Newcastle disease vaccine diluent containing American ginseng root hair polysaccharide and preparation method of Newcastle disease vaccine diluent

Abstract

The invention relates to the technical field of biological medicines, in particular to a Newcastle disease vaccine diluent containing American ginseng root hair polysaccharide and a preparation method of the Newcastle disease vaccine diluent. American ginseng and American ginseng root hair are selected for in-vitro screening, and American ginseng root hair which is lower in cost and better in effect is selected as a main component of the vaccine diluent after screening; the method comprises the following steps: adding American ginseng root hair polysaccharide into a phosphate buffer solution, taking out 1mL of the American ginseng root hair polysaccharide, and diluting the American ginseng root hair polysaccharide with the phosphate buffer solution by 10 times to obtain a diluted solution of the Newcastle disease vaccine containing the American ginseng root hair polysaccharide, so as to prepare the diluted solution of the Newcastle disease vaccine containing the American ginseng root hair polysaccharide with the American ginseng root hair polysaccharide concentration of 0.06-0.24 mg / mL, american ginseng polysaccharide and American ginseng root hair polysaccharide are selected for in-vitro screening, and the American ginseng root hair polysaccharide which is lower in cost and better in effect is selected as a main component of the vaccine diluent after screening, so that the problem of how to apply the polysaccharide in American ginseng root hair to the Newcastle disease freeze-dried vaccine diluent is solved; the immune cell proliferation is promoted, the immune titer is improved, and the release of cell factors is promoted.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a diluent of a chicken Newcastle disease vaccine containing American ginseng root polysaccharide and a preparation method thereof. Background Art

[0002] Newcastle Disease (ND) is a highly contagious viral disease caused by the Newcastle disease virus. It is highly contagious, has diverse symptoms, and has a high mortality rate. It mainly affects poultry, especially chickens, has a global distribution, and has caused serious economic losses to the poultry industry. Vaccination is the key to preventing Newcastle disease, and implementing hygiene management and biosecurity measures can reduce the risk of virus transmission.

[0003] Freeze-dried attenuated vaccines are a type of vaccine obtained through artificial attenuation or natural selection, with low toxicity and antigenicity. Compared with inactivated vaccines, attenuated vaccines can reproduce in the body, thus providing more lasting immune protection. They need to be diluted when used, and the usual diluents are physiological saline or phosphate buffered saline (PBS). These diluents cannot improve the protective effect of the vaccine, but can only dilute the vaccine.

[0004] American ginseng (Panax quinquefolium Linn.), also known as Western ginseng, American ginseng, and foreign ginseng, is a perennial herb belonging to the Araliaceae family. It mainly grows in North America, especially the United States and Canada. The root of American ginseng is widely used as a traditional herbal medicine, which has the effects of nourishing the body, improving immunity, and resisting fatigue. The utilization of its root part is not high. Compared with the root of American ginseng, the root part of American ginseng has the advantage of being cheaper.

[0005] Chinese herbal polysaccharides refer to a class of active polysaccharides extracted from Chinese herbal medicines, generally excluding starch and cellulose. These polysaccharides have a variety of biological activities and are believed to maintain or enhance the physiological functions of the body. Among them, studies have shown that Chinese herbal polysaccharides have immune regulation, anti-tumor, anti-viral, hypoglycemic, anti-aging and other effects. These effects have made Chinese herbal polysaccharides a focus of attention in the fields of traditional medicine and modern medicine, and they are widely used in the production and development of drugs, health products and medical care products. Its unique pharmacological effects provide new directions for exploration of human health and also bring new opportunities for drug research and development. Studies have found that American ginseng polysaccharides can promote immune function and have a significant inhibitory effect on the pro-inflammatory response induced by lipopolysaccharide. Therefore, American ginseng root polysaccharides can be developed as vaccine diluents with immunity enhancement.

[0006] In view of the above-mentioned defects, the inventors of the present invention finally obtained the present invention after a long period of research and practice. Summary of the invention

[0007] The purpose of the present invention is to solve the problem of how to use polysaccharides in American ginseng roots for chicken Newcastle disease freeze-dried vaccine diluent to promote immune cell proliferation, improve immune titer and promote the release of cytokines. A diluent of chicken Newcastle disease vaccine containing American ginseng root polysaccharides and a preparation method thereof are provided.

[0008] In order to achieve the above purpose, the present invention discloses a method for preparing a diluent of a chicken Newcastle disease vaccine containing American ginseng root polysaccharide, wherein American ginseng root polysaccharide is added into a phosphate buffer, and then 1 mL is taken out and diluted 10 times with a phosphate buffer to obtain a diluent of a chicken Newcastle disease vaccine containing American ginseng root polysaccharide.

[0009] The concentration of American ginseng polysaccharide in the diluent of the chicken Newcastle disease vaccine containing American ginseng polysaccharide is 0.06-0.24 mg / mL.

[0010] The preparation method of American ginseng polysaccharide comprises the following steps:

[0011] S1, grinding the roots of American ginseng into powder, mixing with RO water, rotating heating extraction, water bath rotary evaporation and negative pressure concentration;

[0012] S2, adding 9 times the volume of anhydrous ethanol to the polysaccharide concentrated by negative pressure in step S1 and stirring to fully precipitate the polysaccharide, and placing the sugar-ethanol mixture in a refrigerator to precipitate for 12 hours;

[0013] S3, taking out the sugar-ethanol mixture after precipitation in step S2 and centrifuging it to fully precipitate the sugar insoluble in ethanol, discarding the upper layer of anhydrous ethanol, and drying the polysaccharide at 50° C. for 12 hours to obtain dry crude polysaccharide.

[0014] In the step S1, the dosage ratio of American ginseng root polysaccharide to RO water is 1 g:10 mL.

[0015] In step S1, the rotary heating extraction temperature is 98° C. and the extraction time is 2 hours.

[0016] In the step S1, the temperature of the water bath rotary evaporation is 50° C., and the time of the water bath rotary evaporation is 2 hours.

[0017] In the step S1, the volume after negative pressure concentration is 50 mL.

[0018] In step S3, the centrifugal speed is 4000 rpm and the centrifugal time is 20 min.

[0019] The invention also discloses a diluent of chicken Newcastle disease vaccine containing American ginseng polysaccharide prepared by the preparation method.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention uses American ginseng root polysaccharide for Newcastle disease freeze-dried vaccine diluent, and has the effects of promoting immune cell proliferation, improving immune titer, and promoting the release of cytokines, and can greatly improve the immune effect of Newcastle disease vaccine, quickly produce antibodies and cytokines, and improve the body's ability to resist diseases. The American ginseng root part is selected, the raw material price is low (compared with American ginseng decoction pieces), the extraction method is simple, the selected American ginseng polysaccharide has a wide safe concentration and high safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The in vitro lymphocyte proliferation effect of American ginseng root polysaccharide;

[0022] Figure 2 The in vitro lymphocyte proliferation effect of American ginseng polysaccharide;

[0023] Figure 3 This is the result of the hemagglutination inhibition test in the high-dose group of American ginseng polysaccharide;

[0024] Figure 4 This is the result of the hemagglutination inhibition test in the medium-dose group of American ginseng polysaccharide;

[0025] Figure 5 This is the result of the hemagglutination inhibition test in the low-dose group of American ginseng polysaccharide;

[0026] Figure 6 The effect of high dose of American ginseng polysaccharide on chicken cytokines;

[0027] Figure 7 The effect of medium dose of American ginseng polysaccharide on chicken cytokines;

[0028] Figure 8 The effect of low doses of American ginseng polysaccharides on chicken cytokines. DETAILED DESCRIPTION

[0029] The above and other technical features and advantages of the present invention are described in more detail below in conjunction with the accompanying drawings.

[0030] Example 1

[0031] Preparation of polysaccharides

[0032] Take 50g of American ginseng and American ginseng root respectively, grind to powder, mix with 500mL RO water, rotate and heat at 98℃ to extract for 2h, rotate in a 50℃ water bath for 2h, concentrate to 50mL under negative pressure, add 9 times volume of anhydrous ethanol (450mL) and stir to fully precipitate the polysaccharide, place the sugar-ethanol mixture in a refrigerator at 4℃ for 12h, centrifuge at 4000rpm for 20min to fully precipitate the sugar insoluble in ethanol, and discard the upper anhydrous ethanol. Dry the polysaccharide at 50℃ for 12h to obtain dry crude polysaccharide, grind and store in a desiccator. The sugar content was determined by phenol-sulfuric acid method. The moisture content was determined by the drying loss method, and the total saponin content was determined by the vanillin-acetic acid method. The determination results are shown in Table 1.

[0033] Table 1 Test results of ingredients of American ginseng roots and American ginseng slices

[0034] Moisture content Total saponin content Polysaccharide extraction rate Polysaccharide content Sugar cost per gram American Ginseng Root 11.90％ 7.10％ 15.10％ 81.30％ 0.7 yuan American ginseng slices 11.00％ 11.80％ 5.30％ 43％ 7.1 yuan

[0035] Example 2

[0036] In vitro activity test of polysaccharides

[0037] 60-day-old laying roosters were killed by bleeding from the jugular vein. The spleen was removed aseptically, washed three times with Hanks solution, cut into pieces in a plate containing 5 mL of Hanks solution, passed through a 0.45 μm cell sieve, and the cell fluid was collected. Lymphocyte separation solution was added, centrifuged at 2000 rpm for 10 min, washed twice with Hanks solution, and the cells were counted using a cell counting plate. The cell concentration was adjusted to 1×10 with complete RPMI 1640. 7 / mL. The calculation formula is as follows:

[0038]

[0039] Add 100 μL to each well of a 96-well cell culture plate, and then add 100 μL of 2-fold gradient dilutions of American ginseng polysaccharides and polysaccharides from American ginseng roots filtered through a 0.22 μm filter membrane, respectively, and repeat 6 wells for each concentration. Place the cell plate in a 37°C, 5% CO 2 After 44 h of incubation in an incubator, 20 μL of MTT (5 mg / mL) was added to each well. After 4 h of incubation, the cells were taken out and centrifuged at 2500 rpm for 10 min. 100 μL of DMSO was removed and 100 μL of DMSO was added to each well to dissolve the precipitate. The absorbance at 570 nm (A) was measured by an enzyme-linked immunosorbent assay. 570 value), as an indicator of lymphocyte proliferation, the results are as follows Figure 1 , 2As shown. This figure shows the in vitro activity test of American ginseng polysaccharides, which shows that American ginseng polysaccharides at 1000μg / mL~250μg / mL and polysaccharides in American ginseng roots at 3.7411μg / mL~0.4157μg / mL have a significant promoting effect on the proliferation of chicken spleen lymphocytes (P<0.01). It can be seen that American ginseng polysaccharides and polysaccharides in American ginseng roots have immune promoting activity in vitro, but the activity of polysaccharides in American ginseng roots is stronger than that of American ginseng polysaccharides, and the cost is low, so American ginseng roots are selected as the main raw material of vaccine diluent.

[0040] Example 3

[0041] Preparation of vaccine diluent

[0042] Dilute with 0 mg / mL, 0.06 mg / mL, 0.12 mg / mL, and 0.24 mg / mL of American ginseng polysaccharide respectively.

[0043] a. Add 6 mg of American ginseng polysaccharide to 10 mL of phosphate buffer, and then take out 1 mL and dilute it 10 times with phosphate buffer to obtain an animal vaccine diluent with an American ginseng polysaccharide concentration of 60 μg / mL. American ginseng polysaccharide is prepared according to Example 1.

[0044] b. Add 12 mg of American ginseng polysaccharide to 10 mL of phosphate buffer, and then take out 1 mL and dilute it 10 times with phosphate buffer to obtain an animal vaccine diluent with an American ginseng polysaccharide concentration of 120 μg / mL. American ginseng polysaccharide is prepared according to Example 1.

[0045] 24 mg of American ginseng polysaccharide was added to 10 mL of phosphate buffer, and 1 mL was taken out and diluted 10 times with phosphate buffer to obtain an animal vaccine diluent with an American ginseng polysaccharide concentration of 240 μg / mL. American ginseng polysaccharide was prepared according to Example 1.

[0046] Example 4

[0047] Immunity test of Newcastle disease vaccine in chickens by diluent of American ginseng polysaccharide vaccine

[0048] Newcastle disease vaccine (La Sota strain, batch number: 202409) was purchased from Qingdao Lijian Biotechnology Co., Ltd. The vaccine was diluted and shaken well with the above-prepared vaccine diluent.

[0049] 100 14-day-old Dawu Jinfeng laying hens were randomly divided into 5 groups, namely, high-dose group (AGPH), medium-dose group (AGPM), low-dose group (AGPL), vaccine control group (VC), and blank control group (BC), with 20 chickens in each group. Except for the blank control group (normal saline for nasal and eye drops), the other four groups of chickens were inoculated with 0mg / mL, 0.06mg / mL, 0.12mg / mL, and 0.24mg / mL of Newcastle disease vaccine diluted with ginseng polysaccharide by nasal drops and eye drops, and a second immunization was performed 14 days after immunization. Blood was collected from the subwing vein of the chickens at 7d, 14d, 21d, 28d, 35d, and 42d after immunization, and the serum was separated, and the antibody titer was detected by hemagglutination (HA) and hemagglutination inhibition (HI) tests.

[0050] Example 5

[0051] Antibody titer testing

[0052] 1. Preparation of 1% chicken red blood cell suspension

[0053] Take a rooster free of specific pathogens, collect 3 mL of blood from the wing vein, add 3 times the volume of normal saline, mix well, centrifuge at 1000r / min for 8 minutes at 4°C, discard the supernatant; add 3 times the volume of normal saline, repeat the above operation for 3 times, and draw 500 μL of red blood cell sediment into a sterile centrifuge tube containing 45.5 mL of PBS to make a red blood cell suspension. When using, gently shake the centrifuge tube to suspend the red blood cells.

[0054] 2. Add 25 μL PBS to each well of a 96-well plate with a pointed bottom, 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, let it stand for 30 minutes before determining the result.

[0055] Result determination: 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, which is the titer of the antigen.

[0056] 3. Verification of the four-unit antigen

[0057] According to the results of the HA test, use PBS to prepare four-unit antigen; add 25μL of PBS to the 1st to 5th wells of the conical bottom 96-well plate, add 25μL of the prepared four-unit antigen to the 1st well, mix and dilute to the 5th well, discard 25μL, add 25μL of 1% chicken red blood cell suspension to each well, and observe the results after standing for 30 minutes; interpret the results: the first 2 wells are completely aggregated, half of the red blood cells in the 3rd well immediately become streamlined, and all the red blood cells in the 4th and 5th wells immediately become streamlined. This result shows that the four-unit antigen is accurate, that is, the verification is successful.

[0058] 4. Hemagglutination inhibition test (HI)

[0059] 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 25 μL 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.

[0060] Read the result: 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. Figure 3 , 4 , as shown in Figure 5.

[0061] Statistical analysis

[0062] One-way ANOVA was performed using Graphpad Pism 9.5 software. All data were expressed as mean ± standard deviation. P>0.05 indicated no significant difference, P<0.05 indicated significant difference, and P<0.01 indicated extremely significant difference.

[0063] like Figure 3 As shown in the figure, after the first immunization, the level of NDV-specific antibody in the high-dose American ginseng polysaccharide (AGPH) group of chickens increased. At 7d and 14d after the first immunization (P<0.05) and 7d and 14d after the second immunization, the level of NDV-specific antibody in the AGPH group was significantly higher than that in the immunization control group.

[0064] like Figure 4 As shown in the figure, it can be concluded that after the first immunization, the level of NDV-specific antibodies in the immunized group and the medium-dose American ginseng polysaccharide (AGPM) group increased. At 7d and 14d after the first immunization (P<0.05) and 7d and 14d after the second immunization, the level of NDV-specific antibodies in the AGPM group was higher than that in the immunized control group.

[0065] like Figure 5As shown in the figure, it can be concluded that after the first immunization, the level of NDV-specific antibodies in the immunized group and the low-dose American ginseng polysaccharide (AGPL) group increased. At 7d and 14d after the first immunization (P<0.05) and 7d and 14d after the second immunization, the level of NDV-specific antibodies in the AGPL group was higher than that in the immunized control group.

[0066] The serum antibody level of the American ginseng polysaccharide group was significantly higher than that of the blank control group and the vaccine control group on days 14, 21, 28, and 35 after immunization (P<0.05). The American ginseng polysaccharide medium-dose group showed a better immune effect on days 28, 35, and 42.

[0067] Example 6

[0068] Effects of American ginseng polysaccharide vaccine diluent on chicken cytokines

[0069] One week after the second immunization, the double antibody sandwich enzyme-linked immunosorbent assay (ELISA) method was used to detect IL-2, IL-4, IL-6, and INF-γ in the experimental chickens.

[0070] Set up standard wells, 0 value wells, blank wells and sample wells. Add 50 μL of standards of different concentrations to the standard wells, add 50 μL of sample diluent to the 0 value wells, add nothing to the blank wells, and add 50 μL of the sample to be tested to the sample wells.

[0071] In addition to the blank wells, 100 μL of horseradish peroxidase (HRP)-labeled detection antibody was added to the standard wells, 0 value wells and sample wells.

[0072] Cover the reaction plate with a sealing film and incubate in a 37°C water bath or incubator in the dark for 60 minutes.

[0073] Remove the sealing film, discard the liquid, pat dry on absorbent paper, fill each well with washing solution, let stand for 20 seconds, shake off the washing solution, pat dry on absorbent paper, and repeat this 5 times. After washing the plate, before adding the substrate, pat the reaction plate dry on a clean, non-chipping paper.

[0074] Mix substrate A and B in a 1:1 ratio and add 100 μL of the substrate mixture to all wells. Cover the reaction plate with a sealing film and incubate in a 37°C water bath or incubator in the dark for 15 minutes.

[0075] Add 50 μL of stop solution to all wells, and read the absorbance (OD value) of each well on a microplate reader.

[0076] With the concentration of the standard as the horizontal axis (6 standard wells, plus 1 0 value well, a total of 7 concentration points), and the corresponding absorbance (OD value) as the vertical axis, computer software was used to use four-parameter logistic curve fitting (4-pl) to create a standard curve equation, and the concentration value of the sample was calculated using the equation through the absorbance (OD value) of the sample. The measured value is as follows Figure 5 , 6 , as shown in 7.

[0077] like Figure 6 As shown, it can be concluded that the IL-2, IL-4, IL-6, and INF-γ of the chickens after the vaccine was diluted with the American ginseng polysaccharide vaccine diluent were significantly higher (P < 0.01) than those of the common vaccine group and the blank control group, indicating that high-dose American ginseng polysaccharides can promote the production of IL-2, IL-4, IL-6, and INF-γ in chicken cells.

[0078] like Figure 7 As shown in the figure, it can be concluded that the IL-2, IL-4, IL-6, and INF-γ of the chickens after the vaccine was diluted with the American ginseng polysaccharide vaccine diluent were significantly higher (P < 0.01) than those of the common vaccine group and the blank control group, indicating that the medium dose of American ginseng polysaccharide can promote the production of IL-2, IL-4, IL-6, and INF-γ in chicken cells.

[0079] like Figure 8 As shown, it can be concluded that the IL-2, IL-4, IL-6, and INF-γ of chickens after being vaccinated with American ginseng polysaccharide vaccine diluent were significantly higher (P < 0.01) than those of the common vaccine group and the blank control group, indicating that low-dose American ginseng polysaccharide can promote the production of IL-2, IL-4, IL-6, and INF-γ in chicken cells.

[0080] The above description is only a preferred embodiment of the present invention, which is only illustrative and not restrictive of the present invention. Those skilled in the art understand that many changes, modifications, and even equivalences may be made to the present invention within the spirit and scope defined by the claims of the present invention, but all of them will fall within the scope of protection of the present invention.

Claims

1. A method for preparing a diluent of a chicken Newcastle disease vaccine containing American ginseng root polysaccharide, characterized in that: Add American ginseng root polysaccharide to phosphate buffer, and then take out 1 mL and dilute it 10 times with phosphate buffer to obtain a dilution of chicken Newcastle disease vaccine containing American ginseng root polysaccharide.

2. A method for preparing a diluent of a chicken Newcastle disease vaccine containing American ginseng root polysaccharide as claimed in claim 1, characterized in that: The concentration of American ginseng polysaccharide in the diluent of the chicken Newcastle disease vaccine containing American ginseng root polysaccharide is 0.06-0.24 mg / mL.

3. The method for preparing a diluent of a chicken Newcastle disease vaccine containing American ginseng root polysaccharide as claimed in claim 1, characterized in that: The preparation method of American ginseng polysaccharide comprises the following steps: S1, grinding the roots of American ginseng into powder, mixing with RO water, rotating heating extraction, water bath rotary evaporation and negative pressure concentration; S2, adding 9 times the volume of anhydrous ethanol to the polysaccharide concentrated by negative pressure in step S1 and stirring to fully precipitate the polysaccharide, and placing the sugar-ethanol mixture in a refrigerator to precipitate for 12 hours; S3, taking out the sugar-ethanol mixture after precipitation in step S2 and centrifuging it to fully precipitate the sugar insoluble in ethanol, discarding the upper layer of anhydrous ethanol, and drying the polysaccharide at 50° C. for 12 hours to obtain dry crude polysaccharide.

4. A method for preparing a diluent of a chicken Newcastle disease vaccine containing American ginseng root polysaccharide as claimed in claim 3, characterized in that: In the step S1, the dosage ratio of American ginseng root polysaccharide to RO water is 1 g:10 mL.

5. The method for preparing a diluent of a chicken Newcastle disease vaccine containing American ginseng root polysaccharide as claimed in claim 3, characterized in that: In step S1, the rotary heating extraction temperature is 98° C. and the extraction time is 2 hours.

6. The method for preparing a diluent of a chicken Newcastle disease vaccine containing American ginseng root polysaccharide as claimed in claim 3, characterized in that: In the step S1, the temperature of the water bath rotary evaporation is 50° C., and the time of the water bath rotary evaporation is 2 hours.

7. The method for preparing a diluent of a chicken Newcastle disease vaccine containing American ginseng root polysaccharide as claimed in claim 3, characterized in that: In the step S1, the volume after negative pressure concentration is 50 mL.

8. The method for preparing a diluent of a chicken Newcastle disease vaccine containing American ginseng root polysaccharide as claimed in claim 3, characterized in that: In step S3, the centrifugal speed is 4000 rpm and the centrifugal time is 20 min.

9. A diluent of a chicken Newcastle disease vaccine containing American ginseng root polysaccharide prepared by the preparation method according to any one of claims 1 to 8.

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