Preparation method of polysaccharide vaccine diluent and application of polysaccharide vaccine diluent in Newcastle disease vaccines

Abstract

The invention provides a preparation method of a polysaccharide vaccine diluent and application of the polysaccharide vaccine diluent to a Newcastle disease vaccine, and belongs to the technical field of biological pharmacy. The invention provides an Acanthopanax southern polysaccharide vaccine diluent and a preparation method thereof, and solves the problems that the titer uniformity of an antibody generated by diluting a vaccine by using normal saline or a phosphate buffer solution is not high, a stress reaction and an anaphylactic reaction are generated on an organism after inoculation, the immune effect of the vaccine is influenced, and the like. The acanthopanax southern polysaccharide vaccine diluent can be applied to animal vaccines, including Newcastle disease vaccines, and solves the problem that no polysaccharide diluent for Newcastle disease vaccines exists in the prior art.

Description

Technical Field

[0001] The invention belongs to the technical field of biopharmaceuticals, and particularly relates to application of an animal vaccine diluent of Acanthopanax acanthopanax polysaccharide in Newcastle disease vaccines. Background Art

[0002] Polysaccharides are one of the effective ingredients of traditional Chinese medicine. They are usually polymerized sugars and high-molecular carbohydrates formed by multiple monosaccharide molecules connected by glycosidic bonds. They are widely present in plants, animals and microorganisms, and have biological activities such as immunomodulation, anti-tumor, anti-inflammatory and antioxidant effects. Polysaccharides have low toxicity and less toxic side effects. Therefore, traditional Chinese medicine polysaccharides are ideal for the development of new immune adjuvants. Acanthopanax senticosus belongs to the genus Acanthopanax of the Araliaceae family. It is mainly distributed in Heilongjiang, Jilin, Hebei and Shanxi, and is more common in northeastern China. Acanthopanax senticosus polysaccharide (ASP) is an immunologically active polysaccharide isolated from Acanthopanax senticosus, a traditional Chinese medicine, and has the effects of regulating animal immune function, anti-oxidation and anti-tumor.

[0003] Newcastle Disease (ND) is an acute, febrile, septicemic and highly contagious disease of poultry caused by Newcastle Disease Virus (NDV), which can lead to high mortality and economic losses in poultry. Newcastle Disease Vaccine is an important means of preventing the disease, which can help protect chickens from the threat of Newcastle Disease. Live vaccine is one of the commonly used vaccine types. Live vaccine is a vaccine that contains live virus but has been attenuated, which can stimulate the body to produce a strong immune response. Newcastle Disease live vaccine is classified according to the different virus strains and the strength of virulence, mainly including B1, LaSota and V4.

[0004] Most live vaccines are stored in a freeze-dried form, and vaccine diluents are required when used, usually physiological saline or phosphate buffer. However, the uniformity of antibody titers produced by vaccines diluted with them is not high, and stress and allergic reactions are produced in the body after vaccination, affecting the immune effect of the vaccine. Current studies have shown that astragalus polysaccharides, lentinan polysaccharides, and epimedium polysaccharides can be used as vaccine diluents to stimulate immune responses and enhance immune effects after vaccination. However, there are currently no reports on polysaccharide vaccine diluents for Newcastle disease vaccines. Therefore, in order to ensure the immune effect of Newcastle disease vaccines, a polysaccharide vaccine diluent for Newcastle disease vaccines is needed. Summary of the invention

[0005] The invention provides a polysaccharide vaccine diluent of Acanthopanax acanthopanax. The polysaccharide vaccine diluent is used as a diluent of a Newcastle disease vaccine of chickens. The diluent solves the problems that the uniformity of antibody titer produced by diluting the vaccine with physiological saline or phosphate buffer solution is not high, stress reaction and allergic reaction are generated in the body after vaccination, and the immune effect of the vaccine is affected. The invention also solves the problem that there is no polysaccharide diluent for the Newcastle disease vaccine of chickens in the prior art.

[0006] The invention provides an acanthopanax acanthopanax polysaccharide vaccine diluent, comprising acanthopanax acanthopanax polysaccharide and a phosphate buffer solution, wherein the acanthopanax acanthopanax polysaccharide concentration is 400-1600 μg / mL.

[0007] Preferably, the concentration of Acanthopanax acanthopanax polysaccharide is 400, 800 or 1600 μg / mL.

[0008] The invention provides a preparation method for diluting an Acanthopanax acanthopanax polysaccharide vaccine, which comprises mixing Acanthopanax acanthopanax polysaccharide with a phosphate buffer solution.

[0009] Preferably, the preparation method of Acanthopanax acanthopanax polysaccharide is to grind Acanthopanax acanthopanax, then add pure water, soak in a hot water bath, filter to remove the filter residue, concentrate using a rotary evaporator, add anhydrous ethanol for alcohol precipitation, let stand at 4°C for 12 hours, collect the precipitate by centrifugation, and finally freeze-dry.

[0010] Preferably, the volume of pure water added is 10 times the weight of Aconitum carmichaelii.

[0011] Preferably, the extraction is carried out in a hot water bath at 90°C for 2 hours.

[0012] Preferably, the concentration is to 1 / 10 of the total volume.

[0013] Preferably, 9 times the volume of anhydrous ethanol is added as compared to the concentrated liquid.

[0014] The invention provides application of a polysaccharide vaccine diluent of Acanthopanax acanthopanax in animal vaccines.

[0015] The invention provides a chicken Newcastle disease vaccine, which comprises the acanthopanax acanthopanax polysaccharide vaccine diluent.

[0016] Beneficial Effects

[0017] 1. The present invention provides a kind of Acanthopanax radix polysaccharide animal vaccine diluent, which has the effect of proliferation on chicken spleen lymphocytes, and the proliferation effect is best when Acanthopanax radix polysaccharide is 250 and 500 μg / mL. It can enhance the body's immune function, and can cooperate with the vaccine to improve the antibody titer, and reduce the body's stress response and allergic reaction after vaccination.

[0018] 2. The present invention provides a Newcastle disease vaccine diluted with Acanthopanax radix polysaccharide vaccine diluent, which effectively stimulates immune response and enhances immune effect, and the immune effect is better than that of the Newcastle disease vaccine.

[0019] 3. The present invention provides a new design idea for developing a diluent for live Newcastle disease vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the effect of Acanthopanax acanthopanax polysaccharide on the proliferation of chicken spleen lymphocytes.

[0021] Figure 2 This study is the effect of high dose of Acanthopanax acanthopanax polysaccharide on NDV serum antibody levels.

[0022] Figure 3 This is the effect of medium dose of Acanthopanax acanthopanax polysaccharide on NDV serum antibody level.

[0023] Figure 4 This study is the effect of low dose of Acanthopanax acanthopanax polysaccharide on NDV serum antibody level.

[0024] Figure 5 This study is the effect of high dose of Acanthopanax acanthopanax polysaccharide on NDV serum cytokine levels.

[0025] Figure 6 This is the effect of medium dose of Acanthopanax acanthopanax polysaccharide on NDV serum cytokine levels.

[0026] Figure 7 This study is the effect of low dose of Acanthopanax acanthopanax polysaccharide on NDV serum cytokine levels. DETAILED DESCRIPTION

[0027] Sources of materials used in the experiment:

[0028] Acanthopanax acanthopanax was purchased from Anguo Pharmaceutical City, Hebei Province; anhydrous ethanol, phenol, and sulfuric acid were purchased from Sinopharm Reagent Company; fetal bovine serum and RPMI 1640 culture medium were purchased from Gibco; 0.22 um microporous filter membrane was purchased from Shanghai Anpu Company; Newcastle disease vaccine (LaSota strain, batch number: 202409) was purchased from Harbin Pharmaceutical Group Biological Vaccine Co., Ltd.; IL-2, IL-4, IL-6, and INF-γ detection kits were purchased from Quanzhou Ruixin Biotechnology Co., Ltd.; HA antigen was purchased from Qingdao Lijian.

[0029] Example 1. Preparation of Acanthopanax acanthopanax polysaccharide and determination of sugar content.

[0030] 1. Preparation of Acanthopanax Acanthopanax Polysaccharide

[0031] Weigh 50g of Acanthopanax australis and grind it, add 10 times of pure water, soak it in a hot water bath at 90℃ for 2h, then filter and remove the residue to obtain a filtrate. The filtrate is concentrated to 1 / 10 of the total volume using a rotary evaporator, and 9 times the volume of anhydrous ethanol is added for alcohol precipitation. After standing at 4℃ for 12h, the precipitate is collected by centrifugation at 4000rpm for 20min, and then freeze-dried to obtain Acanthopanax australis polysaccharide (crude polysaccharide).

[0032] 2. The phenol-sulfuric acid method was used to determine the polysaccharide content of Acanthopanax acanthopanax.

[0033] Preparation of glucose with a concentration of 0.04 mg / mL: Accurately weigh 20 mg of glucose standard dried to constant weight at 105°C, dissolve it in pure water and make up to 500 mL, shake well and store at 4°C.

[0034] Preparation of 5% phenol solution: Weigh 100g of phenol, distill in an oil bath, collect the fractions at 180-182 degrees to obtain refined phenol. Weigh 5.0g of refined phenol, dilute to 100mL, shake well, and store at 4℃ for one month.

[0035] Accurately pipette 0mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL of 0.04mg / mL glucose solution into 25mL test tubes, add 0mL, 1.8mL, 1.6mL, 1.4mL, 1.2mL, 1mL of pure water to 2.0mL, add 1.0mL of phenol solution, mix well on a rotary mixer, carefully add 5mL of concentrated sulfuric acid to the rotary mixer, mix well, boil in a boiling water bath for 30min, cool and use a spectrophotometer at a wavelength of 490nm to measure the absorbance with a blank solution as a control. According to the concentration of the standard solution and the absorbance, the regression equation is: Y = 6.9823X-0.0029, and the correlation coefficient is r = 0.9914. Where Y is the absorbance value, X is the mass, unit: mg.

[0036] Weigh 20 mg of Acanthopanax polysaccharide, add pure water to 100 mL, shake well, and obtain Acanthopanax polysaccharide solution. Take 1.0 mL of Acanthopanax polysaccharide solution and 1.0 mL of pure water in a 25 mL test tube, add 1.0 mL of phenol solution, mix well on a rotary mixer, carefully add 5.0 mL of concentrated sulfuric acid, mix well on a rotary mixer, place in a water bath and boil for 30 minutes, cool to room temperature, and use a spectrophotometer at a wavelength of 490 nm with a blank solution as a control to measure the absorbance value. Find the mass of glucose from the standard curve, and then calculate the polysaccharide content in the sample. The result is: the content of Acanthopanax polysaccharide determined by the phenol-sulfuric acid method is 60.5%.

[0037] Example 2. In vitro activity test of Acanthopanax acanthopanax polysaccharide.

[0038] 1. Prepare Acanthopanax radix polysaccharide solution with RPMI 1640 culture medium containing 10% fetal bovine serum and Acanthopanax radix polysaccharide, filter and sterilize through a 0.22um microporous filter membrane, and dilute to different gradient concentrations (μg / mL): 500μg / mL, 250μg / mL, 125μg / mL, 62.5μg / mL, 1.25μg / mL, 15.625μg / mL, 7.813μg / mL, 3.906μg / mL, 1.953μg / mL, and store in a 4°C refrigerator.

[0039] 2. Take 60-day-old Dawu Jinfeng laying hens, bleed them from the jugular vein, and soak them in 75% ethanol for 5 minutes. Remove the spleen aseptically on a clean bench. Add 5 mL of PBS to the cell culture dish to wash the spleen. Take out a cell sieve with a pore size of 0.45 μm and place it in a new cell culture dish. Place the washed spleen in the cell sieve, add 5 mL of PBS, grind the spleen in the cell sieve, transfer it to a 15 mL centrifuge tube, and centrifuge it at 2000 rpm for 10 minutes. After centrifugation, discard the supernatant and retain the precipitate. Add 5 mL of sterile water and gently blow for 15 seconds to lyse the red blood cells. Immediately after the blowing, add 5 mL of Hanks solution and gently blow to mix. Centrifuge at 2000 rpm for 10 minutes and discard the supernatant. Add RPMI 1640 containing 10% fetal bovine serum to resuspend the cells and adjust the cell concentration to 1×10 7 / mL to obtain a cell suspension.

[0040] 3. Add 100 μL / well of cell suspension to a 96-well cell culture plate, and then add 100 μL of each gradient concentration of Acanthopanax acanthopanax polysaccharide. Make 6 replicate wells for each concentration, set up a cell control well, add PBS to the edge wells for moisturizing, and place the 96-well plate at 37°C and 5% CO. 2 After 44 hours of incubation in the incubator, 20 μL of MTT (5 mg / mL) was added to each well. After 4 hours of incubation, the cells were centrifuged at 2000 rpm for 5 minutes, 100 μL of the supernatant was discarded, and 100 μL of dimethyl sulfoxide (DMSO) was added to each well to dissolve the precipitate. The absorbance at 570 nm (A) was measured by enzyme-linked immunosorbent assay. 570 Value), as an indicator of lymphocyte proliferation. The effect of Acanthopanax polysaccharide on the proliferation of chicken spleen lymphocytes. Figure 1 As shown in Figure 2, the A. 570 The values ​​were significantly higher than those of the corresponding cell control group (P<0.01), indicating that Acanthopanax acanthopanax polysaccharide had a proliferation effect on chicken spleen lymphocytes, and the proliferation effect was best when Acanthopanax acanthopanax polysaccharide was 250 and 500 μg / mL.

[0041] Example 3. Preparation of a diluent of Acanthopanax acanthopanax polysaccharide animal vaccine.

[0042] Experimental Example 1: 4 mg of Acanthopanax radix polysaccharide was added to 10 mL of phosphate buffer to obtain an Acanthopanax radix polysaccharide vaccine diluent having an Acanthopanax radix polysaccharide concentration of 400 μg / mL. Acanthopanax radix polysaccharide was prepared according to Example 1.

[0043] Experimental Example 2: 8 mg of Acanthopanax radix polysaccharide was added to 10 mL of phosphate buffer to obtain an Acanthopanax radix polysaccharide vaccine diluent having an Acanthopanax radix polysaccharide concentration of 800 μg / mL. Acanthopanax radix polysaccharide was prepared according to Example 1.

[0044] Experimental Example 3: 16 mg of Acanthopanax radix polysaccharide was added to 10 mL of phosphate buffer to obtain Acanthopanax radix polysaccharide vaccine diluent having an Acanthopanax radix polysaccharide concentration of 1600 μg / mL. Acanthopanax radix polysaccharide was prepared according to Example 1.

[0045] Example 4. Immunization test of Newcastle disease vaccine diluted with Acanthopanax acanthopanax polysaccharide vaccine diluent.

[0046] 1. Materials.

[0047] (1) Vaccine: Newcastle disease vaccine (La Sota strain, batch number: 202409)

[0048] (2) Chicken interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6) ELISA detection kits and interferon-γ (IFN-γ) detection kits were purchased from Quanzhou Ruixin Biotechnology Co., Ltd.

[0049] (3) The vaccine diluent is Test Example 1, Test Example 2, and Test Example 3 in Example 3.

[0050] 2. Animal grouping and immunization.

[0051] 100 14-day-old Dawu Jinfeng laying hens were randomly divided into 5 groups, 20 in each group, namely, the high-dose group (ASPH), the medium-dose group (ASPM), the low-dose group (ASPL), the vaccine control group (VC), and the blank control group (BC). Except for the blank control group (nasal drops and eye drops of phosphate buffer saline), the other four groups of chickens were inoculated with 0ug / mL, 400ug / mL, 800ug / mL, and 1600ug / mL of Newcastle disease vaccine diluted with nasal drops and eye drops, and a secondary immunization was performed 14 days after immunization.

[0052] Blood was collected from the subwing vein of chickens at 7d, 14d, 21d, 28d, 35d, and 42d after immunization, and the serum was separated. The antibody titer was detected by hemagglutination (HA) and hemagglutination inhibition (HI) tests, and the contents of IL-2, IL-4, IL-6, and INF-γ in the serum at 35d were determined.

[0053] 3. Determination of Antibody Titer

[0054] (1) Preparation of 1% chicken red blood cell suspension

[0055] Take a rooster free of specific pathogens, collect 3 mL of blood from the wing vein, add 3 times the volume of PBS and mix well, centrifuge at 1000r / min for 8 minutes at 4°C, discard the supernatant and keep the precipitate; add 3 times the volume of PBS, repeat the above operation to wash the precipitate 3 times, and obtain the red blood cell precipitate. Pipette 500μl of the red blood cell precipitate into a sterile centrifuge tube containing 45.5mL of PBS to make a chicken red blood cell suspension. When using, gently shake the centrifuge tube to suspend the red blood cells.

[0056] (2) Hemagglutination test (HA)

[0057] Add 25 μL PBS to each well of a conical-bottom 96-well plate, add 25 μL antigen to the first well, dilute it in multiples to the 11th well, discard 25 μL of the liquid in the 11th well, add 25 μL 1% chicken red blood cell suspension to each well, and let it stand for 30 minutes before determining the result.

[0058] Result determination method: Tilt the 96-well plate with a pointed bottom at a certain angle and observe the red blood cell sedimentation. If the sedimentation quickly flows down in a linear manner, it indicates that the virus antigen has not or is not completely agglutinated with the red blood cells; if the red blood cell sedimentation does not flow down or does not condense into a sedimentation, and is distributed in a thin layer, it indicates that the virus antigen is completely agglutinated with the red blood cells. Usually, the dilution multiple of the first well where the sediment flows down in a linear manner is taken as the highest dilution multiple of the test antigen, that is, the titer of the antigen. The measured antigen titer is 9log2.

[0059] (3) Verification of the four-unit antigen

[0060] According to the results of the HA test, PBS was used to prepare four-unit antigen; 25 μL of PBS was added to the 1st to 5th wells of the conical bottom 96-well plate, 25 μL of the prepared four-unit antigen was added to the 1st well, mixed and diluted to the 5th well in multiple proportions, 25 μL was discarded, 25 μL of 1% chicken red blood cell suspension was added to each well, and the results were observed after standing for 30 minutes;

[0061] Interpretation results: The first two holes are completely aggregated, half of the red blood cells in the third hole immediately become streamlined, and all the red blood cells in holes 4 and 5 immediately become streamlined. This result indicates that the four-unit antigen is accurate, that is, the verification is successful.

[0062] (4) Hemagglutination inhibition test (HI)

[0063] Add 25 μL PBS to each well of a 96-well plate with a pointed bottom, add 25 μL of serum to be tested to the first well, dilute it in multiples to the 11th well, discard 25uL of liquid from the 11th well, add 25 μL of four units of antigen to each well, let it react for 30 minutes, then add 25 μL of 1% chicken red blood cell suspension to each well, let it react for 30 minutes, and observe the results.

[0064] Read the results: The maximum dilution that makes the red blood cell sediment flow linearly is the hemagglutination inhibition (HI) titer of the serum sample, expressed as Log2.

[0065] The results are as follows Figure 2-4 As shown in the figure, after the first immunization, the serum NDV antibody levels of chickens in the vaccine control group and Acanthopanax serrata polysaccharide (ASP) group increased and were higher than those in the blank control group. At 7d and 14d after the first immunization (P < 0.05) and 7d and 14d after the second immunization, the serum NDV specific antibody levels in the ASP group were higher than those in the vaccine control group.

[0066] The serum antibody level of the Acanthopanax polysaccharide group was significantly higher than that of the blank control group and the vaccine control group at 14d, 21d, 28d, and 35d after immunization (P < 0.05). The Acanthopanax polysaccharide high-dose group and the medium-dose group showed better immune effects at 28d, 35d, and 42d.

[0067] 4. Determination of serum IL-2, IL-4, IL-6, and INF-γ levels

[0068] Refer to the instructions of the kit, and all reagents and components are restored to room temperature first. Set up standard wells, 0 value wells, blank wells and sample wells in a 96-well ELISA plate. Add 50 μL of standard wells of different concentrations, add 50 μL of sample diluent to the 0 value wells, and do not add to the blank wells. Add 50 μL of serum to be tested to the sample wells, and then add 100 μL of monitoring antibody labeled with horseradish peroxidase (HRP) (no addition to the blank wells), and incubate in a 37°C incubator in the dark for 60 minutes. After the incubation, discard the liquid and pat dry on absorbent paper. After each well is filled with washing solution, let it stand for 20 seconds, shake off the washing solution and pat dry on absorbent paper, and repeat the washing 5 times. Add 100 μL of a mixture of substrate A and substrate B (1:1 volume) to all wells and incubate in a 37°C incubator in the dark for 15 minutes. After taking out, add 50 μL of stop solution to all wells and read the OD value at 450nm on the ELISA reader.

[0069] The results of the high-dose group of Acanthopanax acanthopanax polysaccharide were as follows Figure 5 As shown, the contents of IL-2, IL-4, IL-6 and INF-γ were significantly different from those in the vaccine control group and the blank control group (P < 0.01), and the contents of IL-2 and IL-4 in the vaccine control group were significantly different from those in the blank control group.

[0070] The results of the medium dose group of Acanthopanax acanthopanax polysaccharide were as follows Figure 6 As shown, the contents of IL-2, IL-4, IL-6 and INF-γ were significantly different from those in the vaccine control group and the blank control group (P < 0.01), the content of IL-2 in the vaccine control group was significantly different from that in the blank control group (P < 0.01), and the content of IL-4 in the vaccine control group was significantly different from that in the blank control group (P < 0.05).

[0071] The results of the low-dose group of Acanthopanax acanthopanax polysaccharide were as follows Figure 7 As shown, the contents of IL-2, IL-4, IL-6 and INF-γ were significantly different from those in the vaccine control group and the blank control group (P < 0.01), the content of IL-2 in the vaccine control group was significantly different from that in the blank control group (P < 0.01), and the content of IL-4 in the vaccine control group was significantly different from that in the blank control group (P < 0.05).

[0072] Therefore, the Newcastle disease vaccine diluted with Acanthopanax acutiloba polysaccharide vaccine diluent can effectively stimulate the immune response and enhance the immune effect.

Claims

1. A polysaccharide vaccine diluent, characterized in that: The vaccine diluent comprises Acanthopanax acanthopanax polysaccharide and phosphate buffer, and the concentration of Acanthopanax acanthopanax polysaccharide is 400-1600 μg / mL.

2. The diluent according to claim 1, characterized in that The concentration of Acanthopanax acanthopanax polysaccharide is 400, 800 or 1600 μg / mL.

3. A method for preparing a diluent of Acanthopanax acanthopanax polysaccharide vaccine, characterized in that: The preparation method is to mix Acanthopanax acanthopanax polysaccharide with phosphate buffer.

4. The preparation method according to claim 3, characterized in that: The preparation method of the Acanthopanax acanthopanax polysaccharide comprises the following steps: grinding Acanthopanax acanthopanax, adding pure water, soaking in a hot water bath, filtering to remove the filter residue, concentrating using a rotary evaporator, adding anhydrous ethanol for alcohol precipitation, standing at 4° C. for 12 hours, collecting the precipitate by centrifugation, and finally freeze-drying.

5. The preparation method according to claim 4, characterized in that: The volume of pure water added is 10 times the weight of Aconitum carmichaelii.

6. The preparation method according to claim 4, characterized in that: The hot water bath immersion is 90° C. hot water bath immersion for 2 hours.

7. The preparation method according to claim 4, characterized in that: The concentration is to concentrate to 1 / 10 of the total volume.

8. The preparation method according to claim 4, characterized in that: The anhydrous ethanol is anhydrous ethanol added with 9 times the volume of concentrated liquid.

9. Application of a Acanthopanax acanthopanax polysaccharide vaccine diluent in animal vaccines.

10. A Newcastle disease vaccine, characterized in that: The vaccine comprises the Acanthopanax acanthopanax polysaccharide vaccine diluent as described in claim 1 or 2.

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