CN111925416A - A class of peptides that promote the production of broad-spectrum acquired immune responses in pigs and their applications - Google Patents

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 can promote the proliferation of mononuclear macrophages of healthy pig organisms at high level and remarkably promote CD8 of the healthy pig organisms⁺T cell and B cellThe cells secrete IFN-gamma so as to enhance the generation of broad-spectrum acquired immune response of a pig organism, and the cell can be used as an effective component of a swine disease vaccine or a vaccine immunopotentiator.

Description

A class of peptides that promote the production of broad-spectrum acquired immune responses in pigs and their applications

technical field

The invention relates to the technical field of biomedicine, in particular to a class of polypeptides for promoting the body of pigs to produce a broad-spectrum acquired immune response and applications thereof.

Background technique

Immune enhancers are used alone or in combination with antigens to enhance the activity of animals by enhancing the activity of macrophages, enhancing the immunogenicity and stability of antigenic substances, and promoting the synthesis and secretion of various immune factors and specific antibodies. Substances in the body's immune response.

Immune enhancers can stimulate the body to produce humoral and cell-mediated immune responses, so as to eliminate invading pathogens and protect animals from pathogens.

There are many kinds of immune enhancers, which are generally divided into three categories according to their composition characteristics: Chinese herbal medicine extracts, chemical compounds and cytokines. There are few immune enhancers currently used for swine disease vaccines. It is reported that CVC1320 is used as the immune enhancer of foot-and-mouth disease vaccine (the study of immune enhancer improving the immune efficacy of swine foot-and-mouth disease inactivated vaccine, Yu Xiaoming, 2019), but this immune enhancer is a compound preparation, the composition is complex, and the dosage is large, Production costs are relatively high. Moreover, the preparation is only verified to be applicable to FMDV vaccine, and there is no data to prove that it has a broad-spectrum effect. Tang Bo et al. (Research on the immune potency of inactivated pig vaccines enhanced by immune enhancers, 2016) Using VA5-containing immune enhancers as partners of pig small and JE double inactivated vaccines can improve the antibody titer, but there is no data to prove its efficacy. Has a broad spectrum effect. At present, there is an urgent need for an immunopotentiator that induces a broad-spectrum adaptive immune response in pigs.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a class of polypeptides that promote the production of a broad-spectrum acquired immune response in pigs and applications thereof. The polypeptides of the present invention can significantly promote the proliferation of healthy pig lymphocytes, thereby enhancing the production of a broad-spectrum acquired immune response in pigs. As an effective component of swine disease vaccine or vaccine immune enhancer.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The present invention provides a class of polypeptides for promoting the production of broad-spectrum acquired immune responses in pigs; the polypeptides include one of a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide and a fifth polypeptide species or several;

The amino acid sequence of the first polypeptide is shown in SEQ ID NO.1;

The amino acid sequence of the second polypeptide is shown in SEQ ID NO.2;

The amino acid sequence of the third polypeptide is shown in SEQ ID NO.3;

The amino acid sequence of the fourth polypeptide is shown in SEQ ID NO.4;

The amino acid sequence of the fifth polypeptide is shown in SEQ ID NO.5;

The polypeptide enhances the swine body to produce a broad-spectrum acquired immune response by promoting the proliferation of lymphocytes in the healthy swine body.

The present invention provides a class of polypeptide polymers obtained by polymerizing the polypeptides described in the above scheme.

The present invention provides the application of the polypeptide or the polypeptide polymer described in the above scheme in preparing a preparation for promoting a broad-spectrum acquired immune response in pigs.

The present invention provides a swine disease vaccine swine disease vaccine immune enhancer and/or swine African swine fever subunit vaccine, and the active components of the swine disease vaccine immune enhancer and/or swine African swine fever subunit vaccine include the above solutions. the polypeptide or the polypeptide polymer.

Preferably, the dosage form of the swine disease vaccine immune enhancer or the swine African swine fever subunit vaccine includes injection.

Preferably, the content of the polypeptide or the polypeptide polymer in the swine disease vaccine immune enhancer or the swine African swine fever subunit vaccine is 100 μg/component/head serving.

Beneficial effects of the present invention: The present invention provides a class of polypeptides that promotes a swine body to produce a broad-spectrum acquired immune response; the polypeptides include a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide and a third polypeptide. One or more of the five polypeptides; the amino acid sequences of the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide and the fifth polypeptide are shown in SEQ ID NO. 1 to SEQ ID, respectively NO.5 is shown. The polypeptide of the invention can significantly promote the proliferation of healthy swine lymphocytes to enhance the swine body to produce a broad-spectrum acquired immune response, and can be used as an effective component of a swine disease vaccine or a vaccine immune enhancer.

Description of drawings

Fig. 1 is the identification chromatogram of AP1;

Fig. 2 is the identification chromatogram of AP2;

Fig. 3 is the identification chromatogram of AP3;

Fig. 4 is the identification chromatogram of AP4;

Fig. 5 is the identification chromatogram of AP5;

Figure 6 is a flow statistic diagram of AP1-AP5 promoting the proliferation of ASFV-sensitized lymphocytes and monocyte-macrophages;

Figure 7 is a flow statistic chart showing that AP1-AP5 promotes the proliferation of healthy porcine lymphocytes and monocyte-macrophages;

Figure 8 is the level of AP1 promoting the secretion of IFN-γ by different subtypes of immune cells;

Figure 9 is the level of AP2 promoting the secretion of IFN-γ by different subtypes of immune cells;

Figure 10 is the level of AP3 promoting the secretion of IFN-γ by different subtypes of immune cells;

Figure 11 is the level of AP4 promoting the secretion of IFN-γ by different subtypes of immune cells;

Figure 12 is the level of AP5 promoting the secretion of IFN-γ by different subtypes of immune cells;

Figure 13 shows the proportion of B lymphocyte subsets on the 14th day of the second immunity;

Figure 14 shows the proportion of CD8 ⁺ T lymphocyte subsets;

Figure 15 shows the proportion of B lymphocyte subsets on the 14th day of the second immunity;

Figure 16 shows the level of CD8 ⁺ T lymphocyte subset proportions.

Detailed ways

The present invention provides a class of polypeptides for promoting the production of broad-spectrum acquired immune responses in pigs; the polypeptides include one of a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide and a fifth polypeptide one or several species; the polypeptide enhances the swine body to produce a broad-spectrum acquired immune response by promoting the proliferation of lymphocytes in the healthy swine body.

In the present invention, the amino acid sequence of the first polypeptide is shown in SEQ ID NO.1, specifically: ASAINFLLL (Ala-Ser-Ala-Ile-Asn-Phe-Leu-Leu-Leu), the average molecular weight is 961.15 g/mol, the chemical formula is: C ₄₆ H ₇₆ N ₁₀ O ₁₂ . The theoretical isoelectric point of this polypeptide is pH 7, the GRAVY value of this polypeptide is 2, and it is hydrophobic. This feature facilitates its binding to host proteins or cell surfaces, and can more effectively initiate immune responses. Similar compounds have not been found in the prior art.

In the present invention, the amino acid sequence of the second polypeptide is shown in SEQ ID NO.2, specifically: SASAINFLLL (Ser-Ala-Ser-Ala-Ile-Asn-Phe-Leu-Leu-Leu), average The molecular weight is 1048.23 g/mol, and the chemical formula is: C ₄₉ H ₈₁ N ₁₁ O ₁₄ . The theoretical isoelectric point of the polypeptide is pH 7, the GRAVY value of the polypeptide is 1.72, and the polypeptide has hydrophobicity, which facilitates its binding with host proteins or cell surfaces, and can more effectively initiate an immune response. Similar compounds have not been found in the prior art.

In the present invention, the amino acid sequence of the third polypeptide is shown in SEQ ID NO.3, specifically: HSINMGIFL (His-Ser-Ile-Asn-Met-Gly-Ile-Phe-Leu), and the average molecular weight is 1031.22 g/mol, the chemical formula is: C ₄₇ H ₇₄ N ₁₂ O ₁₂ S. The theoretical isoelectric point of the polypeptide is pH 7.88, the GRAVY value of the polypeptide is 1.07, and it has hydrophobicity, which facilitates its binding with host proteins or cell surfaces, and can more effectively initiate an immune response. Similar compounds have not been found in the prior art.

In the present invention, the amino acid sequence of the fourth polypeptide is shown in SEQ ID NO. 4, specifically: LFPMYSHLFTLI(Leu-Phe-Pro-Met-Tyr-Ser-His-Leu-Phe-Thr-Leu- Ile), the average molecular weight is 1481.79 g/mol, and the chemical formula is: C ₇₄ H ₁₀₈ N ₁₄ O ₁₆ S. The theoretical isoelectric point of the polypeptide is pH 7.88, the GRAVY value of the polypeptide is 1.32, and it has hydrophobicity, which facilitates its binding with host proteins or cell surfaces, and can more effectively initiate immune responses. Similar compounds have not been found in the prior art.

In the present invention, the amino acid sequence of the fifth polypeptide is shown in SEQ ID NO.5, specifically: FTGMVPVSEYLI(Phe-Thr-Gly-Met-Val-Pro-Val-Ser-Glu-Tyr-Leu- Ile), the average molecular weight is 1355.59 g/mol, and the chemical formula is: C ₆₄ H ₉₈ N ₁₂ O ₁₈ S. The theoretical isoelectric point of the polypeptide is pH 3.28, the GRAVY value of the polypeptide is 1.09, and it has hydrophobicity, which facilitates its binding with host proteins or cell surfaces, and can more effectively initiate an immune response. Similar compounds have not been found in the prior art.

In the present invention, the polypeptide described in the above technical solution is obtained by determining the sequence of ASFV circulating strains and predicting by computer-aided bioinformatics, and has specificity.

The present invention provides a class of polypeptide polymers obtained by polymerizing the polypeptides described in the above scheme.

The present invention provides the application of the polypeptide or the polypeptide polymer described in the above scheme in preparing a preparation for promoting a broad-spectrum acquired immune response in pigs.

In the present invention, the polypeptide or the polypeptide polymer enhances the swine body to produce a broad-spectrum adaptive immune response by promoting the proliferation of lymphocytes in the healthy swine body.

The present invention provides a swine disease vaccine swine disease vaccine immune enhancer and/or swine African swine fever subunit vaccine, and the active components of the swine disease vaccine immune enhancer and/or swine African swine fever subunit vaccine include the above solutions. The polypeptide or the polypeptide polymer; the dosage form of the swine disease vaccine immune enhancer or the swine African swine fever subunit vaccine preferably includes injection; The content of the polypeptide or the polypeptide polymer is preferably 100 μg/component/head portion, that is, the content of each polypeptide or each polypeptide polymer is 100 μg/head portion.

The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some, but not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Example 1 Solid Phase Synthesis and Purity Detection of Polypeptides

The polypeptides of the present invention 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 specific implementation process of the present invention, the polypeptide is synthesized by Shanghai Sangon Bioengineering Co., Ltd.

The detection wavelength is 214 nm. The purity of the final purified polypeptide product was >98%, and the structure was identified by ESI-MS. The identification results are shown in Figures 1 to 5. Figure 1 is the identification chromatogram of AP1, Figure 2 is the identification chromatogram of AP2, and Figure 3 is the identification chromatogram of AP2. The identification chromatogram of AP3, Figure 4 is the identification chromatogram of AP4, the peak time is 13.329min. Figure 5 is the identification chromatogram of AP5, the peak time is 14.298min. It can be seen from Fig. 1 to Fig. 5 that the present invention successfully synthesized the above-mentioned five polypeptides.

Example 2 Porcine Lymphocyte Proliferation Experiment

1. ASFV inactivated virus immunized pigs.

Five 90-day-old male landrace pigs were immunized with inactivated virus of ASFV circulating strain (from the African Swine Fever Regional Laboratory of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences) (10HID50), boosted once a month later, and administered to the pigs 7 days later Euthanize and remove the spleen after dissection. Unimmunized healthy pigs were used as a negative control group.

2. Preparation, culture and proliferation detection of spleen cells.

1) Aseptically treat the collected pig spleen with 75% alcohol. After washing three times with PBS, the spleen is cut into small pieces, placed in a folded sterile gauze (2 layers), and placed in 5 mL of serum-containing 1640 medium. Grind the spleen in a petri dish.

2) Then suck the liquid into a 15ml centrifuge tube, centrifuge at 1000rpm for 5min.

3) Discard the supernatant and tap the pellet (cells) to suspend uniformly.

4) Add 10 ml of erythrocyte lysis solution, lyse for 10 minutes, then add 6 ml of serum-containing 1640 medium to terminate the lysis, mix well, and centrifuge at 1000 rpm for 5 minutes.

5) Discard the supernatant, tap the precipitate again to make it evenly suspended, then take 10 μL of the suspension to dilute 40 times, count the cells, and adjust the cell concentration to 1×10 ⁶ cells/mL.

6) After CFSE staining, the prepared splenocytes were seeded into a 24-well plate (1×10 ⁶ cells/well).

7) 0.2 μg of the first to fifth polypeptides provided by the present invention were respectively added, placed in a CO ₂ incubator for 60 hours, and the proliferation of ASFV antigen-specific splenocytes was detected by flow cytometry.

The detection results are shown in Figure 6. Figure 6 is a flow statistic diagram of AP1-AP5 promoting the proliferation of ASFV-sensitized lymphocytes and monocyte-macrophages. It can be seen from Figure 6 that the five polypeptides of the present invention can significantly promote the specificity of ASFV. Spleen immune cells proliferate, and the main type of immune cells is mononuclear macrophages. .

The healthy pig spleen lymphocytes were treated in the same way, and the effect of the polypeptide on the proliferation of non-antigen-specific spleen cells in pigs was examined.

The detection results are shown in FIG. 7 , which is a flow statistic diagram of AP1-AP5 promoting the proliferation of healthy porcine lymphocytes and monocyte-macrophages. It can be seen from Figure 7 that the five polypeptides of the present invention can also significantly promote the proliferation of healthy pig spleen immune cells. The main type of immune cells is mononuclear macrophages.

Example 3 Detection of IFN-γ secretion by different subtypes of spleen lymphocytes

The procedure of immunization of pigs and the isolation and culture of spleen cells were the same as those in Example 2. After the dispersed splenocytes were obtained, a single cell suspension was prepared in RPMI1640 complete medium at a concentration of 1×10 ⁶ /ml. It was inoculated into a 24-well plate, and 0.2 μg of the polypeptides (AP1-AP5) provided by the present invention were added to each well, and cultured in a CO ₂ incubator for 60 hours. Cells in each well were collected, labeled with porcine CD3, CD4, CD8 and IFN-γ-specific antibodies, respectively, and then washed twice with PBS buffer containing 1-2% serum, and finally dispersed into a cell suspension with the washing solution. The levels of IFN-γ secreted by total T lymphocytes, B cells, CD4 ⁺ T lymphocytes, CD8 ⁺ T lymphocytes and mononuclear macrophages were determined by flow cytometry.

The test results are shown in Figures 8 to 11, in which Figure 8 shows the levels of IFN-γ that AP1 promotes different subtypes of immune cells; Figure 9 shows the levels of AP2 that promote the secretion of IFN-γ by different subtypes of immune cells; Figure 10 shows that AP3 promotes different immune cells to secrete IFN-γ. The levels of IFN-γ secreted by immune cells of different subtypes; Figure 11 is the level of IFN-γ secretion by different subtypes of immune cells promoted by AP4; Figure 12 is the level of IFN-γ secretion by different subtypes of immune cells promoted by AP5; 12 It can be seen that the five polypeptides in the present invention can significantly enhance the ability of B lymphocytes and CD8 ⁺ T lymphocytes to secrete IFN-γ.

Example 4: Immune cell typing experiment of immunizing animals with polypeptides as active ingredients of ASFV vaccine

Three 90-day-old male landrace pigs were immunized with 5 polypeptides in the present invention mixed (100 μg/head of each polypeptide), boosted once a month later, and flow cytometry was used to detect the level of immune cells in peripheral blood 14 days after the second immunization. Proportion. The same volume of PBS immunization group served as a control.

The detection results are shown in Figures 13 to 14, wherein Figure 13 shows the proportion of B lymphocyte subsets on 14 d of the second immunity; Figure 14 shows the proportion of CD8 ⁺ T lymphocyte subsets. It can be seen from Fig. 13 to Fig. 14 that the mixed immunization of the five polypeptides in the present invention can promote the immune response of lymphocytes in pigs and enhance the immunity of pigs.

Example 6: Immune cell typing experiment of immunizing animals with polypeptides as immune enhancers

Immunize three 90-day-old male Landrace pigs with foot-and-mouth disease O/MYA98/BY/2010 strain inactivated vaccine or inactivated vaccine plus 5 polypeptides of the present invention mixed (100 μg/head of each polypeptide), and boost after one month After the first immunization, the proportion of immune cells in peripheral blood was detected by flow cytometry 14 days after the second immunization. The same volume of PBS immunization group served as a control.

The detection results are shown in Figures 15 to 16, wherein Figure 15 shows the proportion of B lymphocyte subsets on the 14th day of the second immunity; Figure 16 shows the proportion of CD8 ⁺ T lymphocyte subsets. It can be seen from Fig. 15 to Fig. 16 that the five polypeptides in 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 above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

sequence listing

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Claims (6)

1. A polypeptide that promotes the production of a broad-spectrum acquired immune response in pigs; the polypeptide comprises one of a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide and a fifth polypeptide or several; The amino acid sequence of the first polypeptide is shown in SEQ ID NO.1; The amino acid sequence of the second polypeptide is shown in SEQ ID NO.2; The amino acid sequence of the third polypeptide is shown in SEQ ID NO.3; The amino acid sequence of the fourth polypeptide is shown in SEQ ID NO.4; The amino acid sequence of the fifth polypeptide is shown in SEQ ID NO.5; The polypeptide enhances the production of broad-spectrum adaptive immune responses in pigs by promoting the proliferation of mononuclear macrophages in healthy pigs and/or promoting the secretion of IFN-γ by CD8 ⁺ T cells and B cells in healthy pigs. 2. A class of polypeptide polymers obtained by polymerizing the polypeptide of claim 1. 3. Use of the polypeptide according to claim 1 or the polypeptide polymer according to claim 2 in the preparation of a preparation for promoting a swine body to produce a broad-spectrum adaptive immune response. 4. a swine disease vaccine swine disease vaccine immune enhancer and/or swine African swine fever subunit vaccine, the active ingredient of said swine disease vaccine immune enhancer and/or swine African swine fever subunit vaccine comprises the described in claim 1 The polypeptide or the polypeptide polymer of claim 2. 5. swine disease vaccine immune enhancer and/or swine African swine fever subunit vaccine according to claim 4, is characterized in that, the dosage form of described swine disease vaccine immune enhancer or swine African swine fever subunit vaccine comprises injection . 6. swine disease vaccine immune enhancer and/or swine African swine fever subunit vaccine according to claim 5, is characterized in that, described polypeptide in described swine disease vaccine immune enhancer or swine African swine fever subunit vaccine Or the content of the polypeptide polymer is 100 μg/component/head portion.

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