CN114044806A - Polypeptide for promoting pig body to generate broad-spectrum immune response and application thereof - Google Patents
Polypeptide for promoting pig body to generate broad-spectrum immune response and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
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- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
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- Pharmacology & Pharmacy (AREA)
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Abstract
The invention provides a polypeptide for promoting a pig organism to generate a broad-spectrum immune response and application thereof, belonging to the technical field of biological medicines; the polypeptide comprises one or more of a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide and a fifth polypeptide; the amino acid sequences of the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide and the fifth polypeptide are respectively shown as SEQ ID NO. 1-SEQ ID NO. 5. The polypeptide can obviously promote the proliferation of lymphocytes of healthy pigs so as to enhance the generation of broad-spectrum immune response of pig organisms, and can be used as an effective component of a swine disease vaccine or a vaccine immunopotentiator.
Description
The application is a divisional application with the application date of 26/08/2020 and the application number of CN202010873533.6, and the invention name of the application is 'polypeptide for promoting pig organism to generate broad-spectrum immune response and application thereof'.
Technical Field
The invention relates to the technical field of biomedicine, in particular to a polypeptide for promoting a pig organism to generate a broad-spectrum immune response and application thereof.
Background
The immunopotentiator is a substance which is used alone or in combination with an antigen and can enhance the immune response of an animal body by enhancing the activity of macrophages, enhancing the immunogenicity and stability of antigen substances, promoting the synthesis and secretion of various immune factors and specific antibodies and the like.
The immunopotentiator can stimulate the body to generate humoral-mediated and cell-mediated immune response, so as to eliminate invading pathogens and protect animals from the pathogens.
Immunopotentiators are widely available and are mainly classified into three main groups according to their component characteristics: herbal extracts, chemical compositions and cytokines. Currently, there are few immunopotentiators used in swine disease vaccines. CVC1320 is reported to be used as an immunopotentiator of a foot-and-mouth disease vaccine (the immunopotentiator is used for improving the immunity efficacy of a swine foot-and-mouth disease inactivated vaccine, as is known in the introduction, 2019), but the immunopotentiator is a compound preparation, has complex components, large using dosage and relatively high production cost. Moreover, the preparation is only verified to be applicable to FMDV vaccines, and no data is available to demonstrate that it has a broad spectrum of effects. Tangbo et al (research on enhancing the immune efficacy of inactivated vaccines for pigs by using immunopotentiators, 2016) use VA 5-containing immunopotentiators as porcine parvo-encephalitis bivalent inactivated vaccine partners, and can improve the antibody titer, but no data prove that the inactivated vaccines have a broad-spectrum effect. At present, an immunopotentiator which can promote the pig body to generate a broad-spectrum immune response is urgently needed.
Disclosure of Invention
The polypeptide can obviously promote healthy pig lymphocytes to proliferate so as to enhance the pig organism to generate broad-spectrum immune response, and can be used as an effective component of a swine disease vaccine or a vaccine immunopotentiator.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a polypeptide for promoting a pig body to generate broad-spectrum immune response; the polypeptide comprises one or more of a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide and a fifth polypeptide;
the amino acid sequence of the first polypeptide is shown as SEQ ID NO. 1;
the amino acid sequence of the second polypeptide is shown as SEQ ID NO. 2;
the amino acid sequence of the third polypeptide is shown as SEQ ID NO. 3;
the amino acid sequence of the fourth polypeptide is shown as SEQ ID NO. 4;
the amino acid sequence of the fifth polypeptide is shown as SEQ ID NO. 5;
the polypeptide can enhance the pig organism to generate broad-spectrum immune response by promoting the lymphocyte proliferation of the healthy pig organism.
The invention provides a polypeptide polymer obtained by polymerizing the polypeptide in the scheme.
The invention provides application of the polypeptide or the polypeptide polymer in the scheme in preparing a preparation for promoting a pig organism to generate a broad-spectrum immune response.
The invention provides a pig immunopotentiator, and the active ingredient of the immunopotentiator comprises the polypeptide or the polypeptide polymer in the scheme.
Preferably, the formulation of the immunopotentiator includes an injection.
Preferably, the content of the polypeptide or polypeptide polymer in the immunopotentiator is 100 μ g/fraction/head part.
The invention has the beneficial effects that: the invention provides a polypeptide for promoting a pig body to generate broad-spectrum immune response; the polypeptide comprises one or more of a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide and a fifth polypeptide; the amino acid sequences of the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide and the fifth polypeptide are respectively shown as SEQ ID NO. 1-SEQ ID NO. 5. The polypeptide can obviously promote the proliferation of lymphocytes of healthy pigs so as to enhance the generation of broad-spectrum immune response of pig organisms, and can be used as an effective component of a swine disease vaccine or a vaccine immunopotentiator.
Drawings
FIG. 1 is an identification chromatogram of AP 1;
FIG. 2 is a mass spectrum of AP 1;
FIG. 3 is an identification chromatogram of AP 2;
FIG. 4 is a mass spectrum of AP 2;
FIG. 5 is an identification chromatogram of AP 3;
FIG. 6 is a mass spectrum of AP 3;
FIG. 7 is an identification chromatogram of AP 4;
FIG. 8 is a mass spectrum of AP 4;
FIG. 9 is an identification chromatogram of AP 5;
FIG. 10 is a mass spectrum of AP 5;
FIG. 11 is a flow chart showing that AP 1-AP 5 promote proliferation of ASFV-sensitized lymphocytes and mononuclear macrophages;
FIG. 12 is a data statistics diagram of FIG. 11;
FIG. 13 is a statistical chart of flow data of AP 1-AP 5 promoting proliferation of lymphocytes and monocytes macrophages in healthy pigs;
FIG. 14 is a graph of hyperimmune 14d, B lymphocyte subpopulation fraction levels;
FIG. 15 shows CD4+T lymphocyte subpopulation proportion level;
FIG. 16 is the radioimmunoassay 14d, the level of B lymphocyte subpopulations;
FIG. 17 shows CD8+T lymphocyte subpopulation ratio levels.
Detailed Description
The invention provides a polypeptide for promoting a pig body to generate broad-spectrum immune response; the polypeptide comprises one or more of a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide and a fifth polypeptide; the polypeptide can enhance the pig organism to generate broad-spectrum immune response by promoting the lymphocyte proliferation of the healthy pig organism.
In the invention, the amino acid sequence of the first polypeptide is shown as SEQ ID NO.1, and specifically comprises the following steps: MAAKIFIVL (Met-Ala-Ala-Lys-Ile-Phe-Ile-Val-Leu); the first polypeptide has an average molecular weight of 1005.31g/mol and has a chemical formula: c49H84N10O10And S. The polypeptide has a theoretical isoelectric point of pH 10.09, a GRAVY value of 2.38, and hydrophobicity. This property facilitates its binding to host proteins or cell surfaces, enabling a more efficient initiation of immune responses. In the prior art, no analogous compounds are known.
In the invention, the amino acid sequence of the second polypeptide is shown as SEQ ID NO.2, and specifically comprises: AGAQLTALF (Ala-Gly-Ala-Gln-Leu-Thr-Ala-Leu-Phe); the second polypeptide has an average molecular weight of 891.02g/mol and has a chemical formula: c41H66N10O12. The polypeptide has a theoretical isoelectric point of pH7, a GRAVY value of 1.24, hydrophobicity, and favorable binding with host protein or cell surface, and can more effectively initiate immune response. In the prior art, no analogous compounds are known.
In the invention, the amino acid sequence of the third polypeptide is shown as SEQ ID NO.3, and specifically comprises: STMYYCYVL (Ser-Thr-Met-Tyr-Tyr-Cys-Tyr-Val-Leu); the third polypeptide has an average molecular weight of 1142.34g/mol and has a chemical formula: c53H75N9O15S2. The polypeptide has a theoretical isoelectric point of pH5.25, a GRAVY value of 0.78, hydrophobicity,this property facilitates its binding to host proteins or cell surfaces, enabling a more efficient initiation of immune responses. In the prior art, no analogous compounds are known.
In the invention, the amino acid sequence of the fourth polypeptide is shown as SEQ ID NO.4, and specifically comprises: FAGGEIPAG (Phe-Ala-Gly-Gly-Glu-Ile-Pro-Ala-Gly); the fourth polypeptide has an average molecular weight of 817.88g/mol and has a chemical formula: c37H55N9O12. The theoretical isoelectric point of the polypeptide is pH3.28, the GRAVY value of the polypeptide is 0.51, the polypeptide has hydrophobicity, and the characteristic is favorable for the binding with host protein or cell surface, so that the immune response can be more effectively started. In the prior art, no analogous compounds are known.
In the invention, the amino acid sequence of the fifth polypeptide is shown as SEQ ID NO.5, and specifically comprises: ACEDAFIEF (Ala-Cys-Glu-Asp-Ala-Phe-Ile-Glu-Phe); the average molecular weight of the fifth polypeptide is 1044.13g/mol, and the chemical formula is as follows: c47H65N9O16And S. The theoretical isoelectric point of the polypeptide is pH2.94, the GRAVY value of the polypeptide is 0.63, the polypeptide has hydrophobicity, and the characteristic is favorable for the binding with host protein or cell surface, so that the immune response can be more effectively started. In the prior art, no analogous compounds are known.
In the invention, the polypeptide in the technical scheme is obtained by determining the sequence of an ASFV epidemic strain and predicting through computer-assisted bioinformatics, and has specificity.
The invention provides a polypeptide polymer obtained by polymerizing the polypeptide in the scheme.
The invention provides application of the polypeptide or the polypeptide polymer in the scheme in preparing a preparation for promoting a pig organism to generate a broad-spectrum immune response.
In the invention, the polypeptide or the polypeptide polymer can enhance the pig body to generate a broad-spectrum immune response by promoting the lymphocyte proliferation of a healthy pig body.
The invention provides a pig immunopotentiator, the active component of which comprises the polypeptide or the polypeptide polymer in the scheme; the formulation of the immunopotentiator preferably includes an injection; the content of the polypeptide or polypeptide polymer in the immunopotentiator is preferably 100. mu.g/fraction, i.e., the content of each polypeptide or each polypeptide polymer is 100. mu.g/fraction.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
EXAMPLE 1 solid phase Synthesis and purity measurement of the polypeptide
The polypeptide of the invention is as follows:
SEQ ID NO.1：MAAKIFIVL(AP1)；
SEQ ID NO.2：AGAQLTALF(AP2)；
SEQ ID NO.3：STMYYCYVL(AP3)；
SEQ ID NO.4：FAGGEIPAG(AP4)；
SEQ ID NO.5：ACEDAFIEF(AP5)；
in the practice of the present invention, the polypeptide is synthesized by Shanghai Biotechnology Inc.
The detection wavelength was 214 nm. Obtaining the final polypeptide purification product with purity of more than 98%, identifying the structure by ESI-MS, and referring to FIG. 1-FIG. 10 for the identification result, wherein FIG. 1 is the identification chromatogram of AP1, and the peak-off time is 9.26 min; FIG. 2 is a mass spectrum of AP1, identifying a molecular weight of 1005.65g/mol, consistent with theoretical values; FIG. 3 is an identification chromatogram of AP2, FIG. 4 is a mass spectrum of AP2, the identification molecular weight is 890.50g/mol, which is consistent with the theoretical value; FIG. 5 is an identification chromatogram of AP3, and FIG. 6 is a mass spectrum of AP3, the identification molecular weight is 1141.50g/mol, which is consistent with the theoretical value; FIG. 7 is an identification chromatogram of AP4 with a peak time of 9.26 min; FIG. 8 is a mass spectrum of AP4, identifying a molecular weight of 817.45g/mol, consistent with theoretical values; FIG. 9 is an identification chromatogram of AP5 with a peak time of 10.307 min; FIG. 10 is a mass spectrum of AP5, identifying a molecular weight of 1044.6g/mol, consistent with theoretical values; as can be seen from FIGS. 1 to 10, the present invention successfully synthesized the above 5 polypeptides.
Example 2 porcine lymphocyte proliferation assay
1. ASFV inactivated virus immunized pigs.
5 male long white pigs of 90 days old were immunized with an inactivated virus of an ASFV epidemic strain (from African swine fever regional laboratory, Lanzhou veterinary institute of Chinese academy of agricultural sciences) (10HID50), boosted once a month later, euthanized 7 days later, and the spleens were removed after dissection. Non-immunized healthy pigs were used as negative control group.
2. Preparation, culture and proliferation condition detection of splenocytes.
1) The collected spleens of pigs were aseptically treated with 75% alcohol, washed three times with PBS, cut into small pieces, placed in a folded sterile gauze (2 layers), and ground in a plate containing 5mL of serum 1640 medium.
2) Then, the liquid was aspirated into a 15ml centrifuge tube, and centrifuged at 1000rpm for 5 min.
3) The supernatant was discarded and the pellet (cells) was knocked to make uniform suspension.
4) Adding 10ml erythrocyte lysate, cracking for 10min, adding 6ml culture medium containing serum 1640 to stop cracking, mixing uniformly, centrifuging for 5min at 1000 rpm.
5) The supernatant was discarded, the precipitate was again knocked to be uniformly suspended, and then 10. mu.L of the suspension was diluted 40-fold and subjected to cell counting to adjust the cell concentration to 1X 106Individual cells/mL.
6) After CFSE staining, prepared splenocytes were plated in 24-well plates (1X 10)6Individual cells/well).
7) Respectively adding 0.2 mu g of the first polypeptide to the fifth polypeptide provided by the invention, and placing in CO2Culturing for 60h in an incubator, and detecting the proliferation condition of ASFV antigen-specific splenocytes by flow cytometry.
The detection results are shown in FIGS. 11 to 12, wherein FIG. 11 is a flow chart showing AP1 to AP5 promoting proliferation of ASFV-sensitized lymphocytes and mononuclear macrophages; FIG. 12 is a data statistics map of FIG. 11. As can be seen from FIGS. 11 to 12, all of the 5 polypeptides of the present invention significantly promoted proliferation of immune cells of ASFV-sensitized spleen, and the main types of immune cells were lymphocytes and mononuclear macrophages.
Healthy pig splenic lymphocytes were treated in the same manner and the effect of the polypeptide on the proliferation of non-antigen specific splenic cells in the pig organism was examined.
The detection results are shown in FIG. 13, and FIG. 13 is a flow data statistical chart of AP 1-AP 5 for promoting the proliferation of lymphocytes and monocytes and macrophages of healthy pigs. As can be seen from fig. 13, 5 polypeptides of the present invention significantly promoted proliferation of splenic immune cells.
EXAMPLE 3 polypeptide immune animal immunocytotyping experiments
3 male Changbai pigs with the age of 90 days are immunized by 5 polypeptide mixtures (each polypeptide is 100 mu g/head part) of the invention, the immunization is strengthened once after one month, and the proportion of immune cells in peripheral blood is detected by a flow cytometry at 14d after the second immunization. The same volume PBS immunized group served as control.
The detection results are shown in FIGS. 14-15, in which FIG. 14 shows the proportion of the B lymphocyte subpopulation at the level of the hyperimmune 14 d; FIG. 15 shows CD4+T lymphocyte subpopulation ratio levels. From fig. 14 to fig. 15, it is known that the 5 polypeptide mixed immunity of the present invention can promote the generation of lymphocyte immune response in pig body and enhance the immunity of pig.
EXAMPLE 4 polypeptide as immunopotentiator immune animal immunocyte typing experiment
A foot-and-mouth disease O/MYA98/BY/2010 strain inactivated vaccine or a mixture of the inactivated vaccine and 5 polypeptides (each polypeptide is 100 mu g per head part) is used for immunizing 3 male Changbai pigs with the age of 90 days, the immunization is performed once after one month, and the proportion of immune cells in peripheral blood is detected BY a 14d flow cytometer after two-immunization. The same volume PBS immunized group served as control.
The detection results are shown in FIGS. 16-17, in which FIG. 16 shows the proportion of the B lymphocyte subpopulation at the level of the hyperimmune 14 d; FIG. 17 shows CD8+T lymphocyte subpopulation ratio levels. From FIGS. 16 to 17, it is clear that 5 polypeptides of the present invention can enhance the level of lymphocyte immune response of swine foot-and-mouth disease vaccine, and improve the immunogenicity of the vaccine.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Lanzhou veterinary research institute of Chinese academy of agricultural sciences
<120> polypeptide for promoting pig organism to generate broad-spectrum immune response and application thereof
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Met Ala Ala Lys Ile Phe Ile Val Leu
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Ala Gly Ala Gln Leu Thr Ala Leu Phe
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Ser Thr Met Tyr Tyr Cys Tyr Val Leu
1 5
<210> 4
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Phe Ala Gly Gly Glu Ile Pro Ala Gly
1 5
<210> 5
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Ala Cys Glu Asp Ala Phe Ile Glu Phe
1 5
Claims (5)
1. A polypeptide for promoting a pig body to generate a broad-spectrum immune response, wherein the polypeptide is a first polypeptide or a fourth polypeptide;
the amino acid sequence of the first polypeptide is shown as SEQ ID NO. 1;
the amino acid sequence of the fourth polypeptide is shown as SEQ ID NO. 4;
the polypeptide can enhance the pig organism to generate broad-spectrum immune response by promoting the proliferation of lymphocytes and mononuclear macrophages of the healthy pig organism.
2. Use of a polypeptide according to claim 1 in the preparation of a formulation for promoting a broad-spectrum immune response in a pig.
3. An immunopotentiator for swine, the active ingredient of which comprises the polypeptide of claim 1.
4. The immunopotentiator according to claim 3, wherein the dosage form of the immunopotentiator comprises an injection.
5. The immunopotentiator according to claim 4, wherein the amount of the polypeptide in the immunopotentiator is 100 μ g/fraction.
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| CN202111340467.7A CN114044806B (en) | 2020-08-26 | 2020-08-26 | Polypeptide for promoting pig organism to generate broad-spectrum immune response and application thereof |
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| CN202010873533.6A CN112300248B (en) | 2020-08-26 | 2020-08-26 | Polypeptide for promoting pig body to generate broad-spectrum immune response and application thereof |
| CN202111340467.7A CN114044806B (en) | 2020-08-26 | 2020-08-26 | Polypeptide for promoting pig organism to generate broad-spectrum immune response and application thereof |
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| CN202010873533.6A Active CN112300248B (en) | 2020-08-26 | 2020-08-26 | Polypeptide for promoting pig body to generate broad-spectrum immune response and application thereof |
| CN202111340455.4A Active CN114057834B (en) | 2020-08-26 | 2020-08-26 | Polypeptide for promoting pig organism to generate broad-spectrum immune response and application thereof |
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| CN110862435A (en) * | 2019-12-05 | 2020-03-06 | 中国农业大学 | African swine fever CTL epitope polypeptide and application thereof |
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| CN108117583B (en) * | 2018-01-03 | 2020-06-09 | 中牧实业股份有限公司 | Immunopotentiator and application thereof |
| WO2020264312A1 (en) * | 2019-06-28 | 2020-12-30 | Phibro Animal Health Corporation | African swine fever vaccine |
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| CN110862435A (en) * | 2019-12-05 | 2020-03-06 | 中国农业大学 | African swine fever CTL epitope polypeptide and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114057834A (en) | 2022-02-18 |
| CN112300248A (en) | 2021-02-02 |
| CN112300248B (en) | 2021-12-03 |
| CN114057834B (en) | 2023-10-27 |
| CN114044806B (en) | 2023-10-24 |
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