culture production poison in the fluid nutrient medium for being conducive to production poison, is used
The method of filtering harvests toxin, made of formaldehyde detoxification.Although its immune effect is preferable, there are still some problems：(1)
Clostridium tetani can form spore form, and toxicity is high, and there is certain risk in when production；(2) formaldehyde treated toxoid easily causes dirt
Dye residual, easily causes side reaction when horses are immunized；(3) toxoid after being chemically treated is it may also happen that Poison Reverse, to horses
Generate toxicity.
Invention content
In order to solve it is of the existing technology it is long-term the use of tetanus toxoid is largely immunogene, on immune length
Toxic side effects are led to the problem of, the present invention provides a kind of horse source tetanus antibodies, low, easy with immunity height, price
The features such as production, nontoxicity.
The object of the present invention is to provide a kind of horse source tetanus antibodies.
According to a kind of horse source tetanus antibody of the specific embodiment of the invention, following steps is used to be prepared：
(1) mixture of lockjaw Asia toxin, tetanus toxoid or lockjaw Asia toxin and tetanus toxoid is used
Fundamental immunity is carried out to horses；
(2) mixture of lockjaw Asia toxin, tetanus toxoid or lockjaw Asia toxin and tetanus toxoid is used
Booster immunization is carried out to the horses after fundamental immunity；
(3) after booster immunization, using lockjaw Asia toxin, tetanus toxoid or lockjaw Asia toxin with it is broken
It is immune that the mixture of wind toxoid carries out the second journey to horses；
After (4) second journeys are immune, lockjaw Asia toxin, tetanus toxoid or lockjaw Asia toxin and lockjaw are used
It is immune that the mixture of toxoid carries out third journey to horses；
(5) after third journey is immune, the blood plasma and serum of horses are extracted.
1315 amino acid of tetanus toxin overall length, molecular weight 150kDa are made of A, B, C three parts altogether, per part
Molecular weight is 50kDa.C segments are the C-terminals of heavy chain, have the function of combining nerve cell.B segments are the N-terminals of heavy chain, tool
There is importing effect.A segments are light chains, there is proteinase activity, can inhibit the release of transmitter substance, have paralysis effect.It is broken
A, B, C segment general name " lockjaw Asia toxin " of cold toxin.
The present invention by using lockjaw Asia toxin as immunogene, be used alone by lockjaw Asia toxin or with
TT is used in combination, it was confirmed that its effective immunogenicity, and substitute the feasibility that TT does antigen.
High toxicity antigen, with molecular biology method, can be resolved into sub- toxin Fragment by the present invention with concrete instance explanation,
It can reduce to leading to the problem of toxic side effects with immune animal.Its immunogenicity can be such as kept, what vaccine and antitoxin can be used
It produces and uses.Sub- toxin Fragment can such as be simplified with E. coli system great expression, production process, it is not necessary to complex and expensive
Safeguard, and it produces anti-pure keeping degree height, small toxicity, whole production prices are greatly reduced.
According to a kind of horse source tetanus antibody of the specific embodiment of the invention, in step (1), fundamental immunity 2 is carried out
It is secondary, it is spaced 7 days between being immunized twice.
According to a kind of horse source tetanus antibody of the specific embodiment of the invention, with Fu Shi Freund's incomplete adjuvants and lockjaw
0.6~0.7mg/ml is made in the mixture mixing of sub- toxin, tetanus toxoid or lockjaw Asia toxin and tetanus toxoid
After lotion, neck and back to horses carry out intramuscular injection.
According to a kind of horse source tetanus antibody of the specific embodiment of the invention, in step (1), tetanus poison is used
Element carries out fundamental immunity to horses.
According to a kind of horse source tetanus antibody of the specific embodiment of the invention, in step (2), carries out 7 reinforcements and exempt from
Epidemic disease, every minor tick 7 days.
According to a kind of horse source tetanus antibody of the specific embodiment of the invention, in step (2), muscle is carried out to horses
The dosage of injection is respectively 2~3ml of first time, for the second time 4~6ml, for the third time 6~9ml, the 4th 8~12ml, the 5th time 8
~12ml, the 6th 12~18ml, the 7th 16~24ml.
According to a kind of horse source tetanus antibody of the specific embodiment of the invention, the is carried out within 16 days after booster immunization
Two journeys are immune, and the second journey is immunized altogether three times, every minor tick 7 days.
According to a kind of horse source tetanus antibody of the specific embodiment of the invention, in step (3), muscle is carried out to horses
The dosage of injection is respectively first time 6ml, second of 12ml, third time 24ml.
According to a kind of horse source tetanus antibody of the specific embodiment of the invention, carry out within 18 days after the second journey is immune
Third journey is immune, and it includes 3 times that third journey is immune, every minor tick 7 days.
According to a kind of horse source tetanus antibody of the specific embodiment of the invention, in step (4), muscle is carried out to horses
The dosage of injection is respectively first time 6ml, second of 12ml, third time 24ml.
Because tetanus toxoid and sub- toxin immunity effect and drug amount are using difference, immunization ways of the invention are with broken
The sub- toxin of cold and its mixture specially design allotment as antigen.
Beneficial effects of the present invention are：
The present invention is used in combination using lockjaw Asia toxin separately as antigen or with tetanus toxoid, to horses
It carries out after being immunized, has detected it and it is shown by the comparison of antibody titer and blood plasma potency in horses vivo immunogenicity
With preferable immunogenicity.
The present invention is immunized horses by lockjaw Asia toxin, almost non-toxic, and horses drying lesion will not be caused dead
It dies.The survival of experiment horses has been more than between averagely breaking anti-immunity horse Shi Yong Time now, and herds of horses are still healthy, can be continued long-term
Immunization experiment.
Tetanus Toxin Fragment C production is simple, and purity is high, almost non-toxic, does not need complicated, expensive protection, whole raw
It is the 50% or less of toxoid to produce price.
For production horse source tetanus antibody, lockjaw Asia toxin can be used with tetanus toxoid with mixed and alternate, use it
The antibody of production is suitable with the horse source tetanus antibody effect being immunized with tetanus toxoid.
Description of the drawings
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, below will to embodiment or
Attached drawing needed to be used in the description of the prior art is briefly described, it should be apparent that, the accompanying drawings in the following description is only
Some embodiments of the present invention, for those of ordinary skill in the art, without creative efforts, also
It can be obtain other attached drawings according to these attached drawings.
Fig. 1 is non-reduced (A) and restores the SDS-PAGE analyses of (B) horse blood plasma.Swimming lane M：Molecular weight marker；Swimming lane 1 to
6：The blood plasma of horse from 1 to 6 group；Swimming lane 7：The blood plasma for the horse being immunized from conventional TT；
Fig. 2 is the serum antibody titer after Different immunizing schedule.A. the anti-TT IgG titres in serum；B. in serum
Anti- TeNT-Hc IgG titres；C. the anti-TT IgM titres in serum；D. serum moderate resistance TeNT-Hc IgM potency；
Fig. 3 is the plasma antibody potency after different immunologic processes.A. the anti-TT IgG titres in the blood plasma from 1-6 groups；
B. the anti-TeNT-Hc IgG titres in blood plasma；C. anti-TT IgG after immune programme and TT routine immunization blood plasma in blood plasma three times
Titre；D. the anti-TeNT-Hc IgG titres after immunologic process three times and TT routine immunization blood plasma in blood plasma；
Fig. 4 is to test blood plasma antibody titer by agar diffusion method；
Fig. 5 is the antitoxin titre measured by flocculoreaction method in blood plasma；
Fig. 6 is the neutralization tetanus toxin effect of corresponding antibodies in the blood plasma from embodiment 1-6 groups；A. for the first time
Blood plasma is collected after immunologic process；B. blood plasma is collected after the second journey；C. blood plasma is collected after third journey.
Specific implementation mode
To make the object, technical solutions and advantages of the present invention clearer, technical scheme of the present invention will be carried out below
Detailed description.Obviously, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Base
Embodiment in the present invention, those of ordinary skill in the art obtained institute without making creative work
There is other embodiment, belongs to the range that the present invention is protected.
Experiment material
Horses：Family is herded purchased from Datong of Qinghai area, the healthy stallion without lockjaw natural antibody is selected, 4~10 years old, puts down
Equal 6.5 years old age, 250~400kg of weight, average weight 328.3kg.
Incomplete Freund's adjuvant：Atoleine (being purchased from upper marine Qin's chemical reagent Co., Ltd) is (medicinal with lanolin
Grade, purchased from Chinese Huating lanolin factory) according to volume 2:1 ratio is mixed with.
Antigen and adjuvant：
TeNT-Hc antigens, molecular weight 45KD, by Beijing prepared by Bioengineering Research Institute, with incomplete Freund's adjuvant 1:1
It is mixed into the water-in-oil emulsion of protein content 0.625mg/ml；
Tetanus toxoid (TT) antigen is purchased from Chengdu Olymvax Biopharmaceuticals Inc., with incomplete Freund
Agent is mixed into the water-in-oil emulsion of protein content 0.625mg/ml；
TeNT-Hc and tetanus toxoid are pressed protein content 1 by hybrid antigen:After 1 mixing, with incomplete Freund's adjuvant
1:1 is mixed into the water-in-oil emulsion of protein content 0.625mg/ml.
Secondary antibody：HRP marks anti-horse IgG, IgM secondary antibody to be purchased from Abcam companies.
Lockjaw normaltoxin, antitetanic serum national standard are purchased from National Institute for Food and Drugs Control.
ICR mouse：17-19g, half male and half female are purchased from Hunan SJA Laboratory Animal Co. , Ltd.
Borate buffer solution：NaCl 8.5g/L, H3BO34.5g/L, Na2B4O7·10H2O 0.5g/L, pH7.0-7.2。
Experimental method
1. horses grouping, immune programme, serum plasma separation
It selects without natural antibody and age, weight, the horses 18 being in a good state of health, is divided into 6 groups, every group 3.
In immune different phase, using TeNT-Hc, TT of various dose or both hybrid antigen (Mix Ag,
Mixed Antigens) the immune of horses is carried out, immunization ways are neck and back multiple spot intramuscular injection, carry out three journeys altogether and exempt from
Epidemic disease, the first journey include fundamental immunity 2 times, are spaced 7 days, carry out within 56 days after fundamental immunity 7 booster immunizations, every minor tick 7 days；
It is immune to carry out within 16 days after last time booster immunization the second journey, totally 3 times, every minor tick 7 days；After last needle of second journey is immune
It is immune to carry out within 18 days third journey, totally 3 times, every minor tick 7 days.Specific antigen type and immunizing dose are shown in Table 1.
The separation acquisition of serum and blood plasma is carried out respectively in immune different phase, and horse serum is through jugular vein blood collection nature
Separation, horse blood plasma are acquired using pulp grinder jugular vein is singly adopted.
1 horses of table are grouped and antigen immunization protocol
2.SDS-PAGE detects plasma antibody ingredient
The blood plasma that each group third journey acquires after immune is mixed, the blood plasma acquired after immune with conventional TT uses physiological saline
10 μ L are taken to be added 2 × loading buffer after 10 times of dilutions, row 12%SDS-PAGE protein electrophoresis after loading is more different
The blood plasma component difference of immune group.
As shown in Figure 1, in terms of SDS-PAGE electrophoretic analysis results, under non-reduced (A) and also original state (B) condition, difference is immune
Horse blood plasma (comparative example 7) is immunized with TT in the Main Components size and ratio of antibody in horse blood plasma (embodiment 1-6) prepared by mode
There was no significant difference.
3. antibody level detects
In fundamental immunity, booster immunization, the second journey last time is immune and latter week is immunized in third journey last time,
Horse serum and blood plasma are acquired respectively, and using ELISA method detection, its anti-TT and anti-TeNT-Hc antibody titers, specific method are：Point
96 hole elisa plates (Costar) are not coated with the TT and TeNT-Hc of 2 μ g/mL, 4 DEG C of coatings are stayed overnight, using PBST (PBS+0.1%
Tween-20 it) washs 4 times, each 5min, is added 1:20000 (for IgG detection) or 1:500(for IgM
Detection) start the horse serum or blood plasma of doubling dilution, 37 DEG C of incubation 1h, PBST washings 4 times, each 5min are added 1:
100000 diluted HRP-anti horse IgG or 1:20000 diluted HRP-anti horse IgM, 37 DEG C of incubations
40min, PBST are washed 4 times, are added after tmb substrate developing solution (Sigma) develops the color and are used 2M H2SO4Color development stopping, 450nm waves
Long detection readings.
As shown in Fig. 2, fundamental immunity latter week, five, six groups of serum moderate resistance TT IgG antibody titres of embodiment are apparent high
In other groups, there is significant difference, but with the increase of immune time, one week after the first, second and third journey is immune
The anti-TT IgG antibodies titre of serum, in addition to example IV, there was no significant difference between each group (p >=0.05), and antibody titer is minimum
The 4th group there is significant difference (p ﹤ 0.05) with highest 5th group of antibody titer.Equally, with immunization schedule, each reality
Apply anti-TeNT-Hc IgG antibody titers between example, anti-TT and TeNT-Hc IgM antibodies titre there are no significant difference (p >=
0.05) the stronger humoral immunity based on IgG type antibody, can be induced.
As shown in figure 3, latter week, horse blood plasma moderate resistance TT and anti-TeNT-Hc IgG antibodies is immunized per journey in six groups of embodiments
Titre.Wherein, embodiment five is higher on the whole for anti-TT IgG antibodies titre, is significantly higher than after the first journey is immune
First and the 4th group (p ﹤ 0.05), but after rear two-way is immune, there is no significant difference (p >=0.05) between each group.It is anti-
Example IV is slightly lower on the whole for TeNT-Hc IgG antibodies titre, and the 5th is substantially less than after the first journey and third journey are immune
Group (p ﹤ 0.05), unknown significance difference (p >=0.05) between other each groups.TeNT-Hc is participated in six groups of immune horses three
The plasma mixtures collected after the anti-TT of blood plasma, anti-TeNT-Hc titres and TT routine immunizations after journey is immune compare, and tie
Fruit shows that there was no significant difference (p >=0.05) for anti-TT IgG antibodies level between each group, but in TT routine immunization group blood plasma
Anti- TeNT-Hc antibody titers are substantially less than TeNT-Hc and participate in immune six groups of horses blood plasma (p ﹤ 0.01).
4. agar diffusion method detects blood plasma potency
2.25g agaroses are weighed, 150ml purified waters are added, plate is poured into after boiling, plum blossom hole is beaten after waiting for its cooling quietly.
It is loaded into hole, the 00 μ L of TeNT-Hc antigen 1s of 0.17mg/mL are added in interstitial hole, around to add successively by extension rate clockwise
Enter 5 ×, 10 ×, 20 ×, 40 ×, 80 ×, 160 ×, 320 ×, 640 × 100 μ L of immune horse serum after dilution.It is put into wet box, 37
DEG C culture 48 hours, observe precipitation line.
From the point of view of the experiment of Fig. 4 is recorded a demerit, after being immunized except the second journey, there are the outer (p of significant difference between embodiment three, four
﹤ 0.05), there was no significant difference (p >=0.05) for the antibody titer between the first journey and the immune rear each group of third journey.
5. flocculoreaction method detects blood plasma potency
With reference to three 3506 methods of Chinese Pharmacopoeia version in 2015：Precision measures the antitoxin of the 100Lf/mL of different volumes
Plain flocculoreaction standard solution, is separately added into flocculoreaction pipe, takes the horse blood plasma 1mL to be measured of appropriate dilutions multiple, quickly accurate
It is really added in above-mentioned each flocculoreaction pipe, shakes up, set in 45-50 DEG C of water-bath, be observed continuously, flocculent deposit occurs at first in record
Reaction tube, accurately repeat 2-3 times, (V is as a result, the cotton-shaped unit of horse blood plasma potency (Lf/mL)=V × n × 100 for record observation
There is the volume of the antitoxin flocculoreaction standard solution used when flocculent deposit, mL earliest；N is the dilute of horse blood plasma to be measured
Release multiple).
It is determined that the potency that TeNT-Hc toxoids are 0.17mg/ml in protein content is in the preliminary experiment of early period
100Lf/ml compares the antitoxic potency of the immune rear different groups of three journeys using it as antigen detection, and the results are shown in Figure 5, real
The cotton-shaped unit of antitoxin for applying example three and embodiment five is slightly above other groups, and minimum compared with embodiment three of example IV has
Significant difference (p ﹤ 0.05), there was no significant difference between other each groups.
6. mouse neutralization test,in vivo method detects blood plasma potency
With reference to three 3508 methods of Chinese Pharmacopoeia version in 2015：By tetanus antitoxin standard items and boric acid salt buffer
The diluted various concentration of liquid horse blood plasma 0.2mL to be measured, mixes with 0.2mL tetanus toxin standard items, after 37 DEG C of reaction 1h, note
It penetrates in mouse peritoneal, every group of 3 mouse, observes mouse invasion and death condition.The potency of horse serum to be measured is and control mice
There is the highest dilution of lockjaw Nervous toxicity symptom most severe one in death simultaneously.
The results are shown in Figure 6.One week after the first journey is immune, except the blood plasma antitoxin of embodiment one, example IV
For mean titre less than outside 1000IU/mL, remaining each group mean titre is above 1000IU/mL；In the immune end of second and third journey
In latter week, in addition to example IV, remaining each group antitoxin mean titre is above 1000IU.According to results of statistical analysis, respectively
There was no significant difference (p >=0.05) between group antitoxin potency.
Tetanus toxoid TT is played an important role as immunogene in the preparation of TAT, due to the spy of TT itself
Property：Amplify dimension-limited using production strain when production, environmental pollution is easily caused using formalin-inactivated, a large amount of repeatedly immune horses
Hepatotoxicity easily is generated to horses, product quality is caused to decline the increase with production cost.
The present invention is used in combination using tetanus toxoid separately as antigen or with TT, after horses are immunized,
It is had detected in horses vivo immunogenicity, by the comparison of antibody titer and blood plasma potency, after showing that it is immunized with conventional TT
Blood plasma be comparable, in addition to the 4th group, the mean titre of remaining each group has reached 1000IU/mL or more.It lengthens and exempts from again
Epidemic disease number and adjustment dosage can meet tetanus antitoxin production potency demand.
From the experimental results, there was no significant difference between Examples 1 to 6 each group, and embodiment 5 first uses TT to carry out two needles
Fundamental immunity, subsequently immune using TeNT-Hc, antibody titer and potency are slightly higher.In addition, being directed to immune programme and immunizing agent
After amount advanced optimizes, TeNT-Hc shows its better immunogenicity.With immune process, TT due to its production technology and
Hypotoxicity caused by self-characteristic can cause horses hepatic disease dead, and TeNT-Hc is the recombinant product of totally nontoxic,
Its advantage can more and more be shown with permanent immunity observation.
The above description is merely a specific embodiment, but scope of protection of the present invention is not limited thereto, appoints
What those familiar with the art in the technical scope disclosed by the present invention, can easily think of the change or the replacement, answer
It is included within the scope of the present invention.Therefore, protection scope of the present invention should be with the scope of the claims
It is accurate.
Claims (10)
1. a kind of horse source tetanus antibody, which is characterized in that horse source tetanus antibody is prepared using following steps：
(1) use lockjaw Asia toxin, tetanus toxoid or the mixture of lockjaw Asia toxin and tetanus toxoid to horse
Carry out fundamental immunity；
(2) use lockjaw Asia toxin, tetanus toxoid or the mixture of lockjaw Asia toxin and tetanus toxoid to base
Horses after plinth is immune carry out booster immunization；
(3) after booster immunization, broken lockjaw Asia toxin, tetanus toxoid or lockjaw Asia toxin and tetanus are used
It is immune that the mixture of toxin carries out the second journey to horses；
After (4) second journeys are immune, lockjaw Asia toxin, tetanus toxoid or lockjaw Asia toxin and tetanus toxoid it is mixed
It is immune to horses progress third journey to close object；
(5) after third journey is immune, the blood plasma and serum of horses are extracted.
2. horse source according to claim 1 tetanus antibody, which is characterized in that in step (1), carry out fundamental immunity 2 times,
It is spaced 7 days between being immunized twice.
3. horse source according to claim 1 tetanus antibody, which is characterized in that lockjaw Asia toxin, tetanus toxoid
Or 0.6~0.7mg/ml lotions are made with Fu Shi Freund's incomplete adjuvant mixings in the mixture of lockjaw Asia toxin and tetanus toxoid
Afterwards, intramuscular injection is carried out to the neck of horses and back.
4. horse source according to claim 1 tetanus antibody, which is characterized in that in step (1), use tetanus toxoid
Fundamental immunity is carried out to horses.
5. horse source according to claim 1 tetanus antibody, which is characterized in that in step (2), 7 booster immunizations are carried out,
Every minor tick 7 days.
6. horse source according to claim 5 tetanus antibody, which is characterized in that in step (2), muscle note is carried out to horses
The dosage penetrated is respectively 2~3ml of first time, for the second time 4~6ml, for the third time 6~9ml, the 4th 8~12ml, the 5th time 8~
12ml, the 6th 12~18ml, the 7th 16~24ml.
7. horse source according to claim 1 tetanus antibody, which is characterized in that carry out second within 16 days after booster immunization
Journey is immune, and the second journey is immunized altogether three times, every minor tick 7 days.
8. horse source according to claim 7 tetanus antibody, which is characterized in that in step (3), muscle note is carried out to horses
The dosage penetrated is respectively first time 6ml, second of 12ml, third time 24ml.
9. horse source according to claim 1 tetanus antibody, which is characterized in that carry out within 18 days after the second journey is immune the
Three journeys are immune, and it includes 3 times that third journey is immune, every minor tick 7 days.
10. horse source according to claim 9 tetanus antibody, which is characterized in that in step (4), muscle is carried out to horses
The dosage of injection is respectively first time 6ml, second of 12ml, third time 24ml.
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