CN102698264A - Bursin-like polypeptide derivative applied as immuno-enhancer of compound hog cholera vaccines - Google Patents
Bursin-like polypeptide derivative applied as immuno-enhancer of compound hog cholera vaccines Download PDFInfo
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Abstract
The invention provides a bursin-like polypeptide derivative applied as an immuno-enhancer of compound hog cholera vaccines, and relates to the field of preparation of animal vaccines. The bursin-like amyloid polypeptide derivative is formed by series connection of multicopy fundamental sequences of Lys-His-Gly through peptide bonds, and a carboxy terminal of the bursin amyloid polypeptide derivative exists in an amide form with a structure of (Lys-His-Gly-)n NH2 (n=2, 4, 6, 8 or 10). The compound hog cholera vaccines are prepared by evenly mixing 1.0 ml of hog cholera virus culture solution with the virus content larger than infective dose of 50000 rabbits per millilitre, 3.75 ml of skim milk powder with mass concentration of 5% and 0.5-10.0 mg of bursin amyloid polypeptide derivative, and freezing and drying. The bursin-like polypeptide derivative, serving as the immuno-enhancer, is applied to the compound hog cholera vaccines, and accordingly efficient immune effect can be achieved.
Description
Technical field
The present invention relates to the preparation field of animal vaccine, particularly the plain appearance of capsule polypeptide derivative in preparation compound recipe swine Fever Vaccine as the application of vaccine immunopotentiator.
Background technology
The Audhya report had extracted natural tripeptides from chicken bursa in 1986, and it is Lys-His-Gly-NH that its aminoacid is formed
2, this three Toplink is induced the differentiation of the precursor B cell of birds and mammal, makes B cell selective differentiation hormone, can increase the concentration of cAMP, cGMP in people's the Daudi B cell line, confirms that simultaneously artificial synthetic tripeptides also has identical effect.But this tripeptides can not cause the differentiation of precursor T cell.Research both at home and abroad confirmed already that this three Toplink improved humoral immune reaction, strengthened cellular immune level, and tens of kinds of poultry attenuated vaccines, inactivated vaccine and subunit vaccines such as bird flu, Streptococcus suis are had the effect of significant stimulation antibody-secreting.
Yet natural to separate the polypeptide bioactive substance that obtains the same with other for natural tripeptides, in the drug development application facet certain restriction arranged.At first, the conformation unstability: natural line style peptide has good biological activity external, but the gentle Qu Zaocheng conformation of the molecule of line style peptide is variable, and the intensity and the selectivity of itself and receptors bind are affected; Secondly, bioavailability is low: relatively poor membrane permeability, be prone to by enzyme degraded, easy-clear, indissoluble is separated and is prone to accumulative these characteristics in some cases, causes polypeptide in digestion and blood circulation, to be difficult to exist with stable pharmacologically active form.There are some researches show that the natural tripeptides half-life in vivo is merely 20 min.Therefore, with Lys-His-Gly-NH
2Be the vaccine of immunostimulant preparation, immune effect is undesirable, has received very big restriction in application.
Summary of the invention
The object of the invention provides the application of the plain appearance of capsule polypeptide derivative as compound recipe swine Fever Vaccine immunostimulant, and the plain appearance of said capsule polypeptide can significantly strengthen the immune effect of compound recipe swine Fever Vaccine.
The present invention provides the application as vaccine immunopotentiator in preparation compound recipe swine Fever Vaccine of the plain appearance of seed capsules polypeptide derivative; The plain appearance of said capsule polypeptide derivative is that the basic sequence Lys-His-Gly by multicopy is in series through peptide bond; Its c-terminus exists with amide form, and structure is: (Lys-His-Gly-)
nNH
2, n=2,4,6,8 or 10.
Said compound recipe swine Fever Vaccine adopts the preparation of following method: with 1.0ml swine fever virus culture fluid, 3.75ml mass concentration is 5% defatted milk powder and the plain appearance of 0.5～10.0 mg capsule polypeptide derivative mixing, lyophilization then; The viral level of said swine fever virus culture fluid is every milliliter of rabbit infective dose greater than 50000 families.
The present invention is applied to the compound recipe swine Fever Vaccine with the plain appearance of 5 seed capsules polypeptide derivative as immunostimulant, obtains immune effect efficiently.Zoopery is the result show, with the plain appearance of capsule polypeptide derivative, as the compound recipe swine Fever Vaccine of immunostimulant preparation, the commentaries on classics of antibody sun rate can reach (back 28 days of immunity) more than 80%, is significantly higher than normal conventional vaccine (50%-60%); And the vaccine (more than 90%) of making reinforcing agent with newly-designed capsule element appearance polypeptide derivative also significantly is superior to the plain tripeptides (80%) of natural capsule.In addition, zoopery is the result also show, every the plain appearance of pig injection capsule polypeptide derivative solution 1.0-2.0mL also can significantly increase the immune effect of vaccine, thereby improve pig prophylaxis of viral infections and disease-resistant ability.
The specific embodiment
Further set forth the present invention below in conjunction with embodiment.
Adopt international chemical molecular formula among the embodiment, the chemical name of each molecular formula representative is as follows:
BOC: tertbutyloxycarbonyl;
Cys: cysteine;
DCM: dichloromethane;
DMF:N, dinethylformamide;
DIEA:N, the N-diisopropylethylamine;
EDT: dithioglycol;
The FMOC:9-fluorenylmethyloxycarbonyl;
Gly: glycine;
His: histidine;
HOBt:1-hydroxyl-benzo-triazole;
Lys: lysine;
Phenol: phenol;
Rink-Amide MBHA Resin:2-chloramphenicol Choline Chloride Succinate resin;
The two DMA carbonyl phenylhydrazine triazole tetrafluoride boron salt of TBTU:1-oxygen-3-;
TFA: trifluoroacetic acid;
Thioanisole: thioanisole;
TIS: triisopropyl silicon.
Embodiment 1The plain appearance of preparation capsule polypeptide derivative (solid state chemistry synthetic method)
The plain appearance of 5 seed capsules polypeptide derivative, difference called after below: polypeptide A, polypeptide B, peptide C, polypeptide D, polypeptide E.Its concrete composition as follows:
The sequence of polypeptide A is: Lys-His-Gly-Lys-His-Gly-NH
2
The sequence of polypeptide B is: Lys-His-Gly-Lys-His-Gly-Lys-His-Gly-Lys-His-Gly-NH
2
The sequence of peptide C is:
Lys-His-Gly-Lys-His-Gly-Lys-His-Gly-Lys-His-Gly-Lys-His-Gly-Lys-His-Gly-NH
2。
The sequence of polypeptide D is: Lys-His-Gly-Lys-His-Gly-Lys-His-Gly-Lys-His-Gly-
Lys-His-Gly-Lys-His-Gly-Lys-His-Gly-Lys-His-Gly-NH
2。
The sequence of polypeptide E is: Lys-His-Gly-Lys-His-Gly-Lys-His-Gly-Lys-His-Gly-
Lys-His-Gly-Lys-His-Gly-Lys-His-Gly-Lys-His-Gly-Lys-His-Gly-Lys-His-Gly-NH
2。
(a) pre-treatment of reaction column
Before synthetic reaction, soaked reaction column 3-4 hour that is used to adorn resin with an amount of DCM, consumption with can submergence reaction column volume 2/3 be advisable.
(b) activation of resin
(CAS number: 40615-36-9), with the DMF immersion 30min of 1.5L, DMF is removed in filter pressing, and the DMF that reuse contains 20% piperidines soaks 15min, the dress post to get 200g FMOC Rink-Amide mbha resin.With washed with isopropyl alcohol 2 times, DMF washes 3 times.
(c) link Lys
Get FMOC-Lys (BOC)-OH of 100g, the TBTU of 92g, the DMF of 2.0L and HOBt (2M) mixing of 152mL, add the DIEA mixing of 52mL again, add room temperature in the resin column, nitrogen protection reaction 2.0h down.After reaction finishes,, use the DMF washing resin then three times with washed with isopropyl alcohol resin twice.
(d) link His and Gly successively
According to from the order of C end to N end extension polypeptide, get the FMOC-His-OH of 106g and the FMOC-Gly-OH of 59g respectively, identical with the condition of above-mentioned (C), remove the FMOC blocking group after each reaction.Process to the remaining aminoacid connection that repeats deprotection-washing-condensation finishes, and accomplishes the connection of polypeptide.
(e) synthetic (polypeptide A) of 2 series connection, 6 peptides
The sequence of polypeptide A is: Lys-His-Gly-Lys-His-Gly-NH
2
After operation (d) finishes, again repetitive operation in order (c) with operate (d) once, stopped reaction.Preparation TFA/Phenol/H
2The mixed liquor of O/Thioanisole/EDT/TIS is as cutting liquid, and its volume ratio is 80:5:5:5:3:2.At first with nitrogen polypeptide, resin complexes are dried up, put into the container of 5L, add 4 ℃ of airtight stirring reaction 2h of cutting liquid of 100mL, the ice ether that adds 3.0L again leaves standstill 2h for 4 ℃.Centrifugal collecting precipitation, deposition are with absolute ethanol washing, drying, and reprocessing 3 times is dissolved with sterile pure water then, place-40 ℃ freezing.The ice that will freeze is at last put vacuum freeze drier, and lyophilizing to constant weight promptly gets (polypeptide A).
(f). synthetic (the polypeptide B) of 4 series connection, 12 peptides
The sequence of polypeptide B is: Lys-His-Gly-Lys-His-Gly-Lys-His-Gly-Lys-His-Gly-NH
2
After operation (d) finished, repetitive operation in order (c) and operation (d) were three times again, stopped reaction.Preparation TFA/Phenol/H
2The mixed liquor of O/Thioanisole/EDT/TIS is as cutting liquid, and its volume ratio is 80:5:5:5:3:2.At first polypeptide, resin complexes are dried up, put into the container of 5L, add 4 ℃ of airtight stirring reaction 2h of cutting liquid of 100mL, leave standstill 2h for 4 ℃ at the ice ether that adds 3.0L with nitrogen.Centrifugal collecting precipitation, deposition are with absolute ethanol washing, drying, and reprocessing 3 times is dissolved with sterile pure water then, place-40 ℃ freezing.The ice that will freeze is at last put vacuum freeze drier, and lyophilizing to constant weight promptly gets (polypeptide B).
(g). synthetic (peptide C) of 6 series connection, 18 peptides
The sequence of peptide C is:
Lys-His-Gly-Lys-His-Gly-Lys-His-Gly-Lys-His-Gly-Lys-His-Gly-Lys-His-Gly-NH
2。
After operation (d) finished, repetitive operation in order (c) and operation (d) were five times again, stopped reaction.Preparation TFA/Phenol/H
2The mixed liquor of O/Thioanisole/EDT/TIS is as cutting liquid, and its volume ratio is 80:5:5:5:3:2.At first polypeptide, resin complexes are dried up, put into the container of 5L, add 4 ℃ of airtight stirring reaction 2h of cutting liquid of 100mL, leave standstill 2h for 4 ℃ at the ice ether that adds 3.0L with nitrogen.Centrifugal collecting precipitation, deposition are with absolute ethanol washing, drying, and reprocessing 3 times is dissolved with sterile pure water then, place-40 ℃ freezing.The ice that will freeze is at last put vacuum freeze drier, and lyophilizing to constant weight promptly gets (peptide C).
(h). synthetic (the polypeptide D) of 8 series connection, 24 peptides
The sequence of polypeptide D is: Lys-His-Gly-Lys-His-Gly-Lys-His-Gly-Lys-His-Gly-
Lys-His-Gly-Lys-His-Gly-Lys-His-Gly-Lys-His-Gly-NH
2。
After operation (d) finished, repetitive operation in order (c) and operation (d) were seven times again, stopped reaction.Preparation TFA/Phenol/H
2The mixed liquor of O/Thioanisole/EDT/TIS is as cutting liquid, and its volume ratio is 80:5:5:5:3:2.At first polypeptide, resin complexes are dried up, put into the container of 5L, add 4 ℃ of airtight stirring reaction 2h of cutting liquid of 100mL, leave standstill 2h for 4 ℃ at the ice ether that adds 3.0L with nitrogen.Centrifugal collecting precipitation, deposition are with absolute ethanol washing, drying, and reprocessing 3 times is dissolved with sterile pure water then, place-40 ℃ freezing.The ice that will freeze is at last put vacuum freeze drier, and lyophilizing to constant weight promptly gets (polypeptide D).
(i). synthetic (the polypeptide E) of 10 series connection, 30 peptides
The sequence of polypeptide E is: Lys-His-Gly-Lys-His-Gly-Lys-His-Gly-Lys-His-Gly-
Lys-His-Gly-Lys-His-Gly-Lys-His-Gly-Lys-His-Gly-Lys-His-Gly-Lys-His-Gly-NH
2。
After operation (d) finished, repetitive operation in order (c) and operation (d) were nine times again, stopped reaction.Preparation TFA/Phenol/H
2The mixed liquor of O/Thioanisole/EDT/TIS is as cutting liquid, and its volume ratio is 80:5:5:5:3:2.At first polypeptide, resin complexes are dried up, put into the container of 5L, add 4 ℃ of airtight stirring reaction 2h of cutting liquid of 100mL, leave standstill 2h for 4 ℃ at the ice ether that adds 3.0L with nitrogen.Centrifugal collecting precipitation, deposition are with absolute ethanol washing, drying, and reprocessing 3 times is dissolved with sterile pure water then, place-40 ℃ freezing.The ice that will freeze is at last put vacuum freeze drier, and lyophilizing to constant weight promptly gets (polypeptide E).
Above-mentioned each polypeptide is by the biochemical company limited preparation of Shanghai gill.
Embodiment 2Contain the preparation of the compound recipe swine Fever Vaccine of immunostimulant
(1) preparation of swine fever virus culture fluid
Entrust Nanjing Tianbang Bio-industry Co., Ltd.'s preparation; According to method and the qualified swine fever virus culture fluid of standard fabrication described in " live vaccines of hog cholera manufacturing and inspection procedure (I) or (II) " of " People's Republic of China's veterinary biologics rules " (version in 2000), the viral level >=50000 rabbit infective dose in every milliliter of swine fever virus culture fluid.
The preparation of (2) 5% defatted milk powder
Get commercially available defatted milk powder, being prepared into mass concentration with normal saline or PBS buffer (pH7.2) is 5% solution, and physical method sterilization back is subsequent use.
(3) preparation of compound recipe swine Fever Vaccine
The concrete grammar of compound recipe swine Fever Vaccine preparation is: with the swine fever virus culture fluid (>=50000 rabbit infective doses) of 1.0ml, the 3.75ml mass concentration is 5% defatted milk powder and the plain appearance of capsule polypeptide derivative mixing, lyophilization then.
With the plain appearance of embodiment 1 gained 5 seed capsules polypeptide derivative: polypeptide A, polypeptide B, peptide C, polypeptide D, polypeptide E are respectively as immunostimulant; Every peptide species derivant is pressed 0.5mg, 2.5mg, three kinds of dosage of 10.0 mg respectively and is mixed with 1.0ml swine fever virus culture fluid and 3.75ml defatted milk powder solution (5% mass concentration); Make the compound recipe swine Fever Vaccine of 15 kinds of proportionings altogether, code name is respectively A1, A2, A3; B1, B2, B3; C1, C2, C3; D1, D2, D3; E1, E2, E3; Lyophilization.
Embodiment 3Contain the animal immune test of the compound recipe swine Fever Vaccine of immunostimulant
(1) animal preparation:
180 of healthy piglets, 23～28 ages in days available from the pig farm, Jiangsu, divide circle to raise 10 pigs of every circle.
(2) antibody assay kit:
The hog cholera antibody ELISA detection kit, available from Ai Deshi biotech inc (IDEXX), 4 ℃ of preservations are subsequent use.
(3) test preparation is with compound recipe swine Fever Vaccine and contrast agents:
Test is the code name that embodiment 2 makes with the compound recipe swine Fever Vaccine and is respectively A1, A2, A3; B1, B2, B3; C1, C2, C3; D1, D2, D3; 15 vaccines of E1, E2, E3.
Simultaneously prepare code name again and be respectively the immune matched group compound vaccine of I, J and the non-immune blank group reagent that code name is K.
Wherein, the method for making of compound vaccine I is identical with embodiment 2, and just the polypeptide derivative as immunostimulant is substituted by the plain tripeptides of the capsule of natural extract; The plain tripeptides of the capsule of natural extract is made by oneself method for preparing referenced patent ZL200310105809.2 " a kind of method for distilling of bursopoietin and the application in disease treatment and immunity thereof " by National Research Center of Veterinary Biologicals Engineering and Technology.Compound vaccine J is the compound recipe swine Fever Vaccine that substitutes the plain appearance of capsule polypeptide derivative with normal saline; Non-immune contrast agents K then only is a normal saline.
Compound recipe swine Fever Vaccine and above-mentioned matched group vaccine I, J and the blank reagent K of embodiment 2 prepared 15 kinds of proportionings are listed in table 1 successively by group 1～18, and the dosage of immunostimulant is meant the amount of 50 parts in the table 1.
Table 1Be used for the compound recipe swine Fever Vaccine and the contrast of immunity test
| Group | Vaccine or contrast agents code name | The immunostimulant kind | The immunostimulant agent dose |
| 1 | A1 | Polypeptide A | 0.5mg |
| 2 | A2 | Polypeptide A | 2.5mg |
| 3 | A3 | Polypeptide A | 10.0mg |
| 4 | B1 | Polypeptide B | 0.5mg |
| 5 | B2 | Polypeptide B | 2.5mg |
| 6 | B3 | Polypeptide B | 10.0mg |
| 7 | C1 | Peptide C | 0.5mg |
| 8 | C2 | Peptide C | 2.5mg |
| 9 | C3 | Peptide C | 10.0mg |
| 10 | D1 | Polypeptide D | 0.5mg |
| 11 | D2 | Polypeptide D | 2.5mg |
| 12 | D3 | Polypeptide D | 10.0mg |
| 13 | E1 | Polypeptide E | 0.5mg |
| 14 | E2 | Polypeptide E | 2.5mg |
| 15 | E3 | Polypeptide E | 10.0mg |
| 16 | I | The plain tripeptides contrast of the capsule of natural extract | 2.5mg |
| 17 | J | The swine Fever Vaccine contrast | No immunostimulant |
| 18 | K | Non-immunity contrast | ? |
(4) method is used in test
Seeing table 2, is 1-18 with piglet by the circle random number, and each is numbered a test group, carries out immunity in piglet 30 ages in days.The 1-15 group is used for the immunity test of compound recipe swine Fever Vaccine of the present invention, and the 16th group and the 17th group is used for immune controlled trial, and the 18th group is used for non-immune blank test.Immunity test is following:
With group in the table 1 be the compound recipe swine Fever Vaccine (lyophilized powder) of 1-17 all with after the 50mL normal saline dilution, the test group piglet of corresponding group in the immune table 2 respectively, each organizes every piglet musculi colli injection 1.0mL; Every piggy injection normal saline of the 18th test group 1.0mL; Respectively organize equal isolated rearing then.After immunity, take a blood sample when 2w and 4w respectively, separation of serum is measured the ELISA value of antibody with hog cholera antibody detection kit (available from IDEXX company), and the calculating positive rate is listed in table 2.
(5) result of the test:
Visible by the result of table 2, add 4 weeks after the compound recipe swine Fever Vaccine immunity of the plain appearance of capsule of the present invention polypeptide derivative immunostimulant preparation, can arrive more than 80% even 100% protection, be significantly higher than 50% of common vaccine (vaccine J).Relatively plain with the capsule of natural extract, newly-designed 5 peptide species derivants all have active preferably.Compare 3 application doses, visible, 0.5-10.0mg all can significantly strengthen the effect of vaccine, and certain dose dependent is arranged.
Table 2The antibody that immunity back each degree of 2 and 4 weeks is tested group changes positive rate
| Group | Vaccine | 2 all positive rates | 4 all positive rates |
| 1 | A1 | 30% | 90% |
| 2 | A2 | 40% | 100% |
| 3 | A3 | 50% | 100% |
| 4 | B1 | 30% | 90% |
| 5 | B2 | 30% | 100% |
| 6 | B3 | 30% | 100% |
| 7 | C1 | 20% | 90% |
| 8 | C2 | 40% | 100% |
| 9 | C3 | 50% | 100% |
| 10 | D1 | 20% | 100% |
| 11 | D2 | 40% | 100% |
| 12 | D3 | 50% | 100% |
| 13 | E1 | 30% | 90% |
| 14 | E2 | 40% | 100% |
| 15 | E3 | 50% | 100% |
| 16 | I | 10% | 80% |
| 17 | J | 0 | 50% |
| 18 | K | 0 | 0 |
Embodiment 4The diluent of the plain appearance of capsule polypeptide derivative is to the adjuvant function test of swine Fever Vaccine
(1) animal preparation:
80 of healthy piglets, 23～28 ages in days available from the pig farm, Jiangsu, divide circle to raise 10 on every circle.
(2) preparation of the plain appearance of capsule polypeptide derivative diluent:
Use normal saline to be diluted to the solution that concentration is 50 μ g/mL respectively the plain tripeptides of the capsule of plain appearance polypeptide derivative of 5 seed capsules among the embodiment 1 and natural extract, the filtering with microporous membrane degerming of reuse 0.22 μ m, 4 ℃ of preservations, subsequent use as diluent later on.
(3) preparation of vaccine:
Swine Fever Vaccine, 10 part/bottles, lot number: 099010, available from Nanjing Tianbang Bio-industry Co., Ltd., consistent among wherein contained virus and the embodiment 2.
The control vaccine J that does not contain immunostimulant of preparation among the classical swine fever virus vaccine embodiment 2.
(4) antibody assay kit is prepared:
The hog cholera antibody ELISA detection kit, available from IDEXX company, 4 ℃ of preservations are subsequent use.
(5) test method:
By circle test group is divided into 11 groups (seeing table 3) at random, group 1-5 uses the swine Fever Vaccine (lyophilized powder) of 10 parts of the corresponding capsule of 10mL plain appearance polypeptide derivative diluted respectively, carries out immunity in piglet 30 ages in days, and each organizes every incidence intramuscular injection 1.0mL; The swine Fever Vaccine of 10 parts of group 6 usefulness 10ml natural extract capsule cellulose solutions dilution, the swine Fever Vaccine of 10 parts of group 7 usefulness 10ml normal saline dilution is in the every incidence intramuscular injection of piglet 30 ages in days 1.0mL immunity; Group 8 is non-immune blank group (10 of a healthy piglet), every injecting normal saline 1.0mL; Each organizes equal isolated rearing (grouping and processing method are seen table 3).2w and 4w blood sampling after immunity, separation of serum with the ELISA value of hog cholera antibody detection kit mensuration antibody, calculates positive rate.
(6) result of the test:
Organize 8 right and wrong immunity blank group, 2 all positive rates and 4 all positive rates are 0, and other group experimental results are seen table 3.Visible by table 3, similar with the result of embodiment 2, the plain appearance of 5 seed capsules polypeptide derivative all can significantly improve the immunne response of common vaccine (group 7) as the immunostimulant diluent, is promoted to more than 80% by 50%.This shows that the plain appearance of the capsule of chemosynthesis polypeptide derivative is active high, has huge clinical value.
The activity of the plain appearance of table 3 capsule polypeptide derivative diluent
| Group | Number | Vaccine dose | The diluent kind | The immunostimulant agent dose | 2 all positive rates | 4 all positive rates |
| 1 | 10 | 1 part | Polypeptide A | 50μg/1.0 mL | 40% | 90% |
| 2 | 10 | 1 part | Polypeptide B | 50μg/1.0 mL | 40% | 90% |
| 3 | 10 | 1 part | Peptide C | 50μg/1.0 mL | 40% | 90% |
| 4 | 10 | 1 part | Polypeptide D | 50μg/1.0 mL | 40% | 90% |
| 5 | 10 | 1 part | Polypeptide E | 50μg/1.0 mL | 40% | 90% |
| 6 | 10 | 1 part | The plain tripeptides of the capsule of natural extract | 50μg/1.0 mL | 30% | 80% |
| 7 | 10 | 1 part | Normal saline | Do not have | 0 | 50% |
Claims (2)
- The plain appearance of one seed capsules polypeptide derivative in preparation compound recipe swine Fever Vaccine as the application of vaccine immunopotentiator; The plain appearance of said capsule polypeptide derivative is that the basic sequence Lys-His-Gly by multicopy is in series through peptide bond; Its c-terminus exists with amide form, and structure is: (Lys-His-Gly-) nNH 2, n=2,4,6,8 or 10.
- 2. application according to claim 1; It is characterized in that the compound recipe swine Fever Vaccine adopts the preparation of following method: with 1.0ml swine fever virus culture fluid, 3.75ml mass concentration is 5% defatted milk powder and the plain appearance of 0.5～10.0 mg capsule polypeptide derivative mixing, lyophilization then; The viral level of said swine fever virus culture fluid is every milliliter of infective dose greater than 50000 rabbit.
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| CN108084251A (en) * | 2017-11-10 | 2018-05-29 | 浙江美保龙生物技术有限公司 | One species specificity improves micromolecule polypeptide and its application of hog cholera vaccine immune effect |
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