CN113845568B

CN113845568B - Polypeptide for promoting pig organism to generate broad-spectrum acquired immune response and application thereof

Info

Publication number: CN113845568B
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: 2023-08-18
Anticipated expiration: 2040-08-26
Also published as: CN113845572A; CN113845568A; CN111925416A; CN113845572B; CN111925416B

Abstract

The application provides a polypeptide for promoting 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 application can promote the proliferation of mononuclear macrophages of healthy pig organisms at a high level, and obviously promote the CD8 of the healthy pig organisms ⁺ The T cells and the B cells secrete IFN-gamma 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 vaccine or a vaccine immunopotentiator.

Description

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

The application relates to a polypeptide for promoting pig organism to generate broad spectrum acquired immune response and application thereof, which are classified as 26 days of application day 2020 and 26 months of application number CN 202010871934.8.

Technical Field

The application 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 can be used singly or in combination with antigen to enhance the immune response of animal organism by enhancing macrophage activity, 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 organism to generate humoral-mediated and cell-mediated immune response, thereby eliminating the invading pathogen and protecting animals from the pathogen.

Immunopotentiators are of various kinds and are largely classified into three main categories according to their component characteristics: chinese herbal extracts, chemical compositions and cytokines. There are few immunopotentiators currently used in swine vaccines. CVC1320 is reported to be used as an immunopotentiator of a foot-and-mouth disease vaccine (research on improving the immunopotentiator for improving the immunopotentiator of a swine foot-and-mouth disease inactivated vaccine, yu Xiaoming and 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 prove that the preparation has a broad-spectrum effect. Tang Bo et al (study of immunopotentiator improving the immunopotentiator of inactivated vaccine for pig, 2016) used VA 5-containing immunopotentiator as a dual inactivated vaccine partner for pig's tiny, japanese encephalitis, and could improve antibody titer, but no data demonstrated broad-spectrum effect. Currently, there is a lack of an immunopotentiator that promotes a broad spectrum of acquired immune responses in the swine organism.

Disclosure of Invention

The application aims to provide a polypeptide for promoting a pig organism to generate a broad-spectrum acquired immune response and application thereof, and the polypeptide can obviously promote healthy pig lymphocyte proliferation so as to enhance the pig organism to generate the 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 application provides the following technical solutions:

the application 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 enhance the pig body to generate broad-spectrum acquired immune response by promoting lymphocyte proliferation of healthy pig body.

The application provides a polypeptide polymer obtained by polymerizing the polypeptide in the scheme.

The application provides application of the polypeptide or the polypeptide polymer in the scheme in preparation of a preparation for promoting a pig organism to generate broad-spectrum acquired immune response.

The application 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.

Preferably, the swine disease vaccine immunopotentiator or swine african swine fever subunit vaccine dosage form 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 mug/component/head.

The application has the beneficial effects that: the application 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 healthy pig lymphocytes 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.

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 of data statistics for the proliferation of AP 1-AP 5-activated lymphocytes and monocytes;

FIG. 7 is a flow chart of statistics of AP 1-AP 5 promoting proliferation of healthy porcine lymphocytes and mononuclear macrophages;

FIG. 8 is a graph showing that AP1 promotes IFN-gamma secretion by immune cells of different subtypes;

FIG. 9 is a graph showing that AP2 promotes IFN-gamma secretion from various subtypes of immune cells;

FIG. 10 shows that AP3 promotes IFN-gamma secretion from various subtypes of immune cells;

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

FIG. 12 is a graph showing that AP5 promotes IFN-gamma secretion by various subtypes of immune cells;

FIG. 13 shows the proportion of the level of the sub-population of B lymphocytes in the secondary claim 14 d;

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

FIG. 15 shows the proportion of the level of the sub-population of B lymphocytes in the secondary claim 14 d;

FIG. 16 is CD8 ⁺ T lymphocyte subpopulation proportion level.

Detailed Description

The application 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 enhance the pig body to generate broad-spectrum acquired immune response by promoting lymphocyte proliferation of healthy pig body.

In the application, 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) has an average molecular weight of 961.15g/mol and a chemical formula: c (C) ₄₆ H ₇₆ N ₁₀ O ₁₂ . The theoretical isoelectric point of the polypeptide is pH7, the GRAVY value of the polypeptide is 2, and the polypeptide has hydrophobicity. This property facilitates binding to host proteins or cell surfaces and enables more efficient initiation of immune responses. In the prior art, no similar compound is found.

In the application, the amino acid sequence of the second polypeptide is shown as SEQ ID NO.2, and specifically comprises the following steps: SASAINFLLL (Ser-Ala-Ser-Ala-Ile-Asn-Phe-Leu-Leu-Leu) has an average molecular weight of 1048.23g/mol and a chemical formula: c (C) ₄₉ H ₈₁ N ₁₁ O ₁₄ . The polypeptide has a theoretical isoelectric point of pH7, a GRAVY value of 1.72, hydrophobicity, and can be combined with host protein or cell surface to effectively start immune response. In the prior art, no similar compound is found.

In the application, the amino acid sequence of the third polypeptide is shown as SEQ ID NO.3, and specifically comprises the following steps: HSINMGIFL (His-Ser-Ile-Asn-Met-Gly-Ile-Phe-Leu) has an average molecular weight of 1031.22g/mol and a chemical formula: c (C) ₄₇ H ₇₄ N ₁₂ O ₁₂ S, S. The theoretical isoelectric point of the polypeptide is pH7.88, the GRAVY value of the polypeptide is 1.07, and the polypeptide has hydrophobicity, which is favorable for binding with host protein or cell surface, and can more effectively start immune response. In the prior art, no similar compound is found.

In the application, the amino acid sequence of the fourth polypeptide is shown as SEQ ID NO.4, and specifically comprises the following steps: LFPMYSHLFTLI (Leu-Phe-Pro-Met-Tyr-Ser-His-Leu-Phe-Thr-Leu-Ile) has an average molecular weight of 1481.79g/mol and a chemical formula: c (C) ₇₄ H ₁₀₈ N ₁₄ O ₁₆ S, S. The polypeptide has a theoretical isoelectric point of pH7.88, a GRAVY value of 1.32, and hydrophobicity, and can be combined with host protein or cell surface to more effectively start immune response. In the prior art, no similar compound is found.

In the application, the amino acid sequence of the fifth polypeptide is shown as SEQ ID NO.5, and specifically comprises the following steps: FTGMVPVSEYLI (Phe-Thr-Gly-Met-Val-Pro-Val-Ser-Glu-Tyr-Leu-Ile) has an average molecular weight of 1355.59g/mol and a chemical formula: c (C) ₆₄ H ₉₈ N ₁₂ O ₁₈ S, S. The polypeptide has a theoretical isoelectric point of pH3.28, a GRAVY value of 1.09, and hydrophobicity, and the characteristic is favorable for combining with host protein or cell surface, so that immune response can be more effectively started. In the prior art, no similar compound is found.

In the application, the polypeptide in the technical scheme is obtained by determining the sequence of an ASFV epidemic strain and by computer-aided bioinformatics prediction, and has specificity.

In the present application, the polypeptide or the polypeptide polymer enhances the generation of a broad spectrum of acquired immune responses in the pig body by promoting lymphocyte proliferation in a healthy pig body.

The application 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 swine disease vaccine immunopotentiator or swine African swine fever subunit vaccine is preferably prepared from 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 mug/component/head, namely the content of each polypeptide or each polypeptide polymer is 100 mug/head.

The technical solutions of the present application will be clearly and completely described in the following in connection with the embodiments of the present application. It will be apparent that the described embodiments are only some, but not all, embodiments of the application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.

Example 1 solid phase Synthesis and purity detection of Polypeptides

The polypeptides of the application are 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 application, the polypeptide is synthesized by Shanghai Biotechnology Co.

The detection wavelength was 214nm. The final purified polypeptide product purity is more than 98%, and the ESI-MS identification structure is adopted, wherein 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 time is 13.329min. FIG. 5 is an identification chromatogram of AP5 with a peak time of 14.298min. As can be seen from FIGS. 1 to 5, the present application successfully synthesizes the 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 ASFV epidemic strain inactivated virus (from african swine fever area laboratory of the institute of veterinarian, department of academy of agricultural sciences, china) (10 HID 50), boosted once a month later, euthanized 7 days later, dissected and spleened out. Non-immunized healthy pigs were used as negative control group.

2. And (5) preparing, culturing and detecting proliferation of spleen cells.

1) The collected pig spleens were subjected to aseptic treatment with 75% alcohol, washed three times with PBS, the spleens were cut into small pieces, placed in folded sterile gauze (2 layers), and the spleens were ground in a dish containing 5mL of medium containing serum 1640.

2) The liquid was then aspirated into a 15ml centrifuge tube, 1000rpm, and centrifuged for 5min.

3) The supernatant was discarded and the pellet (cells) was knocked to suspend it uniformly.

4) 10ml of erythrocyte lysate is added for 10min of lysis, then 6ml of medium containing serum 1640 is added for stopping the lysis, and after being evenly mixed, 1000rpm is carried out for 5min of centrifugation.

5) Removing supernatant, knocking precipitate again to suspend it uniformly, diluting 10 μl of suspension 40 times, counting cells, and adjusting cell concentration to 1×10 ⁶ Individual cells/mL.

6) After CFSE staining, the prepared spleen cells were seeded into 24-well plates (1X 10) ⁶ Individual cells/well).

7) Adding 0.2 mug of the first polypeptide to the fifth polypeptide provided by the application respectively, and placing the mixture in CO ₂ Culturing in an incubator for 60 hours, and detecting the proliferation of ASFV antigen-specific spleen cells by flow cytometry.

Referring to fig. 6, fig. 6 is a flow chart of data statistics of AP 1-AP 5 for promoting proliferation of ASFV sensitized lymphocytes and mononuclear macrophages, and as can be seen from fig. 6, the 5 polypeptides of the application can significantly promote proliferation of ASFV-specific spleen immune cells, and the main type of immune cells is mononuclear macrophages. .

Healthy porcine spleen lymphocytes were treated in the same manner and the effect of the polypeptide on non-antigen specific spleen cell proliferation in the porcine organism was examined.

Referring to fig. 7, fig. 7 is a flow chart of statistics of data of AP1 to AP5 promoting proliferation of healthy porcine lymphocytes and mononuclear macrophages. As can be seen from fig. 7, the 5 polypeptides of the present application can also significantly promote proliferation of spleen immune cells in healthy pigs. The main type of immune cells is mononuclear macrophages.

Example 3 detection of IFN-gamma secretion by spleen lymphocytes of different subtypes

Pig immunization procedure and spleen cell isolation culture were as in example 2. After obtaining dispersed spleen cells, the spleen cells are prepared into single cell suspension with RPMI1640 complete medium, and the concentration is 1 multiplied by 10 ⁶ /ml. Inoculating into 24 hole plate, adding 0.2 mug of polypeptide (AP 1-AP 5) provided by the application into each hole, placing into CO ₂ Culturing in an incubator for 60 hours. Collecting cells of each well, labeling cells with specific antibodies of pig CD3, CD4, CD8 and IFN-gamma, washing with PBS buffer solution containing 1-2% serum for 2 times, dispersing into cell suspension, detecting with flow cytometry, and determining total T lymphocyte, B cell and CD4 ⁺ T lymphocytes, CD8 ⁺ T lymphocytes and monocytes macrophages secrete IFN-gamma levels.

The detection results are shown in fig. 8-11, wherein fig. 8 shows the level of the AP1 for promoting the secretion of IFN-gamma by different subtype immune cells; FIG. 9 is a graph showing that AP2 promotes IFN-gamma secretion from various subtypes of immune cells; FIG. 10 shows that AP3 promotes IFN-gamma secretion from various subtypes of immune cells; FIG. 11 shows that AP4 promotes IFN-gamma secretion by various subtypes of immune cells; FIG. 12 is a graph showing that AP5 promotes IFN-gamma secretion by various subtypes of immune cells; as can be seen from FIGS. 8 to 12, the five polypeptides of the present application can significantly enhance B lymphocytes and CD8 ⁺ T lymphocyte cells secrete IFN- γ.

Example 4 immune animal immunocyte typing experiment Using polypeptide as active ingredient of ASFV vaccine

3 male white pigs of 90 days old were immunized with a mixture of 5 polypeptides of the application (100. Mu.g/head of each polypeptide), boosted once a month, and the proportion of immune cells in peripheral blood was measured by a 14d flow cytometer after two-immunization. The same volume of PBS immunized group served as control.

The detection results are shown in fig. 13-14, wherein fig. 13 shows the proportion level of the secondary-exemption 14d, B lymphocyte subpopulation; FIG. 14 is CD8 ⁺ T lymphocyte subpopulation proportion level. From FIGS. 13 to 14, it is understood that the mixed immunization with 5 polypeptides of the present application can promote the generation of lymphocyte immune response in swine organism and enhance swine immunity.

EXAMPLE 6 immunopotentiator immune cell typing experiment of animals

3 male white pigs of 90 days old are immunized BY 5 polypeptide mixtures (100 mug/head parts of each polypeptide) of the foot-and-mouth disease O/MYA98/BY/2010 strain inactivated vaccine or inactivated vaccine, one month later, the immunization is enhanced, and the proportion of immune cells in peripheral blood is detected BY a 14d flow cytometer after double immunization. The same volume of PBS immunized group served as control.

The detection results are shown in fig. 15-16, wherein fig. 15 shows the proportion level of the secondary-exemption 14d, B lymphocyte subpopulation; FIG. 16 is CD8 ⁺ T lymphocyte subpopulation proportion level. From FIGS. 15 to 16, it is understood that 5 polypeptides of the present application can enhance the level of lymphocyte immune response of the swine foot-and-mouth disease vaccine, and enhance the immunogenicity of the vaccine.

The foregoing is merely a preferred embodiment of the present application and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present application, which are intended to be comprehended within the scope of the present application.

Sequence listing

<110> the animal doctor institute of Lanzhou, china academy of agricultural sciences

<120> polypeptide for promoting pig organism to generate broad spectrum acquired immune response and application thereof

<160> 5

<170> SIPOSequenceListing 1.0

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<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 1

Ala Ser Ala Ile Asn Phe Leu Leu Leu

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<210> 2

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<212> PRT

<213> Artificial sequence (Artificial Sequence)

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Ser 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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<210> 4

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<213> Artificial sequence (Artificial Sequence)

<400> 4

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

1 5 10

<210> 5

<211> 12

<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 organism to generate a 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 can promote proliferation of mononuclear macrophages of healthy pig organisms and/or promote CD8 of healthy pig organisms ⁺ T cells and B cells secrete IFN-gamma to enhance the broad spectrum acquired immune response of the swine organism.

2. Use of the polypeptide of claim 1 for the preparation of a formulation for promoting a broad spectrum of acquired immune responses in a swine organism.