Application of radix pseudostellariae polysaccharide in preparation of avian influenza H9 subtype vaccine immunopotentiator

Abstract

The invention discloses application of radix pseudostellariae polysaccharide in preparation of an avian influenza H9 subtype vaccine immunopotentiator, belongs to the technical field of traditional Chinese medicine application, and provides application of the radix pseudostellariae polysaccharide in preparation of the avian influenza H9 subtype vaccine immunopotentiator. The invention provides an avian influenza H9 subtype vaccine immunopotentiator containing the radix pseudostellariae polysaccharide, and the effective component in the avian influenza H9 subtype vaccine immunopotentiator is only the radix pseudostellariae polysaccharide. The invention provides an application of radix pseudostellariae polysaccharide in preparation of a preparation for improving avian influenza H9 subtype vaccine specific antibody titer. The invention finds that the radix pseudostellariae polysaccharide can obviously improve the specific antibody titer of the avian influenza H9 subtype vaccine, maintain the maternal antibody level, reduce the blank period of the avian influenza H9 subtype vaccine and prolong the duration of the high antibody level.

Description

Technical Field

[0001] The invention belongs to the technical field of application of traditional Chinese medicines, and in particular relates to the application of Pseudostellaria heterophylla polysaccharide in the preparation of an avian influenza H9 subtype vaccine immunopotentiator. Background Art

[0002] Avian influenza is a highly contagious disease caused by influenza A virus, of which the H9N2 subtype is one of the most prevalent avian influenza viruses in the world. Although the pathogenicity of this subtype is relatively low, it has a wide range of infection, a high frequency of mutation, and the risk of cross-species transmission, posing a potential threat to human public health. Therefore, it is crucial to develop effective methods to prevent and control H9 subtype avian influenza. Importance of vaccine adjuvants As the main means of preventing infectious diseases, vaccines play an irreplaceable role in improving the body's immunity. However, a single antigen component is often difficult to stimulate a strong enough immune response, especially when facing new or highly variable pathogens. At this time, the appropriate use of adjuvants becomes one of the effective ways to enhance the effect of vaccines. An ideal adjuvant should be able to enhance the immune response, significantly increase the level of specific antibodies and cell-mediated immunity; prolong the protection time and ensure long-term and stable protection; increase safety and not cause serious side effects or allergic reactions.

[0003] Radix Pseudostellariae is a traditional Chinese medicinal material, which has been found to contain rich active polysaccharide components in recent years. Studies have shown that these polysaccharides not only have good biocompatibility and low toxicity, but also have multiple biological functions, such as anti-oxidation, anti-inflammatory, and immune regulation. In particular, the regulatory effect on the immune system is particularly prominent, and it can promote the body's ability to fight foreign pathogens by activating multiple immune cells such as macrophages, NK cells, and T / B lymphocytes. In addition, some studies have also pointed out that Radix Pseudostellariae polysaccharides may induce DCs maturation and secrete cytokines such as IL-12 through the TLR4 signaling pathway, further enhancing the adaptive immune response. Advantages of Radix Pseudostellariae polysaccharides as vaccine adjuvants Based on the above characteristics, the application of Radix Pseudostellariae polysaccharides in the field of vaccine adjuvants shows great potential. Compared with traditional aluminum salt adjuvants, it is more attractive in practical applications due to its safety advantage of natural sources.

[0004] In summary, considering the current severe situation of avian influenza prevention and control, as well as the unique physicochemical properties of Pseudostellaria heterophylla polysaccharide and its excellent immunomodulatory properties, exploring its use as an adjuvant for H9 subtype avian influenza vaccine has important theoretical significance and broad application prospects. Summary of the invention

[0005] In order to solve the above technical problems, the present invention proposes the use of Pseudostellariae Polysaccharide in the preparation of avian influenza H9 subtype vaccine immune enhancer. Pseudostellariae Polysaccharide can effectively improve the specific antibody titer of avian influenza H9 subtype vaccine in chickens and enhance the body's humoral immune function.

[0006] To achieve the above-mentioned purpose, the present invention provides the use of Pseudostellaria heterophylla polysaccharide in the preparation of an avian influenza H9 subtype vaccine immunopotentiator.

[0007] Preferably, the Radix Pseudostellariae polysaccharide can promote the specific immune response of the avian influenza H9 subtype vaccine and increase the specific antibody level.

[0008] Preferably, the avian influenza H9 subtype vaccine is an avian influenza virus type A H9 subtype strain.

[0009] Preferably, the avian influenza H9 subtype vaccine is injected into 9-day-old chicks, and Pseudostellariae polysaccharide is fed to the injected chicks at a dosage of 100-400 mg / kg starting from 7 days of age and stopping feeding at 49 days of age.

[0010] The present invention also provides an avian influenza H9 subtype vaccine immunopotentiator comprising the Pseudostellariae Radix Polysaccharide, wherein the active ingredient of the avian influenza H9 subtype vaccine immunopotentiator is only Pseudostellariae Radix Polysaccharide.

[0011] The present invention also provides the use of the Pseudostellariae Radix polysaccharide in the preparation of a preparation for improving the specific antibody titer of avian influenza H9 subtype vaccine.

[0012] Preferably, the Radix Pseudostellariae polysaccharide can significantly increase the titer of specific antibodies of avian influenza H9 subtype vaccine, maintain the level of maternal antibodies, reduce the blank period of avian influenza H9 subtype vaccine, and prolong the duration of high antibody level.

[0013] Preferably, the avian influenza H9 subtype vaccine is an avian influenza virus type A H9 subtype strain.

[0014] Preferably, the avian influenza H9 subtype vaccine is injected into 9-day-old chicks, and Pseudostellariae polysaccharide is fed to the injected chicks at a dosage of 100-400 mg / kg starting from 7 days of age and stopping feeding at 49 days of age.

[0015] The present invention also provides a preparation for improving the titer of specific antibodies of avian influenza H9 subtype vaccine, comprising the Pseudostellariae Pseudostellariae polysaccharide, wherein the effective ingredient of the preparation for improving the titer of specific antibodies of avian influenza H9 subtype vaccine is only Pseudostellariae Pseudostellariae polysaccharide.

[0016] Compared with the prior art, the present invention has the following advantages and technical effects:

[0017] The invention provides an application of Pseudostellariae Radix Polysaccharide in the preparation of an avian influenza H9 subtype vaccine immunopotentiator. The Pseudostellariae Radix Polysaccharide can significantly improve the specific antibody titer of the avian influenza H9 subtype vaccine, maintain the level of maternal antibodies, reduce the blank period of the avian influenza H9 subtype vaccine, and prolong the duration of high antibody levels. The Pseudostellariae Radix Polysaccharide can promote the specific immune response of the avian influenza H9 subtype vaccine, improve the specific antibody level, the immunoglobulin IgG content, the immunoglobulin IgM content, the C3 complement content, and the C4 complement content. The Pseudostellariae Radix Polysaccharide can also promote the development of the spleen and bursa of Fabricius, and promote the expression of humoral immunity-related genes, thereby improving the humoral immune response capability. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 The dynamic changes of serum specific antibody titers at different ages;

[0020] Figure 2 The effect of Radix Pseudostellariae Polysaccharide as an immune enhancer for avian influenza H9 subtype vaccine on serum immunoglobulin, where A is the serum IgG content of chickens in different age groups, and B is the serum IgM content of chickens in different age groups. In the figure, a, b, and c represent the significant difference analysis;

[0021] Figure 3 The effect of Radix Pseudostellariae Polysaccharide as an immunopotentiator of avian influenza H9 subtype vaccine on the immune organ index of chicks, where A is the spleen index of chicks at different ages, and B is the bursa of Fabricius index of chicks at different ages. In the figure, a, b, and c represent the significant difference analysis;

[0022] Figure 4 The effect of Radix Pseudostellariae Polysaccharide as an immunopotentiator of avian influenza H9 subtype vaccine on serum complement, where A is the serum C3 complement content of chicks in different age groups, and B is the serum C4 complement content of chicks in different age groups. In the figure, a, b, and c represent the significant difference analysis;

[0023] Figure 5 The effect of Radix Pseudostellariae Polysaccharide as an immunopotentiator of avian influenza H9 subtype vaccine on serum cytokines, wherein A is the serum IL-4 content of chickens in different age groups, B is the serum IL-6 content of chickens in different age groups, and C is the serum IFN-γ content of chickens in different age groups. In the figure, a, b, and c represent the significant difference analysis;

[0024] Figure 6The figure shows the effect of Radix Pseudostellariae polysaccharide as an immunopotentiator for avian influenza H9 subtype vaccine on the expression of genes related to humoral immunity, where A is the gene expression in spleen tissue, and B is the gene expression in bursa of Fabricius. In the figure, a and b represent the significant difference analysis. DETAILED DESCRIPTION

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] Sources of materials used in the present invention: Radix Pseudostellariae polysaccharide, purchased from Shanyang Lianfeng Biotechnology Co., Ltd., Shaanxi Province, with a content of 90%; Newcastle disease vaccine purchased from Qingdao Yibang Bioengineering Co., Ltd.; avian influenza H5 vaccine purchased from Qingdao Yibang Bioengineering Co., Ltd.; avian influenza H9 subtype vaccine purchased from Qingdao Yibang Bioengineering Co., Ltd.

[0031] Example 1

[0032] 1. The effect of Pseudostellariae Polysaccharide on the specific antibody level of different chicken vaccines and the screening of Pseudostellariae Polysaccharide dosage.

[0033] Experimental groups: control group (fed only with basic diet); low, medium and high dose experimental groups of Pseudostellariae Polysaccharide (on the basis of feeding the basic diet, the corresponding amount of Pseudostellariae Polysaccharide was added into each kilogram of feed, and the Pseudostellariae Polysaccharide was fed at 100mg / kg, 200mg / kg and 400mg / kg respectively).

[0034] Experimental method: 7-day-old chicks were fed according to the above groupings, and the vaccines were injected at 9 days of age. The sera of chicks at 7, 13, 19, 25, and 31 days of age were collected to detect the hemagglutination inhibition titer of Newcastle disease vaccine, avian influenza H5 vaccine, and avian influenza H9 subtype vaccine to indicate the antibody titer.

[0035] Method for detecting the hemagglutination inhibition titer of Newcastle disease vaccine, avian influenza H5 vaccine, and avian influenza H9 subtype vaccine.

[0036] HI test, or hemagglutination inhibition test, is a common method for determining serum antibodies to hemagglutinating antigens such as avian influenza, Newcastle disease, and egg drop syndrome. All poultry disease laboratories and research institutions use this method. Before the test, all materials for the HI test should be prepared, including the serum to be tested, standard negative serum and positive serum, PBS buffer, 1% chicken red blood cells, 96-well hemagglutination plate, pipette and pipette tip.

[0037] 1. Determine the HA titer of the antigen: HA is the hemagglutination test, which is a necessary step for the HI test. First, the hemagglutination titer of the antigen used must be determined to facilitate the subsequent configuration of the four-unit antigen.

[0038] 2. Preparation and verification of four-unit antigen: After the hemagglutination titer of the antigen is determined, the four-unit antigen can be prepared. After preparation, the four-unit antigen must be verified to ensure the accuracy of the preparation.

[0039] 3. Dilute the serum to be tested in multiple proportions: First, add PBS to the 96-well blood coagulation plate, 25 microliters per well, then mark the sample number in the first vertical row and set it as a negative serum control and a positive serum control. Then use an eight-channel pipette to repeatedly blow the first vertical row of holes for about 8 times, draw 25 microliters and add it to the second vertical row, blow and dilute again, draw 25 microliters and add it to the third vertical row, and so on, until the last hole, draw 25 microliters and discard, and the serum multiple dilution is completed.

[0040] 4. Add four units of antigen: Add 25 microliters of verified four-unit antigen solution to each well of the diluted plate, from low concentration to high concentration, to ensure that the wells are not contaminated.

[0041] 5. Antigen-antibody interaction: After adding the antigen, the antibodies in the serum will react with the antigen, and the reaction will take place at 25°C for 20 minutes. If there are antibodies, the antibodies will bind to the antigen, and the antigen will not be able to bind to the red blood cells.

[0042] 6. Add 1% chicken red blood cells: After the antibody and antigen have finished reacting, you can add 1% chicken red blood cells, 25 microliters per well. When adding, make sure the pipette tip is as vertical as possible to the blood coagulation plate, 0.5 cm above it, to avoid contamination. After adding red blood cells, the whole system should be allowed to react for 20 minutes. At this time, the red blood cells will bind to the unbound antigens and agglutinate.

[0043] 7. Read the result: After the reaction is completed, you can read the result. Stand the blood coagulation plate upright and read the number on the back. The hole through which the red blood cells can completely flow out is taken as the HI titer of the serum. The premise is that both negative and positive serum must be established, that is, the negative serum well shows complete agglutination, and the positive serum titer is within 1 titer of the marked one.

[0044] Table 1 Dynamic changes in Newcastle disease vaccine antibody titer

[0045]

[0046] Table 2 Dynamic changes in antibody titers of avian influenza H5 vaccine

[0047]

[0048] Table 3 Dynamic changes in antibody titers of avian influenza H9 subtype vaccine

[0049]

[0050] The results are shown in Tables 1, 2 and 3. Adding appropriate concentrations of Pseudostellaria heterophylla polysaccharide to the diet can effectively increase the specific antibody titer of chickens to Newcastle disease vaccine, avian influenza H5 vaccine and avian influenza H9 subtype vaccine, and enhance the body's humoral immune function; among them, the improvement in the specific antibody titer of avian influenza H9 subtype vaccine was the most significant, and at the age of 19 days, it showed a significant effect of improving the specific antibody titer of avian influenza H9 subtype vaccine.

[0051] 2. Determine the effect of Pseudostellaria heterophylla polysaccharide on the humoral immunity of chicks after immunization with avian influenza H9 subtype vaccine.

[0052] The test groups are shown in Table 4 below.

[0053] Table 4 Experimental groups

[0054]

[0055]

[0056] Detection indicators:

[0057] 1. Effect on serum antibody levels: HA-HI detected antibody levels at different times.

[0058] 2. Effect on immune organ index: Weigh the spleen, bursa of Fabricius and body weight, and calculate the immune organ index.

[0059] 3. Effect on serum immunoglobulin: ELISA was used to detect IgG, IgM, and IgA. The kits were purchased from Jiangsu ELISA Industrial Co., Ltd.

[0060] 4. Effect on the complement system: ELISA was used to detect complement C3 and C4. The kits were purchased from Jiangsu ELISA Industrial Co., Ltd.

[0061] 5. Effect on cytokines: ELISA was used to detect IL-4, IL-6, and IFN-γ. The kits were purchased from Jiangsu ELISA Industrial Co., Ltd.

[0062] 6. Impact on the expression of related genes: quantitative detection of related genes such as TNF-α, IFN-γ, CD40, etc.

[0063] The results are as follows Figure 1 As shown, during the entire test period, the average antibody titer level of the Radix Pseudostellariae Polysaccharide test group was higher than that of the vaccine control group and the blank control group. Among them, the antibody titer level of the Radix Pseudostellariae Polysaccharide test group from 28 days to 49 days old was significantly higher than that of the vaccine control group (P<0.05), and the antibody level of the vaccine control group was significantly higher than that of the blank control group (P<0.05). This shows that Radix Pseudostellariae Polysaccharide, as an immune enhancer for avian influenza H9 subtype vaccine, can significantly increase the specific antibody titer of avian influenza H9 subtype vaccine, maintain the level of maternal antibodies, reduce the "blank period" of avian influenza H9 subtype vaccine, and prolong the duration of high antibody levels.

[0064] like Figure 2 Middle A and Figure 2 As shown in B, it is the effect of Pseudostellariae polysaccharide as an immune enhancer of avian influenza H9 subtype vaccine on serum immunoglobulin. The IgG content of Pseudostellariae polysaccharide test group from 21 to 49 days old chicks was significantly higher than that of vaccine control group (P<0.05), and the IgG content of vaccine control group chicks at 21 and 35 days old was significantly higher than that of blank control group (P<0.05), and there was no significant difference at other time points (P>0.05). The IgM content of Pseudostellariae polysaccharide test group at 21 and 28 days old was significantly higher than that of vaccine control group, and the IgM content of Pseudostellariae polysaccharide test group at 35 and 42 days old was significantly higher than that of blank control group (P<0.05).

[0065] like Figure 3 Middle A and Figure 3As shown in B, the effect of Pseudostellariae polysaccharide as an immune enhancer for avian influenza H9 subtype vaccine on the immune organ indexes of chicks. The spleen index of chicks in the Pseudostellariae polysaccharide test group at 35, 42, and 49 days of age was significantly higher than that in the immunized control group, and the spleen index of the immunized control group at 35 days of age was significantly higher than that in the blank control group (P<0.05). At 21 and 35 days of age, the bursal index of chicks in the Pseudostellariae polysaccharide test group was significantly higher than that in the immunized control group, and the bursal index of chicks in the Pseudostellariae polysaccharide test group was significantly higher than that in the blank control group at 35 days of age (P<0.05).

[0066] like Figure 4 Middle A and Figure 4 As shown in B, the effect of Pseudostellariae polysaccharide as an immune enhancer for avian influenza H9 subtype vaccine on serum complement. The serum C3 complement content of chicks in the Pseudostellariae polysaccharide test group at 21, 28 and 35 days of age was significantly higher than that in the vaccine control group, and the serum C3 complement content of chicks in the vaccine control group was significantly higher than that in the blank control group (P<0.05). At 42 and 49 days of age, the serum C3 complement content of chicks in the Pseudostellariae polysaccharide test group was significantly higher than that in the blank control group (P<0.05), and there was no significant difference between the Pseudostellariae polysaccharide test group and the vaccine control group (P>0.05). The serum C4 complement content of chicks in the Pseudostellaria polysaccharide test group at 21, 28 and 42 days of age was significantly higher than that in the vaccine control group, and the serum C4 complement content of chicks in the vaccine control group was significantly higher than that in the blank control group (P<0.05); at 35 days of age, the serum C4 complement content of chicks in the Pseudostellaria polysaccharide test group was significantly higher than that in the blank control group (P<0.05); at 49 days of age, the serum C4 complement content of chicks in the Pseudostellaria polysaccharide test group was significantly higher than that in the vaccine control group (P<0.05).

[0067] like Figure 5 Middle A, Figure 5 Medium B and Figure 5 As shown in C, the effect of Pseudostellariae polysaccharide as an immunopotentiator of avian influenza H9 subtype vaccine on serum cytokines. The serum levels of cytokines IL-4, IL-6 and IFN-γ in the Pseudostellariae polysaccharide test group were higher than those in the vaccine control group and the blank control group, among which the serum levels of the vaccine control group on days 21, 28, 42 and 49 were significantly higher than those in the blank control group. On days 21, 28 and 35, the Pseudostellariae polysaccharide test group exceeded the vaccine control group. Similarly, the serum IL-6 content of the vaccine control group was significantly higher than that of the blank control group on days 21 and 28, while the Pseudostellariae polysaccharide test group exceeded the vaccine control group on days 21, 28, 35 and 42. In addition, the serum IFN-γ level of chicks in the vaccine control group was significantly higher than that in the blank control group on days 21, 42 and 49. In contrast, the Pseudostellariae polysaccharide test group was significantly higher than the vaccine control group on days 21, 28, 35 and 49 (P<0.05).

[0068] like Figure 6 Middle A and Figure 6 As shown in B, the effect of Pseudostellariae polysaccharide as an immunopotentiator of avian influenza H9 subtype vaccine on the expression of humoral immunity-related genes, and qRT-PCR was used to detect the effect of PHP on the expression levels of humoral immunity-related mRNA in the spleen and bursa of Fabricius of chicks. At 35 days of age, the gene expressions of IFN-γ, CD40, CD80 and CD86 in the spleen tissue of the Pseudostellariae polysaccharide test group were significantly higher than those in the immunization control group, and the gene expressions of TNF-α and IL-6 in the spleen tissue of the Pseudostellariae polysaccharide test group were significantly higher than those in the blank control group; the gene expressions of IL-4, IFN-γ, CD40 and CD86 in the bursa tissue of the Pseudostellariae polysaccharide test group were significantly higher than those in the immunization control group, and the gene expression of IL-6 in the bursa tissue of the Pseudostellariae polysaccharide test group was significantly higher than that in the blank control group (P<0.05), and the other differences were not significant.

[0069] In summary, Pseudostellariae polysaccharide can be added to the diet as an immune enhancer for avian influenza H9 subtype vaccine, which can promote the specific immune response of avian influenza H9 subtype vaccine, increase the level of specific antibodies, increase the content of immunoglobulins IgG, IgM and C3, C4 complement content; promote the development of spleen and bursa of Fabricius, promote the expression of humoral immunity-related genes, and thus improve the humoral immune response ability.

[0070] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of Radix Pseudostellariae Polysaccharide in the preparation of avian influenza H9 subtype vaccine immune enhancer.

2. The application according to claim 1, characterized in that: The Pseudostellaria heterophylla polysaccharide can promote the specific immune response of the avian influenza H9 subtype vaccine and increase the specific antibody level.

3. The application according to claim 1, characterized in that: The avian influenza H9 subtype vaccine is a type A avian influenza virus H9 subtype strain.

4. The use according to claim 1, characterized in that: The avian influenza H9 subtype vaccine was injected into 9-day-old chicks, and Pseudostellariae polysaccharide was fed to the injected chicks at a dosage of 100-400 mg / kg starting from the 7th day of age and stopped at the 49th day of age.

5. An avian influenza H9 subtype vaccine immunopotentiator comprising the Radix Pseudostellariae polysaccharide of claim 1, characterized in that: The active ingredient in the avian influenza H9 subtype vaccine immunopotentiator is only Pseudostellaria heterophylla polysaccharide.

6. Use of Pseudostellariae Radix Polysaccharide as claimed in claim 1 in the preparation of a preparation for improving the titer of specific antibodies of avian influenza H9 subtype vaccine.

7. The use according to claim 6, characterized in that: The Pseudostellariae Radix polysaccharide can significantly increase the specific antibody titer of the avian influenza H9 subtype vaccine, maintain the level of maternal antibodies, reduce the blank period of the avian influenza H9 subtype vaccine, and prolong the duration of high antibody levels.

8. The use according to claim 6, characterized in that: The avian influenza H9 subtype vaccine is a type A avian influenza virus H9 subtype strain.

9. The use according to claim 6, characterized in that: The avian influenza H9 subtype vaccine was injected into 9-day-old chicks, and Pseudostellariae polysaccharide was fed to the injected chicks at a dosage of 100-400 mg / kg starting from the 7th day of age and stopped at the 49th day of age.

10. A preparation for improving the specific antibody titer of avian influenza H9 subtype vaccine comprising the Radix Pseudostellariae polysaccharide of claim 1, characterized in that: The active ingredient in the preparation for improving the specific antibody titer of the avian influenza H9 subtype vaccine is only Pseudostellaria heterophylla polysaccharide.

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