CN109180784A - A kind of bursa of farbricius activity hexapeptide and application promoting bird flu and/or the immune response of newcastle disease vaccine - Google Patents
A kind of bursa of farbricius activity hexapeptide and application promoting bird flu and/or the immune response of newcastle disease vaccine Download PDFInfo
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Abstract
It identifies and applies the present invention relates to a kind of promotion bird flu and/or with the separation of the bursa of farbricius activity hexapeptide of the immune response of newcastle disease bigeminy vaccine.Bursa of farbricius activity hexapeptide of the present invention, amino acid group become Lys Gly Asn Arg Val Tyr, and structure is simple, and immunogenicity is extremely weak.Active peptide of the present invention has the function of promoting vaccine immunity reaction, in mouse immune experiment, not only mouse is promoted to generate the antibody response for being directed to AIV antigen, and mouse is promoted to generate the antibody response for being directed to ndv antigen, furthermore, also promote cell immune response, the enhancing of lymphocyte vigor, the reaction of antigen submission and raising vaccine immunity effect effect, it can be used as vaccine adjuvant or immunopotentiator be applied to animal vaccine application study, to improve the immune response ability that animal body is directed to specific antigen, improve the immune efficacy of vaccine, to improve the ability of the anti-epidemic disease infection of animal body.
Description
Technical field
The invention belongs to veterinary biological product technical fields, and in particular to one kind have promote vaccine bird flu and/or with
The bursa of farbricius activity hexapeptide of the immune response of newcastle disease bigeminy vaccine and its application.
Background technique
Bird flu (Avian influenza, AI) is drawn by avian influenza virus (Avian influenza virus, AIV)
A kind of infection or disease syndrome of hair, after poultry infects AIV, some does not show clinical symptoms, and some shows as laying eggs down
Drop and respiratory disease, even result in systemic disease, the death rate is up to 100%.Therefore, according to pathogenic height by bird flu
It is divided into low pathogenicity bird flu (Low pathogenic avian influenza, LPAI) and highly pathogenic bird flu
(Highly pathogenic avian influenza,HPAI).H9N2 subtype avian influenza virus is most wide as whole world prevalence
One of general Low Pathogenic Avian Influenza Virus, the influence to poultry production and human health are on the rise, and are increasingly becoming influenza
One of focus of virus monitor.
In poultry, different subtype avian influenza viruses have different popularities in different regions, various years, to poultry
Different degrees of harm is caused, it is common with hypotypes such as H3, H4, H6, H5, H7, H9.In recent years, due to subtype avian influenzas such as H5, H7
Virus infection people simultaneously causes dead event to emerge one after another, and seriously endangers human health and civil order, bird flu have become
The focus of various countries' Field of Animal Epidemic Disease Control.What is attracted people's attention is the H7N9 bird flu that China breaks out in 2013, can infect people and lead
Cause human death.By the genetic analysis discovery to its each segment, finds its internal gene and H9N2 very high homology, also confirm that
The virus has occurred that recombination.These a large amount of report avian influenza virus have directly been broadcast to the thing of the mankind across inter-species obstacle
Part has more and more caused the concern of society, and its corresponding public health meaning also increasingly highlights.
In China, unique geographical environment and aquaculture model thus sick generation, propagate and provide advantage.And it is immunized
The presence in pressure and live-bird market provides necessary condition again for the recombination and variation of H9N2 subtype avian influenza virus.Vaccine immunity
It is not only the major measure of current livestock and poultry Contagion prevention, and is also the important measure of China's Field of Animal Epidemic Disease Control planning.Face
The most of vaccines used on bed effect good with combination adjuvant competence exertion.Therefore, safety, noresidue, effective Novel free
The research of epidemic disease reinforcing agent and technological innovation have become the important measure that aviculture develops prevention and control planning.
The concept of avian influenza virus broad-spectrum vaccine is the vaccine that all avian influenza virus subtypes are generated with Vaccine effectiveness.Consider
To the epidemiology and genetic diversity of avian influenza virus, vaccine used at present can only generally have specific avian influenza strain
There is Vaccine effectiveness, once having new fowl influenza virus strain or hypotype to occur, original vaccine will lose its Vaccine effectiveness.Cause
This, develops a kind of with inducing the wide spectrum avian influenza vaccine of extensive cross reaction immune response to have special significance.
Vaccine immunity is not only the major measure of current livestock and poultry Contagion prevention.The most of vaccines cooperation clinically used
The good effect of adjuvant competence exertion.Thus, provide a kind of low price, safety, noresidue efficient promotion immune effect of vaccine
Novel active peptide, can be used as vaccine adjuvant or immunopotentiator and be applied to animal vaccine application study, to improve animal body
For the immune response ability of specific antigen, the immune efficacy of vaccine is improved, to improve the energy of the anti-epidemic disease infection of animal body
Power can be applied to the fields such as fundamental immunity research, clinical application research.
Immunity inoculation is the main means of China's prevention and control of fowl influenza and newcastle disease.However, can be consumed greatly using single seedling is immune
Manpower and material resources are measured, the increase of immune time while can reinforce the stress reaction of chicken group.
Summary of the invention
Low, safety that the purpose of the present invention is to provide a kind of prices, noresidue efficient promotion immune effect of vaccine it is new
Type active peptide, can be used as vaccine adjuvant or immunopotentiator is applied to animal vaccine application study, is directed to improving animal body
The immune response ability of specific antigen, improves the immune efficacy of vaccine, so that the ability of the anti-epidemic disease infection of animal body is improved, it can
Applied to fields such as fundamental immunity research, clinical application research.
Bursa of farbricius polypeptide of the present invention can be used for H9N2 subtype avian influenza virus, newcastle disease classical strains (La
Sota bivalent inactivated vaccine), H9N2 subtype avian influenza inactivated vaccine, newcastle disease inactivated vaccine etc., and to their immune guarantor
It protects effect and carries out pre-test, to provide foundation for subsequent inactivated vaccine development.
Purpose of the present invention is realized by following scheme:
A kind of bursa of farbricius polypeptide BP6, amino acid sequence as shown in SEQ ID No.1, are as follows: 5 '-Lys Gly Asn Arg
Val Tyr-3’。
Bursa of farbricius polypeptide BP6 of the present invention is preparing the application in immune drug, is preferably preparing fowl poultry immune medicine
Application in object, particularly preferred, the application in the vaccine adjuvant for preparing pre- avian influenza-prevention and/or newcastle disease.
A kind of immune composition of pre- avian influenza-prevention, the vaccine comprising avian influenza vaccine and/or newcastle disease, and institute of the present invention
The bursa of farbricius polypeptide BP6 stated.
Bursa of farbricius polypeptide BP6 of the present invention is preferably present in immune composition with 2~255 μ g/mL metering, more preferably
10~250 μ g/mL, further preferred 10~50 μ g/mL.In a kind of specific embodiment, the bursa of farbricius of the present invention
Polypeptide BP6 can be present in immune composition with following metering: 10ug/mL, 20ug/mL, 30ug/mL, 40ug/mL, 50ug/
mL。
The present invention is concentrated by ultrafiltration technology, and recycling molecular weight is less than the effective component (i.e. crude extract) of 1kDa.Through vacuum
Dry concentration, is then separated, is purified using reversed high performance liquid chromatography (RP-HPLC), is obtained elution time 11.258 and is divided
The bursa of farbricius active constituent of clock (peak value is high).Through MODIL-TOF mass spectral analysis, the molecular weight for measuring the Synthetic bursin is
735.35 (m/z), and obtain complete amino acid sequence, KGNRVY (i.e. 5 '-Lys Gly Asn Arg Val Tyr-3 ', sequence
Column 1), and it is named as bursa of farbricius hexapeptide (BP6).
The present invention also provides the preparations of more specifically bursa of farbricius polypeptide BP6 a kind of:
Using 50g health chicken bursa tissue as raw material, after cutting off adipose tissue, freezing, ultrasonication, high speed centrifugation, liquid
The technologies such as body supernatant molecule sieve separation obtain the bursa of farbricius crude extract of small-molecular-weight (< 1KDa).Through vacuum freeze drying, with super
Pure water dilution bursa of farbricius crude extract sample carries out reversed high performance liquid chromatography (RP-HPLC) and isolates and purifies, harvest elution peak value ratio
Higher delay peak component.The residence time of sample is 11.258 minutes in the present invention.Utilize gal4 amino acid mass spectral analysis
System (MALDI-TOF-MS) analyzes the amino acid sequence of the sample, and the molecular weight for measuring the Synthetic bursin is 735.35
(m/z), and MS/MS secondary analysis is used, obtains the complete amino acid sequence of the bursa of farbricius polypeptide, i.e. KGNRVY.By artificial
BP6 is synthesized, purity 99.936% then carries out immunization experiment and verifies its immunoloregulation function and application.
Bursa of farbricius polypeptide BP6 of the present invention promotes the immunization experiment of bird flu and newcastle disease bigeminy vaccine to prove: will
Bursa of farbricius polypeptide BP6 is added in bird flu and newcastle disease bigeminy vaccine according to 10,50 and 250 μ g/mL Three doses to be joined
Close Mice Inoculated；AIV antibody level, the NDV antibody subtype of mouse after immune are higher than vaccine group, and T cell hypotype and lymph
Cell Proliferation vigor is changed, and surface of dendritic cells developed by molecule level is higher than vaccine group.In mouse immune,
The experimental group AIV vaccine immunity effect for adding 10 μ g/mL dosage BP6 is significantly higher than the vaccine control group for not adding the component, and adds
The antibody level of the experimental group NDV vaccine of 50 μ g/mL dosage BP6 is added to be significantly higher than the vaccine control group for not adding the component.
Positive effect of the invention:
The present invention separates from the Immune Organs of Chicken bursa of farbricius, identifies a new immune-active peptides BP6, and structure is simple,
It is made of six amino acid, immunogenicity is extremely weak, can be used as veterinary vaccines immunopotentiator, such as promotes AIV inactivated vaccine, new city
The immunocompetence of epidemic disease inactivated vaccine not only promotes mouse to generate the antibody response for being directed to AIV antigen in mouse immune experiment,
And mouse is promoted to generate the antibody response for being directed to ndv antigen, in addition, cell immune response, lymphocyte vigor is also promoted to increase
By force, the reaction of antigen submission and raising vaccine immunity effect effect, can be used as vaccine adjuvant or immunopotentiator are applied to animal epidemic disease
Seedling application study improves the immune efficacy of vaccine, to mention to improve the immune response ability that animal body is directed to specific antigen
The ability of the high anti-epidemic disease infection of animal body, can be applied to the fields such as fundamental immunity research, clinical application research.It is of the present invention
Active peptide be a kind of micromolecule polypeptide from the bursa of farbricius, safety, noresidue, has and promotes widely to exempt from Small side effects
Epidemic disease humidification has stimulation antibody tormation, adjusts cell immune response and improves the effect of vaccine immunity effect to a variety of livestock and poultry,
The amino acid sequence of the polypeptide are as follows: 5 '-KGNRVY-3 '.
Detailed description of the invention
The separation and purifying of Fig. 1 bursa of farbricius hexapeptide.In reversed high-efficient liquid phase chromatogram, arrow is signified, the eluting peak of BP6
11.258min。
Fig. 2: polypeptide BP6 MALDI-TOF-MS/MS analysis.
Fig. 3: 4 weeks after immune, mouse AIV ELISA Specific antibody, with difference between each group of different alphabetic flags
Significantly (p < 0.05).
Fig. 4: 4 weeks after immune, mouse AIV HI antibody level, with significant difference between each group of different alphabetic flags (p <
0.05)。
Fig. 5: 4 weeks after immune, mouse NDV ELISA Specific antibody, with difference between each group of different alphabetic flags
Significantly (p < 0.05).
Fig. 6: 4 weeks after immune, mouse NDV HI Specific antibody, with significant difference between each group of different alphabetic flags
(p<0.05)。
Fig. 7: two exempt from 1 week latter, mouse T cell hypotype, with significant difference (p < 0.05) between each group of different alphabetic flags.
Fig. 8: two exempt from 1 week latter, mouse spleen lymphocyte activity, with significant difference (p between each group of different alphabetic flags
<0.05)。
Fig. 9: two exempt from 1 week, mouse dcs CD40+CD11c+ afterwards, with difference between each group of different alphabetic flags
Significantly (p < 0.05).
Figure 10: two exempt from 1 week, mouse dcs MHCII+CD11c+ afterwards, poor between each group with different alphabetic flags
Different significant (p < 0.05).
Specific embodiment
The technical solution of the embodiment of the present invention to further describe the present invention, but be not construed as limiting the invention.Under
The experimental method in embodiment is stated, is conventional method unless otherwise specified.Test material as used in the following examples, such as
It is that conventional biochemical reagent company is commercially available without specified otherwise.
Embodiment 1
The separation and identification of 1.BP6
The 50gAA broiler chicken bursa of farbricius of no fascia and adipose tissue is washed with 0.85% physiological saline (being cooled to 4-10 DEG C in advance)
Tissue is primary.After draining, bursa of farbricius tissue is placed in tissue mashing machine, adds the physiological saline of pre-cooling, high-speed homogenization three
Secondary, 30 seconds every time, homogenization process was maintained at 0-10 DEG C.Then ultrasound cracking processing 2 times under the conditions of 4 DEG C by homogenate,
5min/ times.Then lysate is heated to 80 DEG C, keeps the temperature 5min, is immediately placed in later on ice, is cooled to 10 DEG C.Then it will split
Solve liquid refrigerated centrifuge 30 minutes (4000 × g/min).Supernatant is collected, multigelation is twice.Then by supernatant high speed centrifugation,
Condition is 12000g/min, 4 DEG C, 30min.It collects supernatant and carries out ultrafiltration, collect the ultrafiltration of 1000Da or less molecular weight
Liquid, i.e. bursa of farbricius crude extracts.After freeze-drying, ultrapure water dilution, then 0.22um membrane filtration, purifies through reversed efficient liquid phase
Analysis, the Peak Activity (see Fig. 1) that harvest elution time to peak is 11.258min, is analyzed, molecular weight through MALDI-TOF-MS/MS
It is 735.35, amino acid sequence KGNRVY (see Fig. 2).
Embodiment 2
1. artificial synthesized BP6
According to KGNRVY sequence (SEQ ID No.1) synthesis polypeptide, purity is for commission commercialization Peptide systhesis company
99.936%.
2. vaccine
Bird flu and newcastle disease bivalent inactivated vaccine (being purchased from Nanjing Tianbang Bio-industry Co., Ltd.).
3. experimental animal mice group
BALB/C mice is randomly divided into five groups, every group 10: (I) PBS control group (every immune 0.2ml PBS)；
(II) AIV+NDV bivalent inactivated vaccine immune group (every immunological sterilization vaccine 0.2ml)；The inactivation of (III~V) AIV+NDV bigeminy
Vaccine+BP6 group (BP6 concentration is respectively 10,50,250 μ g/mL)；By the way of intraperitoneal injection to respective sets mouse respectively into
Row is immunized twice.Immunization interval two weeks, 0.2ml/ every/every time (table 1).
1 BP6 of table and AIV+NDV bivalent inactivated vaccine mixed immunity program
| 0 week (immune for the first time) | 2 weeks (second immune) | |
| 1 | PBS | PBS |
| 2 | Bigeminy vaccine | Bigeminy vaccine |
| 3 | BP6 (10 μ g/mL)+bigeminy vaccine | BP6 (10 μ g/mL)+bigeminy vaccine |
| 4 | BP6 (50 μ g/mL)+bigeminy vaccine | BP6 (50 μ g/mL)+bigeminy vaccine |
| 5 | BP6 (250 μ g/mL)+bigeminy vaccine | BP6 (250 μ g/mL)+bigeminy vaccine |
4. immune mouse antibodies detection
Mouse two exempt from after two weeks, eye socket blood sampling, 8000 × g be centrifuged 10min separate serum, be respectively adopted elisa technique with
Blood clotting Inhibition test detects the special antibody of avian influenza virus special IgG antibody and HI antibody level and newcastle disease virus
IgG and HI antibody level, all testing results are for statistical analysis.
5. immune mouse cell hypotype detection
And it is immune 1 week latter in second, it is sterile to adopt immune mouse spleen, separating spleen lymphocyte, using fluidic cell
Art detects the situation of cell T cell hypotype, lymphocyte activity, Dendritic cells subsets etc., and all testing results are counted
Analysis.
6. result
(1) the bird flu IgG antibody and the horizontal testing result of HI of mouse is immunized
The antibody level for being directed to AIV antigen after mouse immune in 4 weeks serum, knot are determined using indirect ELISA method
Fruit sees Fig. 3.Experiment display, the 4th week, compared with only immune vaccine group mouse, 10 μ g/mL BP6 combine to exempt from BP6 combined immunization group
Epidemic disease group antibody level is significantly higher than vaccine control group, and the antibody level of 50 and 250 μ g/mL BP6 combined immunizations is also above AIV epidemic disease
Seedling control, but it is lower than 10 μ g/mL BP6 combined immunization groups (Fig. 3).At the same time, with blood clotting suppressing method to each group mice serum
Middle AIV special antibody subtype detects detection.The result shows that compared with vaccine group, the AIV of 10 μ g/mL BP6 combined immunization groups
HI antibody level be apparently higher than vaccine control group, and the HI antibody level of the AIV of 50 and 250 μ g/mL BP6 combined immunizations with
Vaccine group is similar (Fig. 4).
(2) the newcastle disease IgG antibody and the horizontal testing result of HI of mouse is immunized
Blood sampling in 4 weeks separates serum after immune, is detected in immune Mice Body using indirect ELISA method and blood clotting suppressing method
IgG antibody and HI antibody level.The result shows that the IgG of 50 and 250 μ g/mL BP6 combined immunization groups is anti-compared with vaccine group
Body level is apparently higher than vaccine group, and the horizontal highest of IgG antibody (Fig. 5) of 250 μ g/mL BP6 combined immunization group newcastle diseases.And
And compared with vaccine group, 50 μ g/mL BP6 combined immunization groups significantly improve HI antibody level (Fig. 6)；This illustrates that BP6 promotes fowl
The antibody level that influenza and newcastle disease bivalent inactivated vaccine are immunized.
(3) the T cell hypotype of mouse is immunized
Latter week is immunized for the second time, separating spleen lymphocyte analyzes T cell hypotype, knot using Tris-clolr flow cytometry
Fruit is as shown in Figure 7.Compared with vaccine group, the CD3+CD4+T cell and CD3+CD8+T cell proportion of BP6 combined immunization group are bright
It is aobvious to increase.But, as the increase of BP9 dosage, CD3+CD4+T cell and CD3+CD8+T cell proportion reduce instead.All
In experimental group, the CD3+CD4+T cell and CD3+CD8+T cell proportion highest of 10 μ g/mL BP6 combined immunization groups.
(4) the lymphopoiesis result of mouse is immunized
Using MTT colorimetric determination BP6 combined immunization on lymphopoietic influence situation, Fig. 8 is as a result seen.It is real
Test the result shows that, compared with vaccine group, the lymphopoiesis ability of BP6 combined vaccine group is significantly increased.As can be seen that with
Vaccine group is compared, and after LPS stimulation, the lymphocyte vigor of three kinds of BP9 dosage combinations immune groups is significantly raised, and PHA is stimulated
Afterwards, the lymphocyte activity of 50 and 250 μ g/mL BP6 combined immunization groups is significantly raised.LPS be common B cell promote division because
Son is polyclonal activator, and PHA is commonly to stimulate lymphopoietic molecule.Therefore, it may be speculated that BP6 can promote
Into the proliferation of lymphocyte, and promote B lymphocyte proliferation.
(5) the CD11c+ sub-types of cells result of mouse is immunized
Second three weeks immune, separating spleen lymphocyte, using in Flow Cytometry methods detection Dendritic Cells
CD40+ sub-types of cells ratio.As a result as shown in figure 9, the CD11c+CD40+ sub-types of cells of 10 μ g/mL BP6 combined immunization groups
Ratio is apparently higher than vaccine group.Moreover, also having detected the ratio of MHCII+ sub-types of cells, as shown in Figure 10.Compared with vaccine group,
The CD11c+MHCII+ sub-types of cells ratio of Three doses BP6 immune group is significantly raised, and 10 μ g/mL BP6 combined immunization groups
CD11c+MHCII+ sub-types of cells ratio highest.
Mouse immune the experimental results showed that, BP6 as vaccine immunopotentiator potentiality and its dosage have much relations,
Imply that BP6 is a kind of immunological molecule that vaccine immunity can be promoted to react.
Sequence table
<110>Agricultural University Of Nanjing
<120>a kind of bursa of farbricius activity hexapeptide and application for promoting bird flu and/or the immune response of newcastle disease vaccine
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 6
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 1
Lys Gly Asn Arg Val Tyr
1 5
Claims (9)
1. a kind of bursa of farbricius polypeptide BP6, amino acid sequence is as shown in SEQ ID No.1.
2. bursa of farbricius polypeptide BP6 described in claim 1 is preparing the application in immune drug.
3. bursa of farbricius polypeptide BP6 described in claim 1 is preparing the application in fowl poultry immune drug.
4. bursa of farbricius polypeptide BP6 described in claim 1 is in the vaccine adjuvant application for preparing pre- avian influenza-prevention and/or newcastle disease.
5. a kind of immune composition of pre- avian influenza-prevention, which is characterized in that include bird flu and/or newcastle disease vaccine and right
It is required that bursa of farbricius polypeptide BP6 described in 1.
6. immune composition according to claim 5, which is characterized in that the bursa of farbricius polypeptide BP6 is with 2~255 μ g/mL
Metering is present in immune composition.
7. immune composition according to claim 6, which is characterized in that the bursa of farbricius polypeptide BP6 is with 10~250 μ g/
ML metering is present in immune composition.
8. immune composition according to claim 7, which is characterized in that the bursa of farbricius polypeptide BP6 is with 10~50 μ g/mL
Metering is present in immune composition.
9. immune composition according to claim 8, which is characterized in that the bursa of farbricius polypeptide BP6 is measured with 10 μ g/mL
It is present in immune composition.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116813795A (en) * | 2023-05-25 | 2023-09-29 | 华中农业大学 | Recombinant AaLS-BSP fusion peptide, preparation method and application |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101830968A (en) * | 2010-04-23 | 2010-09-15 | 南京农业大学 | Bursa of Fabricius heptapeptide with immune regulation effect |
| CN103467571A (en) * | 2013-09-11 | 2013-12-25 | 江苏农牧科技职业学院 | Livestock and poultry immune enhancer bursa fabricius polypeptide |
| CN103585626A (en) * | 2013-11-19 | 2014-02-19 | 浙江美保龙生物技术有限公司 | Preparation method for newcastle disease and infectious bursal disease bigeminal composite inactivated vaccine |
| CN104402971A (en) * | 2014-07-31 | 2015-03-11 | 青岛农业大学 | Polypeptide with immunomodulatory effects |
| CN104829690A (en) * | 2015-05-06 | 2015-08-12 | 青岛农业大学 | Fabricius bursa undecapeptide capable of promoting immunity |
| CN106317216A (en) * | 2016-09-21 | 2017-01-11 | 南京农业大学 | Active peptide for promoting H9N2 avian influenza vaccine and application |
| CN108314708A (en) * | 2017-01-17 | 2018-07-24 | 南京农业大学 | It is a kind of that there is the bursa of farbricius activity nonapeptide for promoting vaccine immunity reaction and its application |
-
2018
- 2018-10-31 CN CN201811293306.5A patent/CN109180784B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101830968A (en) * | 2010-04-23 | 2010-09-15 | 南京农业大学 | Bursa of Fabricius heptapeptide with immune regulation effect |
| CN103467571A (en) * | 2013-09-11 | 2013-12-25 | 江苏农牧科技职业学院 | Livestock and poultry immune enhancer bursa fabricius polypeptide |
| CN103585626A (en) * | 2013-11-19 | 2014-02-19 | 浙江美保龙生物技术有限公司 | Preparation method for newcastle disease and infectious bursal disease bigeminal composite inactivated vaccine |
| CN104402971A (en) * | 2014-07-31 | 2015-03-11 | 青岛农业大学 | Polypeptide with immunomodulatory effects |
| CN104829690A (en) * | 2015-05-06 | 2015-08-12 | 青岛农业大学 | Fabricius bursa undecapeptide capable of promoting immunity |
| CN106317216A (en) * | 2016-09-21 | 2017-01-11 | 南京农业大学 | Active peptide for promoting H9N2 avian influenza vaccine and application |
| CN108314708A (en) * | 2017-01-17 | 2018-07-24 | 南京农业大学 | It is a kind of that there is the bursa of farbricius activity nonapeptide for promoting vaccine immunity reaction and its application |
Non-Patent Citations (1)
| Title |
|---|
| 李胜楠等: "法氏囊活性肽BP-I对禽流感H9N2灭活疫苗的免疫佐剂特性", 《中国畜牧兽医文摘》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116813795A (en) * | 2023-05-25 | 2023-09-29 | 华中农业大学 | Recombinant AaLS-BSP fusion peptide, preparation method and application |
| CN116813795B (en) * | 2023-05-25 | 2024-01-30 | 华中农业大学 | Recombinant AaLS-BSP fusion peptide, preparation method and application |
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