Preparation method of polysaccharide vaccine diluent and application thereof to newcastle disease vaccine for chicken

Abstract

The application provides a preparation method of polysaccharide vaccine diluent and application to chicken newcastle disease vaccine, and belongs to the technical field of biopharmacy.The application provides a south five canes polysaccharide vaccine diluent and a preparation method thereof, and solves the problems of low uniformity of antibody titer, stress reaction and allergic reaction of the body after inoculation, and influence on the immunization effect of the vaccine caused by the physiological saline or the phosphate buffer solution diluent for diluting the vaccine.The south five canes polysaccharide vaccine diluent can be applied to animal vaccines, including chicken newcastle disease vaccine, and solves the problem that there is no polysaccharide diluent for the chicken newcastle disease vaccine in the prior art.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biopharmaceuticals, and particularly relates to application of an animal vaccine diluent of polysaccharide from Acanthopanax giraldii Harms in a Newcastle disease vaccine. BACKGROUND

[0002] Polysaccharide is one of the effective components of traditional Chinese medicine, is a polymer carbohydrate of polymeric sugar high molecules connected by glycosidic bonds, and widely exists in plants, animals and microorganisms, has biological activities such as immunomodulation, anti-tumor, anti-inflammatory and anti-oxidation, and has low toxicity and small toxic side effects, so that the polysaccharide of traditional Chinese medicine is an ideal choice for developing a new type of immunoadjuvant. Acanthopanax giraldii belongs to the plant of Acanthopanax in the family of Araliaceae, and is mainly distributed in Heilongjiang, Jilin, Hebei and Shanxi, and is relatively common in the northeastern region of China. Acanthopanax giraldii polysaccharide (AGSP) is an immunologically active polysaccharide isolated from Acanthopanax giraldii, and has the effects of regulating animal immune function, anti-oxidation and anti-tumor.

[0003] Newcastle disease (ND) is an acute, febrile, septicemia and highly contagious disease of poultry caused by Newcastle disease virus (NDV), which can cause high mortality and economic losses of poultry. Newcastle disease vaccine is an important means to prevent the disease, which can help protect the chicken population from the threat of Newcastle disease, and live vaccine is one of the commonly used vaccine types. Live vaccine is a vaccine containing live virus but treated by attenuation, which can stimulate the body to produce a strong immune response. Newcastle disease live vaccine is classified according to the difference of virus strain, the strength of virulence, etc., and mainly includes B1, LaSota and V4.

[0004] Most of the live vaccines are stored in the form of freeze-drying, and the vaccine diluent is needed when used, and physiological saline or phosphate buffer is usually used, but the antibody titer uniformity of the vaccine diluted by the physiological saline or phosphate buffer is not high, and the body produces stress response and allergic reaction after vaccination, which affects the immune effect of the vaccine. At present, it has been shown by research that Astragalus polysaccharide, lentinan and icariin can be used as vaccine diluent to stimulate immune response and enhance immune effect after vaccination. However, there is no related report on polysaccharide vaccine diluent for Newcastle disease vaccine at present, and therefore in order to ensure the immune effect of the Newcastle disease vaccine, a polysaccharide vaccine diluent for the Newcastle disease vaccine is needed. SUMMARY

[0005] The application provides a polysaccharide vaccine diluent of Kadsura interior, which is used as a diluent of a newcastle disease vaccine for chickens, solves the problems of low uniformity of antibody titer, stress reaction and allergic reaction of the body after vaccination, and influence on the immune effect of the vaccine caused by using physiological saline or phosphate buffer solution to dilute the vaccine, and solves the problem that there is no polysaccharide diluent for the newcastle disease vaccine in the prior art.

[0006] The application provides a polysaccharide vaccine diluent of Kadsura interior, which comprises Kadsura interior polysaccharide and a phosphate buffer solution, and the concentration of the Kadsura interior polysaccharide is 400-1600 mu g / mL.

[0007] Preferably, the concentration of the Kadsura interior polysaccharide is 400, 800 or 1600 mu g / mL.

[0008] The application provides a preparation method of a Kadsura interior polysaccharide vaccine diluent, which comprises mixing Kadsura interior polysaccharide and a phosphate buffer solution.

[0009] Preferably, the preparation method of the Kadsura interior polysaccharide comprises the following steps: grinding Kadsura interior, adding pure water, hot water bath immersion, filtering to remove filter residues, concentrating by using a rotary evaporator, adding anhydrous ethanol for alcohol precipitation, standing at 4 DEG C for 12 hours, centrifuging to collect the precipitate, and finally freeze-drying.

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

[0011] Preferably, the hot water bath immersion is performed at 90 DEG C for 2 hours.

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

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

[0014] The application provides an application of the Kadsura interior polysaccharide vaccine diluent in an animal vaccine.

[0015] The application provides a newcastle disease vaccine for chickens, which comprises the Kadsura interior polysaccharide vaccine diluent.

[0016] Beneficial effects

[0017] 1. The Kadsura interior polysaccharide animal vaccine diluent provided by the application has a proliferative effect on chicken spleen lymphocytes, and the proliferative effect is best when the Kadsura interior polysaccharide is 250 and 500 mu g / mL.

[0018] 2、The chicken newcastle disease vaccine diluted by the polysaccharide vaccine diluent of Kadsura interior A.C.Simoes et Chiu can effectively stimulate immune response, enhance immune effect, and the immune effect is better than that of the chicken newcastle disease vaccine.

[0019] 3、The application provides a new design idea for the development of the chicken newcastle disease live vaccine diluent. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Effect of Kadsura interior polysaccharide on proliferation of chicken spleen lymphocytes.

[0021] Figure 2 Effect of high-dose Kadsura interior polysaccharide on NDV serum antibody level.

[0022] Figure 3 Effect of medium-dose Kadsura interior polysaccharide on NDV serum antibody level.

[0023] Figure 4 Effect of low-dose Kadsura interior polysaccharide on NDV serum antibody level.

[0024] Figure 5 Effect of high-dose Kadsura interior polysaccharide on NDV serum cytokine level.

[0025] Figure 6 Effect of medium-dose Kadsura interior polysaccharide on NDV serum cytokine level.

[0026] Figure 7 Effect of low-dose Kadsura interior polysaccharide on NDV serum cytokine level. DETAILED DESCRIPTION

[0027] Sources of materials used in the experiment:

[0028] Kadsura interior is purchased from anguo medicine market in Hebei; anhydrous ethanol, phenol and sulfuric acid are purchased from a national medicine reagent company; fetal bovine serum and RPMI 1640 culture solution are purchased from Gibco company; 0.22um micropore filter membrane is purchased from Shanghai Anpu company; newcastle disease vaccine (LaSota strain, batch number: 202409) is purchased from Haerbin Pharmaceutical Group Biological Vaccine Co., Ltd.; IL-2, IL-4, IL-6 and INF-gamma detection kits are purchased from Quanzhou Ruixin Biological Technology Co., Ltd.; HA antigen is purchased from Qingdao Lichen.

[0029] Example 1. Preparation of Kadsura interior polysaccharide and determination of sugar content.

[0030] 1. Preparation of Kadsura interior polysaccharide

[0031] Take 50 g of Kadsura japonica to be ground, add 10 times of pure water, 90 ℃ hot water bath for 2 h, then filter out the residue, obtain the 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 12 h, centrifuge at 4000 rpm for 20 min to collect the precipitate, then freeze-dry to obtain Kadsura japonica polysaccharide (crude polysaccharide).

[0032] 2. The content of Kadsura japonica polysaccharide is determined by the phenol-sulfuric acid method.

[0033] Concentration of 0.04 mg / mL glucose: accurately weigh 20 mg of glucose standard dried at 105 ℃ to constant weight, dissolve in pure water and make up to 500 mL, shake well and store at 4 ℃.

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

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

[0036] Weigh 20 mg of Kadsura japonica polysaccharide, add pure water to make up to 100 mL, shake well to obtain a Kadsura japonica polysaccharide solution. Take 1.0 mL of the Kadsura japonica 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, then carefully add 5.0 mL of concentrated sulfuric acid to the rotary mixer and mix well, then place in a water bath for boiling for 30 min. After cooling to room temperature, measure the absorbance value at 490 nm wavelength with the blank solution as the control using a spectrophotometer. The glucose mass is found from the standard curve, and the polysaccharide content in the sample is calculated. The result is: the content of Kadsura japonica polysaccharide determined by the phenol-sulfuric acid method is 60.5%.

[0037] Example 2. In vitro activity test of Kadsura japonica polysaccharide.

[0038] 1. Prepare polysaccharide solution of Acanthopanax giraldii Harms with RPMI 1640 culture solution containing 10% fetal bovine serum and polysaccharide of Acanthopanax giraldii Harms, filter bacteria through 0.22 um microporous filter membrane, dilute into different gradient concentrations (ug / mL): 500 ug / mL, 250 ug / mL, 125 ug / mL, 62.5 ug / mL, 1.25 ug / mL, 15.625 ug / mL, 7.813 ug / mL, 3.906 ug / mL, 1.953 ug / mL, and store in a refrigerator at 4℃.

[0039] 2. Take 60-day-old Jinfeng egg laying hens, and after killing by neck vein bloodletting, soak in 75% ethanol for 5 min, and take out the spleen under sterile conditions on a clean bench. Add 5 mL PBS to a cell culture dish for washing the spleen. Take a cell sieve with a pore size of 0.45 um and place it in a new cell culture dish, place the washed spleen in the cell sieve, add 5 mL PBS, grind the spleen in the cell sieve, and then transfer to a 15 mL centrifuge tube, centrifuge at 2000 rpm for 10 min. After centrifugation, discard the supernatant and retain the precipitate, add 5 mL sterile water and gently blow for 15 s to lyse red blood cells, immediately after blowing, add 5 mL Hanks' solution and mix gently, centrifuge at 2000 rpm for 10 min, and discard the supernatant. Resuspend the cells with RPMI 1640 containing 10% fetal bovine serum, adjust the cell concentration to 1×10 7 6 / mL, and obtain a cell suspension.

[0040] 3. Add 100 uL of cell suspension per well to a 96-well cell culture plate, then add 100 uL of polysaccharide of Acanthopanax giraldii Harms of different gradient concentrations, 6 repeated wells for each concentration, and set up cell control wells, add PBS to the edge wells for moisturizing, and place the 96-well plate in a 37℃, 5% CO2 incubator for 44 h, then add 20 uL of MTT (5 mg / mL) per well, continue to culture for 4 h, then remove and centrifuge at 2000 rpm for 5 min, discard the supernatant, and add 100 uL of dimethyl sulfoxide (DMSO) per well to dissolve the precipitate. Measure the absorbance (A 570 value) at 570 nm on an enzyme-linked immunoassay instrument as an index of lymphocyte proliferation. The results are shown in Table 1. Figure 1 The A 570 value of polysaccharide of Acanthopanax giraldii Harms at 250 and 500 ug / mL was significantly higher than that of the corresponding cell control group (P<0.01), indicating that polysaccharide of Acanthopanax giraldii Harms has a proliferative effect on chicken spleen lymphocytes, and the proliferative effect is best when the concentration of polysaccharide of Acanthopanax giraldii Harms is 250 and 500 ug / mL.

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

[0042] Test Example 1: 4 mg of Acanthopanax senticosus polysaccharide was added to 10 mL of phosphate buffer, to obtain an Acanthopanax senticosus polysaccharide vaccine diluent with a concentration of 400 μg / mL of Acanthopanax senticosus polysaccharide. The Acanthopanax senticosus polysaccharide was prepared according to Example 1.

[0043] Test Example 2: 8 mg of Acanthopanax senticosus polysaccharide was added to 10 mL of phosphate buffer, to obtain an Acanthopanax senticosus polysaccharide vaccine diluent with a concentration of 800 μg / mL of Acanthopanax senticosus polysaccharide. The Acanthopanax senticosus polysaccharide was prepared according to Example 1.

[0044] Test Example 3: 16 mg of Acanthopanax senticosus polysaccharide was added to 10 mL of phosphate buffer, to obtain an Acanthopanax senticosus polysaccharide vaccine diluent with a concentration of 1600 μg / mL of Acanthopanax senticosus polysaccharide. The Acanthopanax senticosus polysaccharide was prepared according to Example 1.

[0045] Example 4. Chicken Newcastle disease vaccine immunization test with Acanthopanax senticosus polysaccharide vaccine diluent dilution.

[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 kit, gamma interferon (IFN-γ) detection kit were purchased from Quanzhou Ruixin Biological Technology Co., Ltd.

[0049] (3) Vaccine diluent was test example 1, test example 2, test example 3 in Example 3.

[0050] 2. Animal grouping and immunization.

[0051] 14-day-old Da Wu gold phoenix laying hens 100, were randomly divided into 5 groups, 20 in each group, respectively, high-dose Acanthopanax senticosus polysaccharide group (ASPH), medium-dose Acanthopanax senticosus polysaccharide group (ASPM), low-dose Acanthopanax senticosus polysaccharide group (ASPL), vaccine control group (VC), blank control group (BC). Except for the blank control group (nasal and eye drops with phosphate buffer), the remaining four groups of chickens were inoculated with 0 ug / mL, 400 ug / mL, 800 ug / mL, 1600 ug / mL of Acanthopanax senticosus polysaccharide diluted Newcastle disease vaccine by nasal and eye drops, respectively, and were immunized again 14 days after immunization.

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

[0053] 3. Determination of antibody titer

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

[0055] Collect 3 mL of blood from the subwing vein of one specific pathogen-free rooster. Add 3 volumes of PBS and mix well. Centrifuge at 1000 rpm for 8 minutes at 4°C, discard the supernatant, and retain the precipitate. Add another 3 volumes of PBS and repeat the above steps to wash the precipitate three times to obtain red blood cell pellet. Transfer 500 μL of the red blood cell pellet to a sterile centrifuge tube containing 45.5 mL of PBS to prepare 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 of PBS to each well of a 96-well plate with a conical bottom. Add 25 μL of antigen to well 1 and serially dilute to well 11. Discard 25 μL of liquid in well 11 and add 25 μL of 1% chicken red blood cell suspension to each well. After standing for 30 minutes, the results can be determined.

[0058] Result interpretation method: Tilt the 96-well plate with a pointed bottom at a certain angle and observe the red blood cell precipitation. If the precipitation flows down rapidly in a linear fashion, it indicates that the viral antigen has not agglutinated or has not completely agglutinated with the red blood cells. If the red blood cell precipitation does not flow down or does not agglutinate into a precipitate, but is distributed in a thin layer, it indicates that the viral antigen has completely agglutinated with the red blood cells. Usually, the dilution factor of the first well where the precipitation flows down in a linear fashion is taken as the highest dilution factor of the test antigen, which is the antigen titer. The measured antigen titer was 9log2.

[0059] (3) Verification of the tetraunit antigen

[0060] Based on the results of the HA test, prepare four units of antigen using PBS; add 25 μL of PBS to wells 1 to 5 of a 96-well plate with a conical bottom, add 25 μL of the prepared four units of antigen to well 1, mix well, serially dilute to well 5, discard 25 μL, add 25 μL of 1% chicken red blood cell suspension to each well, let stand for 30 min and observe the results.

[0061] Interpretation results: The first two wells showed complete aggregation; in the third well, half of the red blood cells immediately became streamlined; and in the fourth and fifth wells, all red blood cells immediately became streamlined. This result indicates that the four-unit antigen is accurate, meaning the verification was successful.

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

[0063] Add 25 μL PBS to each well of a V-bottom 96-well plate, add 25 μL of the serum to be tested to the first well, and then dilute by a factor of two to the 11th well, discard 25 uL liquid from the 11th well, and add 25 μL of four units of antigen to each well. After 30 min of reaction, add 25 μL of 1% chicken red blood cell suspension to each well, and after 30 min of standing, observe the results.

[0064] Read the results: the maximum dilution at which the red blood cells precipitate in a linear flow is the hemagglutination inhibition (HI) titer of the serum sample, expressed as Log2.

[0065] The results are shown in Table 1. Figures 2-4 As shown in Table 1, after the first immunization, the serum NDV antibody levels of the vaccine control group and the ASP group were increased, and were higher than those of the blank control group. On the 7th day and the 14th day after the first immunization (P<0.05) and on the 7th day and the 14th day after the second immunization, the serum NDV specific antibody levels of the ASP group were higher than those of the vaccine control group.

[0066] The serum antibody levels of the ASP group were significantly higher than those of the blank control group and the vaccine control group on the 14th day, the 21st day, the 28th day, and the 35th day after immunization (P<0.05). Moreover, the high-dose and the medium-dose ASP groups showed better immunization effects on the 28th day, the 35th day, and the 42nd day.

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

[0068] Refer to the kit instructions, and all reagents and components are first restored to room temperature. Set up standard wells, 0-value wells, blank wells, and sample wells in a 96-well enzyme-labeled plate, add 50 μL of different concentrations of standard to each standard well, add 50 μL of sample diluent to the 0-value well, do not add to the blank well, and add 50 μL of the serum to be tested to the sample well. Then add 100 μL of horseradish peroxidase (HRP) labeled monitoring antibody (do not add to the blank well), and incubate in a 37°C constant-temperature incubator for 60 min. After incubation, discard the liquid, and pat dry on a water-absorbing paper. Fill each well with washing liquid, stand for 20 s, shake off the washing liquid, and pat dry on a water-absorbing paper. Repeat the washing for 5 times. Add 100 μL of a mixture of substrate A and substrate B (1:1 volume) to each well, and incubate in a 37°C constant-temperature incubator for 15 min. After taking out, add 50 μL of a stop solution to each well, and read the OD value at 450 nm on an enzyme-labeled instrument.

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

[0070] The results of the medium-dose ASP group are shown in Table 3.Figure 6 As shown in Table 4, the contents of IL-2, IL-4, IL-6 and INF-γ were significantly different (P<0.01) between the vaccine control group and the blank control group, and the content of IL-2 was significantly different (P<0.01) between the vaccine control group and the blank control group, and the content of IL-4 was significantly different (P<0.05) between the vaccine control group and the blank control group.

[0071] The results of the low-dose polysaccharide group are shown in Table 5. Figure 7 As shown in Table 4, the contents of IL-2, IL-4, IL-6 and INF-γ were significantly different (P<0.01) between the vaccine control group and the blank control group, and the content of IL-2 was significantly different (P<0.01) between the vaccine control group and the blank control group, and the content of IL-4 was significantly different (P<0.05) between the vaccine control group and the blank control group.

[0072] Therefore, the newcastle disease vaccine diluted with the polysaccharide vaccine diluent of Acanthopanax giraldii Harms can effectively stimulate immune response and enhance immune effect.

Claims

1. The use of a Klamath weed polysaccharide vaccine diluent in the preparation of a Newcastle disease vaccine in chickens, characterized in that, The vaccine diluent comprises polysaccharide of Kandelia candel and phosphate buffer, and the concentration of the polysaccharide of Kandelia candel is 400-1600 µg / mL.

2. Use according to claim 1, characterized in that, The concentration of the polysaccharide of Kandelia candel is 400, 800 or 1600 µg / mL.

3. Use according to claim 1, characterized in that, The preparation method of the vaccine diluent of the polysaccharide of Kandelia candel is mixing the polysaccharide of Kandelia candel with the phosphate buffer.

4. Use according to claim 3, characterized in that, The preparation method of the polysaccharide of Kandelia candel is grinding Kandelia candel, adding pure water, hot water bath immersion, filtering to remove the residue, concentrating by using a rotary evaporator, adding anhydrous ethanol for alcohol precipitation, standing at 4 ℃ for 12 h, collecting the precipitate by centrifugation, and finally freeze-drying.

5. Use according to claim 4, characterized in that, The volume of the pure water added is 10 times the weight of Kandelia candel.

6. Use according to claim 4, characterized in that, The hot water bath immersion is 2 h of 90 ℃ hot water bath extraction.

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

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

9. A Newcastle disease vaccine for chickens, characterized in that, The vaccine comprises the vaccine diluent of the polysaccharide of Kandelia candel, and the vaccine diluent comprises the polysaccharide of Kandelia candel and the phosphate buffer, and the concentration of the polysaccharide of Kandelia candel is 400-1600 µg / mL.

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