CN113956329A

CN113956329A - Polypeptide for promoting pig body to generate broad-spectrum immune response and application thereof

Info

Publication number: CN113956329A
Application number: CN202111340790.4A
Authority: CN
Inventors: 郑海学; 杨帆; 毛箬青; 周晓丽; 孙德惠; 朱昱茜; 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: 2022-01-21
Anticipated expiration: 2040-08-26
Also published as: CN113956329B; CN113956330B; CN112159459B; CN113956330A; CN112159459A

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 biomedicine; 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 remarkably promote IFN-beta signal path activation in a pig organism, promote mononuclear macrophages, T cells and B cells to secrete IFN-gamma, and promote spleen lymphocytes and the mononuclear macrophages to proliferate, so that the pig organism is promoted to generate broad-spectrum immune response; in addition, the polypeptide of the invention can promote the ASFV antigen specific immune response in pigs, thus being capable of being used as an effective component of ASFV subunit vaccine to enhance the immune response of pigs.

Description

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

The application is a divisional application with the application date of 26/08/2020 and the application number of CN202010873521.3, 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 promote mononuclear macrophages, T cells and B cells in a pig body to secrete IFN-gamma so as to promote the pig body to generate broad-spectrum immune response, and can be used as an effective component of an ASFV subunit vaccine to enhance the immune response of pigs.

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 promotes a pig organism to generate a broad-spectrum immune response by promoting the activation of an IFN-beta signal channel in the pig organism; and/or the presence of a gas in the gas,

the polypeptide promotes a pig body to generate a broad-spectrum immune response by promoting mononuclear macrophages, T cells and B cells in the pig body to secrete IFN-gamma; and/or the presence of a gas in the gas,

the polypeptide promotes a pig body to generate a broad-spectrum immune response by promoting proliferation of mononuclear macrophages, T cells and B cells in the pig body.

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 application of the polypeptide or the polypeptide polymer in the scheme in preparation of a preparation for promoting a swine organism to generate African swine fever virus antigen specific 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 polypeptide or the polypeptide polymer is contained in the immunopotentiator in an amount of 100. mu.g/fraction.

The invention provides an African swine fever virus subunit vaccine which comprises the polypeptide or the polypeptide polymer in the scheme.

Preferably, the content of the polypeptide or the polypeptide polymer in the subunit vaccine is 100 μ g/fraction.

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 remarkably promote IFN-beta signal path activation in a pig organism, promote mononuclear macrophages, T cells and B cells to secrete IFN-gamma, and promote spleen lymphocytes and the mononuclear macrophages to proliferate, so that the pig organism is promoted to generate broad-spectrum immune response; in addition, the polypeptide of the invention can promote the ASFV antigen specific immune response in pigs, thus being capable of being used as an effective component of ASFV subunit vaccine to enhance the immune response of pigs.

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 statistical chart of flow data of AP 1-AP 5 promoting proliferation of ASFV sensitized lymphocytes and mononuclear macrophages;

FIG. 12 is a statistical chart of flow data of AP 1-AP 5 promoting proliferation of lymphocytes and monocytes macrophages in healthy pigs;

FIG. 13 shows that AP1 promotes IFN-. gamma.secretion by different subtypes of immune cells;

FIG. 14 shows that AP2 promotes IFN- γ secretion by different subtypes of immune cells;

FIG. 15 shows that AP3 promotes IFN-. gamma.secretion by different subtypes of immune cells;

FIG. 16 shows that AP4 promotes IFN- γ secretion by different subtypes of immune cells;

FIG. 17 shows that AP5 promotes IFN- γ secretion by different subtypes of immune cells;

FIG. 18 shows levels of IFN- β reporter gene promoted by AP 1-AP 5;

FIG. 19 is the hyperimmune 14d, B lymphocyte subpopulation fraction levels;

FIG. 20 shows CD8⁺T lymphocyte subpopulation proportion level;

FIG. 21 is a graph of hyperimmune 14d, B lymphocyte subpopulation fraction levels;

FIG. 22 shows a 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 promotes a pig organism to generate a broad-spectrum immune response by promoting the activation of an IFN-beta signal channel in the pig organism; and/or the polypeptide promotes the pig body to generate a broad-spectrum immune response by promoting mononuclear macrophages, T cells and B cells in the pig body to secrete IFN-gamma; and/or, the polypeptide promotes the pig body to generate a broad-spectrum immune response by promoting proliferation of mononuclear macrophages, T cells and B cells in the pig body.

In the invention, the amino acid sequence of the first polypeptide is shown as SEQ ID NO.1, and specifically comprises the following steps: MMIFLTYDL (Met-Met-Ile-Phe-Leu-Thr-Tyr-Asp-Leu), average molecular weight 1146.41g/mol, chemical formula: c₅₄H₈₃N₉O₁₄S₂. The polypeptide has a theoretical isoelectric point of pH 3.12, a GRAVY value of 1.47, and hydrophobicity. This property facilitates its binding to host proteins or cell surfaces, enabling a more efficient initiation of immune responses.

In the invention, the amino acid sequence of the second polypeptide is shown as SEQ ID NO.2, and specifically comprises: LSIGYTLLF (Leu-Ser-Ile-Gly-Tyr-Thr-Leu-Leu-Phe), with average molecular weight of 1026.22g/mol, and chemical formula: c₅₁H₇₉N₉O₁₃. The theoretical isoelectric point of the polypeptide is pH 7, the GRAVY value of the polypeptide is 1.72, 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 present invention, the amino acid sequence of the third polypeptide is as shown in SEQ ID NOAnd 3, specifically: HLNGFVSTL (His-Leu-Asn-Gly-Phe-Val-Ser-Thr-Leu), average molecular weight 987.1g/mol, chemical formula: c₄₅H₇₀N₁₂O₁₃. The polypeptide has a theoretical isoelectric point of pH 7.88 and a GRAVY value of 0.67, has hydrophobicity, is favorable for being combined with host protein or cell surface, and can more effectively start immune response.

In the invention, the amino acid sequence of the fourth polypeptide is shown as SEQ ID NO.4, and specifically comprises: ASGGGPAP (Ala-Ser-Gly-Gly-Gly-Pro-Ala-Pro), average molecular weight is 612.63g/mol, and the chemical formula is as follows: c₂₅H₄₀N₈O₁₀. The polypeptide has a theoretical isoelectric point of pH 7, a GRAVY value of-0.2, and hydrophilicity.

In the invention, the amino acid sequence of the fifth polypeptide is shown as SEQ ID NO.5, and specifically comprises: GIALDLSTL (Gly-Ile-Ala-Leu-Asp-Leu-Ser-Thr-Leu), average molecular weight 902.04g/mol, chemical formula: c₄₀H₇₁N₉O₁₄. The theoretical isoelectric point of the polypeptide is pH 3.12, the GRAVY value of the polypeptide is 1.37, 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 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 present invention, the polypeptide is preferably synthesized by Shanghai Bioengineering Co., Ltd.

The invention provides a polypeptide polymer comprising a polypeptide according to the above scheme.

In the invention, the polypeptide or the polypeptide polymer promotes the pig body to generate a broad-spectrum immune response by promoting the activation of an IFN-beta signal channel in the pig body; and/or, promote mononuclear macrophage, T cell and B cell in pig organism to secrete IFN-gamma to promote pig organism to produce broad-spectrum immune response; and/or, the polypeptide promotes the pig body to generate a broad-spectrum immune response by promoting proliferation of mononuclear macrophages, T cells and B cells in the pig body.

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 African swine fever virus antigen specific immune response; the polypeptide or the polypeptide polymer promotes the swine organism to generate African swine fever virus antigen specific immune response by promoting spleen lymphocytes and mononuclear macrophages of the swine organism to proliferate.

The invention provides a pig immunopotentiator, the active component of which comprises the polypeptide or the polypeptide polymer in the scheme; the content of the polypeptide or the polypeptide polymer in the immunopotentiator is preferably 100. mu.g/fraction, that is, the content of each polypeptide or each polypeptide polymer is 100. mu.g/fraction.

The invention provides a subunit vaccine of African swine fever virus, which comprises the polypeptide or the polypeptide polymer of the scheme; the content of the polypeptides or the polypeptide polymers in the subunit vaccine is preferably 100. mu.g/fraction, i.e., the content of each polypeptide or each polypeptide polymer is 100. mu.g/fraction.

The dosage forms of the preparation according to the above-mentioned scheme of the invention or the immunopotentiator according to the above-mentioned scheme or the subunit vaccine according to the above-mentioned scheme respectively comprise injections.

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：MMIFLTYDL；

SEQ ID NO.2：LSIGYTLLF；

SEQ ID NO.3：HLNGFVSTL；

SEQ ID NO.4：ASGGGPAP；

SEQ ID NO.5：GIALDLSTL；

the polypeptide is synthesized by Shanghai biological engineering Co., Ltd.

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 result is shown in figures 1-10, wherein figure 1 is an identification chromatogram of AP1, figure 2 is a mass spectrum of AP1, and the identification molecular weight is 1146g/mol, which is consistent with the theoretical value; FIG. 3 is an identification chromatogram of AP2, FIG. 4 is a mass spectrum of AP2, the identification molecular weight is 1026.6g/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 987.55g/mol, which is consistent with the theoretical value; FIG. 7 is an identification chromatogram of AP4, and FIG. 8 is a mass spectrum of AP4, the identification molecular weight is 612.9g/mol, which is consistent with the theoretical value; FIG. 9 is an identification chromatogram of AP5, and FIG. 10 is a mass spectrum of AP5, identifying the molecular weight of 902.5g/mol, corresponding to the theoretical value; as is clear from FIGS. 1 to 10, the 5 polypeptides were successfully synthesized.

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 results of the measurements are shown in FIG. 11. FIG. 11 is a flow chart showing AP 1-AP 5 as flow charts of the promotion of proliferation of ASFV-sensitized lymphocytes and monocytes and macrophages. As can be seen from FIG. 11, all 5 polypeptides of the present invention significantly promoted proliferation of ASFV-specific splenic immune cells, 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 results of the test are shown in FIG. 12. FIG. 12 is a statistical chart of flow data of AP 1-AP 5 promoting proliferation of lymphocytes and monocytes. As can be seen from FIG. 12, 5 polypeptides of the present invention also significantly promoted the proliferation of splenic immune cells in healthy pigs. The 5 polypeptides are proved to have the function of broad-spectrum promotion of pig immunity.

Example 3 detection of IFN-. gamma.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 into 24-well plate, adding 0.2 μ g per well of the polypeptide provided by the present inventionAnd (3) placing the peptide in a CO2 incubator for 60 hours. Collecting cells in each well, labeling with porcine CD3, CD4, CD8 and IFN-gamma specific antibody, washing with 2% serum-containing PBS buffer solution for 2 times, dispersing into cell suspension, detecting with flow cytometer, and determining CD4⁺T lymphocytes and CD8⁺Levels of IFN- γ secretion by T lymphocytes.

The results of the detection are shown in FIGS. 13 to 17. Wherein FIG. 13 is a graph of the levels of IFN- γ secretion by different subtypes of immune cells promoted by AP 1; FIG. 14 shows that AP2 promotes IFN- γ secretion by different subtypes of immune cells; FIG. 15 shows that AP3 promotes IFN-. gamma.secretion by different subtypes of immune cells; FIG. 16 shows that AP4 promotes IFN- γ secretion by different subtypes of immune cells; FIG. 17 shows that AP5 promotes IFN- γ secretion by different subtypes of immune cells; as can be seen from FIGS. 13-17, the five polypeptides of the present invention all significantly enhanced monocyte macrophages, B cells and CD8⁺The ability of T lymphocyte cells to secrete IFN- γ.

Example 4 detection of the Effect of Polypeptides on innate immune response

HEK293-T cells were seeded at 50% density in 12-well cell culture plates and transfected with luciferase reporter plasmid at around 70% of the cells in around 16 h. After 6h of transfection, the polypeptide of the invention was added and incubated for 24 h. Luciferase reaction intensity was measured 12h after SEV stimulation. DMSO incubated wells served as negative controls.

The results of the measurements are shown in FIG. 18. FIG. 18 shows levels of IFN- β reporter gene promoted by AP 1-AP 5. As can be seen from FIG. 18, AP 1-AP 5 all significantly promoted the level of IFN- β reporter gene and thus promoted the innate immune response.

Example 5 polypeptide immune animal immune cell typing experiment

3 male Changbai pigs of 90 days old are mixed and immunized by 5 polypeptides in the invention, the immunization is strengthened once after one month, and the proportion of immune cells in peripheral blood is detected by 14d flow cytometry after two-immunization. The same volume PBS immunized group served as control.

The results of the detection are shown in FIGS. 19 to 20. Wherein FIG. 19 is the Diavoids 14d, B lymphocyte subpopulation proportion levels; FIG. 20 shows CD8⁺T lymphocyte subpopulation ratio levels. E from FIG. 19EFIG. 20 shows that 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. 21-22, in which FIG. 21 shows the proportion of the B lymphocyte subpopulation at the level of the hyperimmune 14 d; FIG. 22 shows a CD8⁺T lymphocyte subpopulation ratio levels. From FIGS. 21 to 22, 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

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Met Ile Phe Leu Thr Tyr Asp Leu

1 5

<210> 2

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Leu Ser Ile Gly Tyr Thr Leu Leu Phe

1 5

<210> 3

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

His Leu Asn Gly Phe Val Ser Thr Leu

1 5

<210> 4

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Ala Ser Gly Gly Gly Pro Ala Pro

1 5

<210> 5

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Gly Ile Ala Leu Asp Leu Ser Thr Leu

1 5

Claims

1. A polypeptide for promoting a pig body to generate a broad-spectrum immune response, wherein the polypeptide is a third polypeptide or a fifth polypeptide;

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

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

the polypeptide promotes the pig body to generate broad-spectrum immune response by promoting the activation of an IFN-beta signal channel in the pig body, and/or,

the polypeptide promotes the pig body to generate broad-spectrum immune response by promoting mononuclear macrophages, T cells and B cells in the pig body to secrete IFN-gamma, and/or,

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. Use of a polypeptide according to claim 1 in the manufacture of a formulation for promoting the production of an african swine fever virus antigen-specific immune response in a swine organism.

4. An immunopotentiator for swine, the active ingredient of which comprises the polypeptide of claim 1.

5. The immunopotentiator according to claim 4, wherein the amount of the polypeptide in the immunopotentiator is 100 μ g/fraction.

6. A subunit vaccine of african swine fever virus comprising the polypeptide of claim 1.

7. The subunit vaccine of claim 6, wherein the polypeptide is present in the subunit vaccine in an amount of 100 μ g/fraction.