CN113845568A

CN113845568A - Polypeptide for promoting pig body to generate broad-spectrum acquired immune response and application thereof

Info

Publication number: CN113845568A
Application number: CN202111363404.3A
Authority: CN
Inventors: 郑海学; 毛箬青; 孙德惠; 曹伟军; 周晓丽; 朱昱茜; 杨帆; 张克山; 刘华南; 刘湘涛
Original assignee: Lanzhou Veterinary Research Institute of CAAS
Current assignee: Lanzhou Veterinary Research Institute of CAAS
Priority date: 2020-08-26
Filing date: 2020-08-26
Publication date: 2021-12-28
Anticipated expiration: 2040-08-26
Also published as: CN113845572A; CN113845568B; CN111925416A; CN113845572B; CN111925416B

Abstract

The invention provides a polypeptide for promoting a pig organism to generate broad-spectrum acquired immune response and application thereof, belonging to the technical field of biological medicine; 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 of the present invention can promote high levelProliferation of mononuclear macrophage of healthy pig body, and remarkable promotion of CD8 of healthy pig body⁺T cells and B cells secrete IFN-gamma so as to enhance the generation of broad-spectrum acquired immune response of a pig organism, and the IFN-gamma can be used as an effective component of a swine disease vaccine or a vaccine immunopotentiator.

Description

Polypeptide for promoting pig body to generate broad-spectrum acquired immune response and application thereof

The application is a divisional application with the application date of 26/08/2020 and the application number of CN202010871934.8, and the invention name of the application is 'polypeptide for promoting pig organism to generate broad spectrum acquired 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 broad-spectrum acquired 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 broad-spectrum acquired 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 acquired 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 organism to generate broad-spectrum acquired 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 promote the lymphocyte proliferation of healthy pig body to enhance the pig body to generate broad-spectrum acquired immune response.

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 broad-spectrum acquired immune response.

The invention provides a swine disease vaccine immunopotentiator and/or a swine African swine fever subunit vaccine, and the active ingredients of the swine disease vaccine immunopotentiator and/or the swine African swine fever subunit vaccine comprise the polypeptide or the polypeptide polymer in the scheme.

Preferably, the dosage form of the swine disease vaccine immunopotentiator or the swine African swine fever subunit vaccine comprises an injection.

Preferably, the content of the polypeptide or the polypeptide polymer in the swine disease vaccine immunopotentiator or the swine African swine fever subunit vaccine is 100 mu g/component/head.

The invention has the beneficial effects that: the invention provides a polypeptide for promoting a pig organism to generate broad-spectrum acquired 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 acquired 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 an identification chromatogram of AP 2;

FIG. 3 is an identification chromatogram of AP 3;

FIG. 4 is an identification chromatogram of AP 4;

FIG. 5 is an identification chromatogram of AP 5;

FIG. 6 is a flow chart showing statistics of AP 1-AP 5 on ASFV-sensitized lymphocytes and monocyte macrophage proliferations;

FIG. 7 is a statistical chart of flow data of AP 1-AP 5 promoting proliferation of lymphocytes and monocytes macrophages in healthy pigs;

FIG. 8 shows that AP1 promotes IFN- γ secretion by different subtypes of immune cells;

FIG. 9 shows that AP2 promotes IFN- γ secretion by different subtypes of immune cells;

FIG. 10 shows that AP3 promotes IFN- γ secretion by different subtypes of immune cells;

FIG. 11 shows that AP4 promotes IFN-. gamma.secretion by different subtypes of immune cells;

FIG. 12 shows that AP5 promotes IFN- γ secretion by different subtypes of immune cells;

FIG. 13 is a graph of hyperimmune 14d, B lymphocyte subpopulation fraction levels;

FIG. 14 shows CD8⁺T lymphocyte subpopulation proportion level;

FIG. 15 is a graph of hyperimmune 14d, B lymphocyte subpopulation fraction levels;

FIG. 16 shows CD8⁺T lymphocyte subpopulation ratio levels.

Detailed Description

The invention provides a polypeptide for promoting a pig organism to generate broad-spectrum acquired 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 promote the lymphocyte proliferation of healthy pig body to enhance the pig body to generate broad-spectrum acquired immune response.

In the invention, the amino acid sequence of the first polypeptide is shown as SEQ ID NO.1, and specifically comprises the following steps: ASAINFLLL (Ala-Ser-Ala-Ile-Asn-Phe-Leu-Leu-Leu), average molecular weight 961.15g/mol, formula: c₄₆H₇₆N₁₀O₁₂. The polypeptide has a theoretical isoelectric point of pH7, a GRAVY value of 2, 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: SASAINFLLL (Ser-Ala-Ser-Ala-Ile-Asn-Phe-Leu-Leu-Leu), with an average molecular weight of 1048.23g/mol, having the formula: c₄₉H₈₁N₁₁O₁₄. The polypeptide has a theoretical isoelectric point of pH7, a GRAVY value of 1.72, hydrophobicity, and the characteristic of facilitating the 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: HSINMGIFL (His-Ser-Ile-Asn-Met-Gly-Ile-Phe-Leu), average molecular weight 1031.22g/mol, chemical formula: c₄₇H₇₄N₁₂O₁₂And S. The theoretical isoelectric point of the polypeptide is pH7.88, the GRAVY value of the polypeptide is 1.07, 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 fourth polypeptide is shown as SEQ ID NO.4, and specifically comprises: LFPMYSHLFTLI (Leu-Phe-Pro-Met-Tyr-Ser-His-Leu-Phe-Thr-Leu-Ile), with average molecular weight of 1481.79g/mol, and formula: c₇₄H₁₀₈N₁₄O₁₆And S. The theoretical isoelectric point of the polypeptide is pH7.88, the GRAVY value of the polypeptide is 1.32, 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: FTGMVPVSEYLI (Phe-Thr-Gly-Met-Val-Pro-Val-Ser-Glu-Tyr-Leu-Ile), with average molecular weight of 1355.59g/mol and chemical formula: c₆₄H₉₈N₁₂O₁₈And S. TheThe theoretical isoelectric point of the polypeptide is pH3.28, the GRAVY value of the polypeptide is 1.09, the polypeptide has hydrophobicity, and the characteristic is favorable for the 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 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.

In the invention, the polypeptide or the polypeptide polymer can enhance the pig body to generate broad-spectrum acquired immune response by promoting the lymphocyte proliferation of the healthy pig body.

The invention provides a swine disease vaccine immunopotentiator and/or a swine African swine fever subunit vaccine, wherein the active ingredients of the swine disease vaccine immunopotentiator and/or the swine African swine fever subunit vaccine comprise the polypeptide or the polypeptide polymer in the scheme; the dosage form of the swine disease vaccine immunopotentiator or the swine African swine fever subunit vaccine preferably comprises an injection; the content of the polypeptide or the polypeptide polymer in the swine disease vaccine immunopotentiator or the swine African swine fever subunit vaccine is preferably 100 mu g/component/head part, namely the content of each polypeptide or each polypeptide polymer is 100 mu g/head part.

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：ASAINFLLL(AP1)；

SEQ ID NO.2：SASAINFLLL(AP2)；

SEQ ID NO.3：HSINMGIFL(AP3)；

SEQ ID NO.4：LFPMYSHLFTLI(AP4)；

SEQ ID NO.5：FTGMVPVSEYLI(AP5)；

in the practice of the present invention, the polypeptide is synthesized by Shanghai Bioengineering, Inc.

The detection wavelength was 214 nm. The purity of the final polypeptide purification product is more than 98%, and the structure is identified by ESI-MS, and the identification results are shown in figures 1-5, wherein figure 1 is an identification chromatogram of AP1, figure 2 is an identification chromatogram of AP2, figure 3 is an identification chromatogram of AP3, figure 4 is an identification chromatogram of AP4, and the peak-off time is 13.329 min. FIG. 5 is an identification chromatogram of AP5 with a peak time of 14.298 min. As can be seen from FIGS. 1 to 5, 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 (10HID50, from African swine fever regional laboratory, Lanzhou veterinary institute, Chinese academy of agricultural sciences), 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 fig. 6, and fig. 6 is a flow data statistical chart of AP 1-AP 5 promoting proliferation of ASFV sensitized lymphocytes and mononuclear macrophages, and it can be seen from fig. 6 that 5 polypeptides of the present invention can significantly promote proliferation of ASFV specific splenic immune cells, and the main type of immune cells is 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. 7, and FIG. 7 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. 7, 5 polypeptides of the present invention were also able to significantly promote the proliferation of healthy pig spleen immune cells. The main type of immune cell is mononuclear macrophages.

Example 3 detection of IFN-y secretion from different subtypes of splenic lymphocytes

The pig immunization protocol and isolation and culture of splenocytes were the same as in example 2. After obtaining the dispersed spleen cells, the single cell suspension is prepared by RPMI1640 complete culture medium, and the concentration is 1X 10⁶And/ml. Inoculating to 24-well plate, adding 0.2 μ g of the polypeptide (AP 1-AP 5) to each well, and placing in CO₂Culturing for 60h in an incubator. Collecting cells in each well, labeling with porcine CD3, CD4, CD8 and IFN-gamma specific antibody, washing with PBS buffer containing 1-2% serum for 2 times, dispersing into cell suspension, and performing flow cytometryDetecting and respectively determining total T lymphocytes, B cells and CD4⁺T lymphocytes, CD8⁺Levels of IFN- γ secretion by T lymphocytes and monocytes macrophages.

The detection results are shown in FIGS. 8-11, wherein FIG. 8 shows that AP1 promotes IFN-gamma secretion levels of different subtype immune cells; FIG. 9 shows that AP2 promotes IFN- γ secretion by different subtypes of immune cells; FIG. 10 shows that AP3 promotes IFN- γ secretion by different subtypes of immune cells; FIG. 11 shows that AP4 promotes IFN-. gamma.secretion by different subtypes of immune cells; FIG. 12 shows that AP5 promotes IFN- γ secretion by different subtypes of immune cells; as can be seen from FIGS. 8 to 12, all of the five polypeptides of the present invention significantly enhanced B lymphocytes and CD8⁺The ability of T lymphocyte cells to secrete IFN- γ.

Example 4 experiments on typing of immune cells of animals immunized with the polypeptide as an active ingredient of ASFV vaccine

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. 13-14, in which FIG. 13 shows the proportion of the B lymphocyte subpopulation at the level of the hyperimmune 14 d; FIG. 14 shows CD8⁺T lymphocyte subpopulation ratio levels. From fig. 13 to 14, it can be seen 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 6 immunization of animals with Polypeptides as immunopotentiators for typing immune cells

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. 15-16, in which FIG. 15 shows the proportion of the B lymphocyte subpopulation at the level of the hyperimmune 14 d; FIG. 16 shows CD8⁺T lymphocyte subpopulation ratio levels. From the figure15-16, 5 polypeptides in the invention can enhance the level of lymphocyte immune response of the 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 body to generate broad-spectrum acquired immune response and application thereof

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Ala Ser Ala Ile Asn Phe Leu Leu Leu

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<213> Artificial Sequence (Artificial Sequence)

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His Ser Ile Asn Met Gly Ile Phe Leu

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<213> Artificial Sequence (Artificial Sequence)

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Leu Phe Pro Met Tyr Ser His Leu Phe Thr Leu Ile

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Phe Thr Gly Met Val Pro Val Ser Glu Tyr Leu Ile

1 5 10

Claims

1. A polypeptide for promoting a pig body to generate broad-spectrum acquired 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 promotes the proliferation of mononuclear macrophages of a healthy pig body and/or promotes the CD8 of the healthy pig body⁺The T cells and the B cells secrete IFN-gamma so as to enhance the pig body to generate broad-spectrum acquired immune response.

2. Use of a polypeptide according to claim 1 for the preparation of a formulation for promoting a broad spectrum adaptive immune response in a porcine organism.

3. A swine disease vaccine immunopotentiator and/or a swine African swine fever subunit vaccine, wherein an active ingredient of the swine disease vaccine immunopotentiator and/or the swine African swine fever subunit vaccine comprises the polypeptide of claim 1.

4. The swine disease vaccine immunopotentiator and/or the swine African swine fever subunit vaccine of claim 3, wherein the formulation of the swine disease vaccine immunopotentiator or the swine African swine fever subunit vaccine comprises injections.

5. The swine disease vaccine immunopotentiator and/or swine African swine fever subunit vaccine of claim 4, wherein the amount of the polypeptide in the swine disease vaccine immunopotentiator or swine African swine fever subunit vaccine is 100 μ g/fraction.