A class of polypeptides that promote the generation of a broad-spectrum immune response in pigs and their applications

Abstract

The present invention provides a class of polypeptides that promote the generation of broad-spectrum acquired immune responses in pigs, belonging to the field of biomedical technology; the polypeptides include one or several 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 in SEQ ID NO.1 to SEQ ID NO.5. The polypeptides of the present invention can significantly promote the secretion of IFN-γ by monocytes, macrophages, T lymphocytes, and B lymphocytes in the pig body and promote the proliferation of monocytes, macrophages, T cells, and B cells in the pig body, thereby promoting the generation of broad-spectrum immune responses in the body. The polypeptides of the present invention can promote the ASFV antigen-specific immune response in pigs and can thus be used as an effective component of an ASFV subunit vaccine to enhance the immune response of pigs.

Description

[0001] This application is a divisional application with an application date of August 26, 2020, application number CN202010871921.0, and invention name: "A class of polypeptides that promote the production of broad-spectrum acquired immune responses in pigs and their applications." Technical Field

[0002] The present invention relates to the field of biomedical technology, and in particular to a class of polypeptides for promoting a pig body to produce a broad-spectrum acquired immune response and applications thereof. Background Art

[0003] Immunopotentiators are a type of substance that is used alone or in combination with antigens to enhance the immune response of animals by increasing macrophage activity, enhancing the immunogenicity and stability of antigens, and promoting the synthesis and secretion of various immune factors and specific antibodies.

[0004] Immunoenhancers can stimulate the body to produce humoral-mediated and cell-mediated immune responses, thereby eliminating invading pathogens and protecting animals from the harm of pathogens.

[0005] There are many types of immunopotentiators, which can be broadly divided into three categories based on their compositional properties: Chinese herbal extracts, chemical compounds, and cytokines. Currently, there are relatively few immunopotentiators used in swine disease vaccines. There are reports of using CVC1320 as an immunopotentiator for foot-and-mouth disease vaccines (Study on Immunopotentiators to Enhance the Efficacy of Inactivated Foot-and-Mouth Disease Vaccines in Swine, Yu Xiaoming, 2019). However, this immunopotentiator is a compound preparation with complex ingredients and requires a large dosage, resulting in relatively high production costs. Furthermore, this preparation has only been validated for use with FMDV vaccines, with no data demonstrating its broad-spectrum efficacy. Tang Bo et al. (Study on Immunopotentiators to Enhance the Efficacy of Inactivated Vaccines for Swine, 2016) used an immunopotentiator containing VA5 as a companion to a combined inactivated vaccine for porcine parvovirus and Japanese encephalitis, which was found to increase antibody titers, but there is no data demonstrating its broad-spectrum efficacy. Currently, there is a pressing need for an immunopotentiator that can stimulate a broad-spectrum immune response in pigs. Summary of the Invention

[0006] The purpose 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 their applications. The polypeptides of the present invention can promote the secretion of IFN-γ by monocytes, macrophages, T cells and B cells in the pig body, thereby prompting the pig body to produce a broad-spectrum immune response, and the polypeptide can be used as an effective ingredient in the ASFV subunit vaccine to enhance the immune response of pigs.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a class of polypeptides that promote the production of a broad-spectrum acquired immune response in a pig body; the polypeptides include one or more of a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide, and a fifth polypeptide;

[0009] The amino acid sequence of the first polypeptide is shown in SEQ ID NO.1;

[0010] The amino acid sequence of the second polypeptide is shown in SEQ ID NO.2;

[0011] The amino acid sequence of the third polypeptide is shown in SEQ ID NO.3;

[0012] The amino acid sequence of the fourth polypeptide is shown in SEQ ID NO.4;

[0013] The amino acid sequence of the fifth polypeptide is shown in SEQ ID NO.5;

[0014] The polypeptide promotes the pig body to produce a broad-spectrum acquired immune response by promoting the secretion of IFN-γ by mononuclear macrophages, T cells and B cells in the pig body; and / or,

[0015] The polypeptide promotes the pig body to produce a broad-spectrum acquired immune response by promoting the proliferation of mononuclear macrophages, T cells and B cells in the pig body.

[0016] The present invention provides a class of polypeptide polymers obtained by polymerizing the polypeptides described in the above scheme.

[0017] The present invention provides the use of the polypeptide or polypeptide polymer described in the above scheme in preparing a preparation for promoting a broad-spectrum acquired immune response in a pig body.

[0018] The present invention provides the use of the polypeptide or polypeptide polymer described in the above scheme in the preparation of a preparation for promoting the production of an African swine fever virus antigen-specific immune response in a pig body.

[0019] The present invention provides a pig immunopotentiator, wherein the active ingredient of the immunopotentiator comprises the polypeptide or the polypeptide polymer described in the above scheme.

[0020] Preferably, the content of the active ingredient in the immunopotentiator is 100 μg / component / portion.

[0021] The present invention provides a subunit vaccine for African swine fever virus, which comprises the polypeptide or the polypeptide polymer described in the above scheme.

[0022] Preferably, the content of the active component in the subunit vaccine is 100 μg / component / dose.

[0023] Beneficial effects of the present invention: The present invention provides a class of polypeptides that promote the production of a broad-spectrum acquired immune response in pigs; the polypeptides include 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 shown in SEQ ID NO.1 to SEQ ID NO.5, respectively. The polypeptides of the present invention can significantly promote the secretion of IFN-γ by mononuclear macrophages, T cells, and B cells in pigs and promote the proliferation of mononuclear macrophages, T cells, and B cells in pigs, thereby prompting the body to produce a broad-spectrum immune response. The polypeptides of the present invention can promote an ASFV antigen-specific immune response in pigs, and thus can be used as an effective component of an ASFV subunit vaccine to enhance the immune response of pigs. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the identification chromatogram of AP1;

[0025] Figure 2 is the mass spectrum of AP1;

[0026] Figure 3 is the identification chromatogram of AP2;

[0027] Figure 4 is the mass spectrum of AP2;

[0028] Figure 5 This is the identification chromatogram of AP3;

[0029] Figure 6 is the mass spectrum of AP3;

[0030] Figure 7 This is the identification chromatogram of AP4;

[0031] Figure 8 is the mass spectrum of AP4;

[0032] Figure 9 This is the identification chromatogram of AP5;

[0033] Figure 10 is the mass spectrum of AP5;

[0034] Figure 11 The flow cytometry data statistics of AP1-AP5 promoting the proliferation of ASFV-sensitized lymphocytes and monocytes and macrophages;

[0035] Figure 12 The flow cytometry data statistics of AP1-AP5 promoting the proliferation of healthy pig lymphocytes and monocytes and macrophages;

[0036] Figure 13 AP1 promotes the secretion of IFN-γ by different subtypes of immune cells;

[0037] Figure 14 AP2 promotes the secretion of IFN-γ by different subtypes of immune cells;

[0038] Figure 15 AP3 promotes the secretion of IFN-γ by different subtypes of immune cells;

[0039] Figure 16 AP4 promotes the secretion of IFN-γ by different subtypes of immune cells;

[0040] Figure 17 AP5 promotes the secretion of IFN-γ by different subtypes of immune cells;

[0041] Figure 18 14 days after the second immunization, the proportion of B lymphocyte subsets;

[0042] Figure 19 CD8 + T lymphocyte subset ratio level;

[0043] Figure 20 14 days after the second immunization, the proportion of B lymphocyte subsets;

[0044] Figure 21 CD8 + The proportion level of T lymphocyte subsets. DETAILED DESCRIPTION

[0045] The present invention provides a class of polypeptides that promote a pig's body to produce a broad-spectrum acquired immune response; the polypeptides include one or more of a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide, and a fifth polypeptide; the polypeptides promote the pig's body to produce a broad-spectrum acquired immune response by promoting the secretion of IFN-γ by monocytes, macrophages, T cells, and B cells in the pig's body; and / or, the polypeptides promote the pig's body to produce a broad-spectrum acquired immune response by promoting the proliferation of monocytes, macrophages, T cells, and B cells in the pig's body.

[0046] In the present invention, the amino acid sequence of the first polypeptide is shown in SEQ ID NO. 1, specifically: SMMMVFNQLI (Ser-Met-Met-Met-Val-Phe-Asn-Gln-Leu-Ile); the average molecular weight of the first polypeptide is 1213.53 g / mol, and the chemical formula is: C 53 H 88 N 12 O 14 S3. This polypeptide has a theoretical isoelectric point of pH 7 and a Gravity value of 1.32, indicating hydrophobicity. This property facilitates its binding to host proteins or cell surfaces, enabling a more effective immune response. No similar compounds are known in the prior art.

[0047] In the present invention, the amino acid sequence of the second polypeptide is shown in SEQ ID NO. 2, specifically: KLFQIIELL (Lys-Leu-Phe-Gln-Ile-Ile-Glu-Leu-Leu); the average molecular weight of the second polypeptide is 1116.39 g / mol, and the chemical formula is: C 55 H 93 N 11 O 13 The theoretical isoelectric point of this polypeptide is pH 7, and the GRAVY value of this polypeptide is 1.37, indicating its hydrophobicity. This property facilitates its binding to host proteins or cell surfaces, enabling a more effective immune response. No similar compounds have been found in the prior art.

[0048] In the present invention, the amino acid sequence of the third polypeptide is shown in SEQ ID NO. 3, specifically: MRIEIFWEL (Met-Arg-Ile-Glu-Ile-Phe-Trp-Glu-Leu); the average molecular weight of the third polypeptide is 1236.48 g / mol, and the chemical formula is: C 59 H 89 N 13 O 14 The theoretical isoelectric point of the polypeptide is pH 4.27, and the GRAVY value of the polypeptide is 0.57, indicating hydrophilicity. No similar compounds have been found in the prior art.

[0049] In the present invention, the amino acid sequence of the fourth polypeptide is shown in SEQ ID NO. 4, specifically: SNDGISFLL (Ser-Asn-Asp-Gly-Ile-Ser-Phe-Leu-Leu); the average molecular weight of the fourth polypeptide is 965 g / mol, and the chemical formula is: C 43 H 68 N 10 O 15 The theoretical isoelectric point of the polypeptide is pH 3.12, and the GRAVY value of the polypeptide is 0.66, indicating that the polypeptide is hydrophobic. No similar compound has been found in the prior art.

[0050] In the present invention, the amino acid sequence of the fifth polypeptide is shown in SEQ ID NO. 5, specifically: MKNMVSKFL (Met-Lys-Asn-Met-Val-Ser-Lys-Phe-Leu); the average molecular weight of the fifth polypeptide is 1097.39 g / mol, and the chemical formula is: C 49 H 84 N 12 O 12S2. This polypeptide has a theoretical isoelectric point of pH 10.6 and a Gravity value of 0.28, indicating hydrophobicity. This property facilitates its binding to host proteins or cell surfaces, enabling a more effective immune response. No similar compounds have been found in the prior art.

[0051] In the present invention, the polypeptide described in the above technical solution is obtained by measuring the sequence of the ASFV epidemic strain and predicting it through computer-assisted bioinformatics, and is specific.

[0052] In the present invention, the polypeptide is preferably synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0053] The present invention provides a polypeptide polymer comprising the polypeptide described in the above scheme.

[0054] The present invention provides the use of the polypeptide or polypeptide polymer described in the above scheme in preparing a preparation for promoting a broad-spectrum acquired immune response in a pig body.

[0055] In the present invention, the polypeptide or the polypeptide polymer promotes the pig body to produce a broad-spectrum acquired immune response by promoting the secretion of IFN-γ by monocytes, macrophages, T cells and B cells in the pig body; and / or, the polypeptide promotes the pig body to produce a broad-spectrum acquired immune response by promoting the proliferation of monocytes, macrophages, T cells and B cells in the pig body.

[0056] The present invention provides the use of the polypeptide or polypeptide polymer described in the above scheme in the preparation of a preparation for promoting the production of an African swine fever virus antigen-specific immune response in a pig body.

[0057] The present invention provides an immunopotentiator for pigs, wherein the active ingredient of the immunopotentiator includes the polypeptide or the polypeptide polymer described in the above scheme; the content of the active ingredient in the immunopotentiator is preferably 100 μg / component / head portion, that is, the content of each polypeptide or each polypeptide polymer is 100 μg / head portion.

[0058] The present invention provides a subunit vaccine for African swine fever virus, which comprises the polypeptide or polypeptide polymer described in the above scheme; the content of the active component in the subunit vaccine is preferably 100 μg / component / head portion, that is, the content of each polypeptide or each polypeptide polymer is 100 μg / head portion.

[0059] The dosage forms of the preparation described in the above embodiment of the present invention, the immunopotentiator described in the above embodiment, or the subunit vaccine described in the above embodiment include injections.

[0060] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0061] Example 1 Solid Phase Synthesis and Purity Detection of Polypeptides

[0062] The polypeptides of the present invention are as follows:

[0063] SEQ ID NO.1:SMMMVFNQLI;

[0064] SEQ ID NO. 2: KLFQIIELL;

[0065] SEQ ID NO. 3: MRIEIFWEL;

[0066] SEQ ID NO.4: SNDGISFLL;

[0067] SEQ ID NO.5: MKNMVSKFL;

[0068] In the specific implementation of the present invention, the polypeptide is synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0069] The detection wavelength was 214 nm. The final peptide purification product had a purity of >98%, and its structure was identified by ESI-MS. The identification results are shown in Figures 1 to 10 ,in Figure 1 This is the identification chromatogram of AP1. Figure 2 This is the mass spectrum of AP1, and the identified molecular weight is 1212.45 g / mol, which is consistent with the theoretical value; Figure 3 This is the identification chromatogram of AP2. Figure 4 Figure 5 is the mass spectrum of AP2, and the identified molecular weight is 1115.70 g / mol, which is consistent with the theoretical value; Figure 6 is the identification chromatogram of AP3, Figure 6 This is the mass spectrum of AP3, and the identified molecular weight is 1236.65 g / mol, which is consistent with the theoretical value; Figure 7 This is the identification chromatogram of AP4. Figure 8 This is the mass spectrum of AP4, and the identified molecular weight is 962.5 g / mol, which is consistent with the theoretical value; Figure 9 This is the identification chromatogram of AP5. Figure 10 The mass spectrum of AP5 is shown in Figure 1, and the molecular weight is 1095.65 g / mol, which is consistent with the theoretical value. Figures 1 to 10 It can be seen that the above five polypeptides were successfully synthesized.

[0070] Example 2 Pig lymphocyte proliferation experiment

[0071] 1. Immunize pigs with inactivated ASFV virus.

[0072] Five 90-day-old male Landrace pigs were immunized with an inactivated ASFV epidemic strain (10 HID50), followed by a booster vaccination one month later. The pigs were euthanized seven days later and their spleens removed after autopsy. Healthy, unimmunized pigs served as a negative control group.

[0073] 2. Preparation, culture and proliferation detection of spleen cells.

[0074] 1) The pig spleen was aseptically treated with 75% alcohol, cut into small pieces, placed in folded sterile gauze (2 layers), and ground in a dish containing 5 mL of serum-containing 1640 culture medium.

[0075] 2) Then, pipette the liquid into a 15 ml centrifuge tube and centrifuge at 1000 rpm for 5 minutes.

[0076] 3) Discard the supernatant and tap the precipitate (cells) to make them evenly suspended.

[0077] 4) Add 1 ml of red blood cell lysis buffer and lyse for 3 minutes. Then add 6 ml of serum-containing 1640 medium to terminate lysis. Mix well and centrifuge at 1000 rpm for 5 minutes.

[0078] 5) Discard the supernatant and tap the precipitate again to evenly suspend it. Then, dilute the suspension 40-fold and count the cells.

[0079] 6) After CFSE staining, the prepared spleen cells were inoculated into 24-well plates (1×10 6 cells / well).

[0080] 7) Add 0.2 μg of the polypeptide provided by the present invention to each well, place the cells in a CO2 incubator and culture for 60 h, and detect the proliferation of ASFV antigen-specific spleen cells by flow cytometry.

[0081] Test results see Figure 11 . Figure 11 The flow cytometry data of AP1-AP5 promoting the proliferation of ASFV-sensitized lymphocytes and monocyte macrophages is shown in Figure 2. Figure 11 It can be seen that the five polypeptides of the present invention can significantly promote the proliferation of ASFV-specific spleen immune cells, and the main types of immune cells are lymphocytes and mononuclear macrophages.

[0082] Healthy pig spleen lymphocytes were treated in the same manner to detect the effect of the polypeptide on the proliferation of non-antigen specific spleen cells in pigs.

[0083] Test results see Figure 12. Figure 12 The flow cytometry data of AP1-AP5 promoting the proliferation of healthy pig lymphocytes and monocytes and macrophages is shown in Figure 2. Figure 12 It can be seen that the five polypeptides of the present invention can also significantly promote the proliferation of healthy pig spleen immune cells, indicating that the five polypeptides have a broad-spectrum effect of promoting pig immunity.

[0084] Example 3 Detection of IFN-γ secretion by different subtypes of splenic lymphocytes

[0085] The pig immunization procedure and spleen cell isolation and culture were the same as in Example 2. After obtaining dispersed spleen cells, a single cell suspension was prepared in RPMI1640 complete medium at a concentration of 1×10 6 / ml. Inoculate into 24-well plates, add 0.2 μg of the polypeptide provided by the present invention to each well, and culture in a CO2 incubator for 60 hours. Collect cells from each well, label the cells with porcine CD3, CD4, CD8 and IFN-γ specific antibodies, then wash twice with PBS buffer containing 2% serum, and finally disperse into cell suspension with the washing solution. Flow cytometer is used to detect and determine the CD4 + T lymphocytes and CD8 + The level of IFN-γ secretion by T lymphocytes.

[0086] Test results see Figures 13 to 17 .in Figure 13 AP1 promotes the secretion of IFN-γ by different subtypes of immune cells; Figure 14 AP2 promotes the secretion of IFN-γ by different subtypes of immune cells; Figure 15 AP3 promotes the secretion of IFN-γ by different subtypes of immune cells; Figure 16 AP4 promotes the secretion of IFN-γ by different subtypes of immune cells; Figure 17 AP5 promotes the secretion of IFN-γ by different subtypes of immune cells; Figures 13 to 17 It can be seen that the five polypeptides of the present invention can significantly enhance the ability of monocytes, macrophages, B cells and T lymphocytes to secrete IFN-γ.

[0087] Example 4 Immune cell typing experiment in polypeptide-immunized animals

[0088] Five 90-day-old male Landrace pigs were immunized with a mixture of the five polypeptides of the present invention (100 μg / pig of each polypeptide). One month later, a booster immunization was performed. 14 and 21 days after immunization, the proportion of immune cells in peripheral blood was measured by flow cytometry. The same volume of PBS was used as a control.

[0089] Test results see Figures 18 and 19 .in Figure 18 14 days after the second immunization, the proportion of B lymphocyte subsets; Figure 19 CD8 + The proportion level of T lymphocyte subsets. Figures 18 and 19 It can be seen that the mixed immunization of the five polypeptides of the present invention can promote the production of lymphocyte immune response in the pig body and enhance the pig's immunity.

[0090] Example 5: Immune cell typing experiment in animals immunized with polypeptides as immune enhancers

[0091] Three 90-day-old male Landrace pigs were immunized with an inactivated FMD O / MYA98 / BY / 2010 vaccine or a mixture of the inactivated vaccine and the five polypeptides of the present invention (100 μg / pig per pig). One month later, a booster immunization was performed. 14 days after the second immunization, the proportion of immune cells in peripheral blood was measured by flow cytometry. The immunized group was treated with the same volume of PBS as a control.

[0092] Test results see Figures 20 and 21 ,in Figure 20 14 days after the second immunization, the proportion of B lymphocyte subsets; Figure 21 CD8 + The proportion level of T lymphocyte subsets. Figures 20 and 21 It can be seen that the five 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.

[0093] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention. Sequence Listing <110> Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences <120> A type of polypeptide that promotes broad-spectrum immune response in pigs and its application <160> 5 <170> SIPOSequenceListing 1.0 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <400> 1 Ser Met Met Met Val Phe Asn Gln Leu Ile 1 5 10 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <400> 2 Lys Leu Phe Gln Ile Ile Glu Leu Leu 1 5 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <400> 3 Met Arg Ile Glu Ile Phe Trp Glu Leu 1 5 <210> 4 <211> 9 <212> PRT <213> Artificial Sequence <400> 4 Ser Asn Asp Gly Ile Ser Phe Leu Leu 1 5 <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <400> 5 Met Lys Asn Met Val Ser Lys Phe Leu 1 5

Claims

1. A class of polypeptides that promote the generation of a broad-spectrum acquired immune response in pigs, said polypeptides being the first polypeptide or the fourth polypeptide; The amino acid sequence of the first polypeptide is shown in SEQ ID NO.1; The amino acid sequence of the fourth polypeptide is shown in SEQ ID NO.4; Said polypeptides promote the generation of a broad-spectrum acquired immune response in pigs by promoting the secretion of IFN-γ by monocytes, macrophages, T cells and B cells in the pig body, and / or, Said polypeptides promote the generation of a broad-spectrum acquired immune response in pigs by promoting the proliferation of monocytes, macrophages, T cells and B cells in the pig body.

2. Use of the polypeptide according to claim 1 in the preparation of a preparation for promoting the generation of a broad-spectrum acquired immune response in pigs.

3. Use of the polypeptide according to claim 1 in the preparation of a preparation for promoting the generation of an African swine fever virus antigen-specific immune response in pigs.

4. An immune enhancer for pigs, the active ingredient of said immune enhancer comprising 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 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.

5. The immunopotentiator according to claim 4, wherein, The content of the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide and the fifth polypeptide in said immune enhancer is 100 μg / component / dose.

6. A subunit vaccine against African swine fever virus, said subunit vaccine against African swine fever virus comprising 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 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.

7. The subunit vaccine according to claim 6, wherein, The content of the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide and the fifth polypeptide in said subunit vaccine is 100 μg / component / dose.

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