Preparation method and application of bovine viral diarrhea virus egg yolk antibody
By preparing an inactivated vaccine for bovine viral diarrhea virus, immunizing laying hens and extracting yolk antibodies, the problem that existing vaccine immunity cannot effectively prevent bovine viral diarrhea virus is solved, and low-cost and efficient treatment and prevention effects are achieved.
Patent Information
- Application Number
- CN202510496626.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-21
- Publication Date
- 2025-07-22
AI Technical Summary
The existing vaccine immunity cannot effectively prevent new strains and mutant strains of the bovine viral diarrhea virus, causing difficulties in preventing and treating the disease and causing economic losses to the breeding industry.
Prepare an inactivated vaccine for bovine viral diarrhea virus, extract yolk antibodies after immunization of laying hens, purify them through multiple precipitation of ammonium sulfate and lyophilized to obtain efficient yolk antibodies.
It provides a low-cost, simple operation method that can efficiently enrich specific antibodies for the treatment and prevention of bovine viral diarrhea disease, reduces the use of target animals and prolongs the shelf life.
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Figure CN120349402A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of yolk antibody preparation, and particularly relates to a method for preparing yolk antibody against bovine viral diarrhea virus and its application. Background Art
[0002] Bovine viral diarrhea is a febrile, acute and highly contagious disease caused by bovine viral diarrhea virus (BVDV), which mainly causes infected cattle to have fever, diarrhea, reproductive disorders and immune dysfunction. The mortality rate of this disease is relatively high, and the latent infection rate exceeds 50%. At present, the host range of BVDV infection is constantly expanding, and deer, pigs and other wild animals can all be infected, and the prevalence is showing an obvious upward trend, which brings serious challenges to the prevention and control of this disease. This disease is one of the main diseases endangering the global cattle industry and brings serious economic losses to the breeding industry. At present, vaccination is still one of the main strategies for preventing this disease. However, with the emergence of new strains and mutant strains of this disease, vaccine immunization can no longer effectively prevent it. Therefore, exploring more effective new diagnostic products will become a new research direction. Summary of the Invention
[0003] The purpose of the present invention is to provide a yolk antibody against bovine viral diarrhea virus.
[0004] The present invention provides a method for preparing a yolk antibody against bovine viral diarrhea virus. The specific steps of the preparation method are as follows:
[0005] Step 1: Prepare a vaccine against bovine viral diarrhea virus;
[0006] Step 2: Immunize laying hens with the vaccine obtained in Step 1 to obtain egg yolks;
[0007] Step 3: Extract the antibody from the egg yolk obtained in Step 2 to obtain a yolk antibody.
[0008] Further limited, the vaccine obtained in Step 1 is an inactivated vaccine or a live attenuated vaccine.
[0009] Further limited, the preparation method of the inactivated vaccine is: bovine viral diarrhea virus is mixed with BEI inactivator to obtain an inactivated antigen, and the inactivated antigen is mixed with Freund's incomplete adjuvant in a volume ratio of 1:1 to obtain an inactivated vaccine.
[0010] Further limited, the method for immunizing laying hens in Step 2 is intramuscular injection in the legs of the laying hens.
[0011] Further limited, the method for obtaining the yolk antibody in Step 3:
[0012] (1) Dilute the separated egg yolk with 0.05 mol / L acetic acid-sodium acetate buffer solution with a volume 10 times that of the egg yolk, stir until the egg yolk is completely dissolved, and let it stand and precipitate at 2-8°C for 12-24 hours;
[0013] (2) centrifuging the 10-fold acid-diluted solution using a tubular centrifuge at 6000 r / min for 20 min, and collecting the supernatant;
[0014] (3) Performing ammonium sulfate precipitation three times: adding solid ammonium sulfate to the supernatant after the above centrifugation to a saturation of 33%, performing the first precipitation purification, stirring until the ammonium sulfate is completely dissolved, and standing at room temperature for precipitation for 2 to 24 hours. The ammonium sulfate precipitation solution is centrifuged using a tubular centrifuge at 6000 r / min for 20 min. After centrifugation, the precipitate is obtained. After repeating three times, the precipitate obtained is dissolved in physiological saline and stirred until the precipitate is completely dissolved to obtain the egg yolk antibody.
[0015] Furthermore, the obtained egg yolk antibody is freeze-dried and stored.
[0016] The freeze-drying method is further defined as follows: pre-freeze the dryer to -70°C, turn on the vacuum pump after 2 to 3 hours, start the plate layer temperature control to -22°C, set the plate layer temperature control to -8°C after working for 18 hours, and press the cover to turn off the refrigeration and vacuum pump after working for 2 to 3 hours.
[0017] It is further defined that the bovine viral diarrhea virus is a bovine viral diarrhea virus of the CP type.
[0018] The present invention provides an egg yolk antibody of bovine viral diarrhea virus obtained by the above preparation method.
[0019] The present invention provides an application of the egg yolk antibody of bovine viral diarrhea virus in preparing a kit for detecting bovine viral diarrhea virus or preparing a medicine for treating bovine viral diarrhea disease.
[0020] Beneficial effects: The present invention provides a method for preparing bovine viral diarrhea egg yolk antibodies, which is a non-invasive method that reduces the use of target animals and economic costs by inactivating the bovine viral diarrhea virus, making a vaccine, and then immunizing laying hens, so that the specific antibodies produced by laying hens are efficiently enriched in the egg yolk. After the egg yolk antibodies are freeze-dried, the storage time is extended. The method has low cost and simple operation, and can be used for targeted treatment and prevention of bovine viral diarrhea. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 This is a graph showing the results of monitoring the titer of yolk antibodies. DETAILED DESCRIPTION
[0022] The advantages and features of the present invention will be more clearly demonstrated below in conjunction with the specific embodiments of the present invention. Anyone who relies on the inspiration of the present invention and makes modifications to the details and form of the technical solution without making any creative work shall fall within the scope of protection of the present invention.
[0023] Example 1. A method for preparing bovine viral diarrhea yolk antibody
[0024] I. Preparation of bovine viral diarrhea antigen
[0025] The bovine viral diarrhea virus strain NM01 (CP type) was inoculated into confluent monolayer MDBK cells at 2%, 5%, and 10% of the culture medium volume respectively. The cell status was observed daily. On the 2nd day after virus inoculation, CPE appeared in the cells with 5% and 10% virus inoculation amounts. On the 4th day after virus inoculation, CPE appeared in all inoculated cells. When the CPE was about 80%, the virus solution was harvested for virus titer determination. The results are shown in Table 1. The virus content was the highest when inoculated at 10% by volume ratio, which was 10 6.0 TCID 50 / ml. Therefore, the virus inoculation amount was set at 10% of the culture medium volume ratio, and the virus solution was stored at -80 °C for later use.
[0026] Table 1 Optimization results of virus inoculation amount
[0027] Virus inoculation amount Virus harvesting days Virus content 2％ 5 <![CDATA[10 4.5 TCID 50 / ml]]> 5％ 4 <![CDATA[10 5.5 TCID 50 / ml]]> 10％ 4 <![CDATA[10 6.0 TCID 50 / ml]]>
[0028] II. Preparation of bovine viral diarrhea inactivated vaccine
[0029] 1. Antigen inactivation:
[0030] The bovine viral diarrhea virus antigen harvested in step I was centrifuged to remove cell debris, and BEI inactivator was added while stirring to a final concentration of 1%. It was allowed to act at 32 °C for 24 h, and then sodium thiosulfate was added for hydrolysis at 37 °C for 2 h with continuous stirring. After inactivation, the inactivated solution was taken for sterility inspection (the inactivated virus solution was inoculated into confluent monolayer MDBK cells, and whether cell lesions appeared was observed after 96 h. It was blindly passaged 3 times, and no cell lesions indicated successful inactivation).
[0031] 2. Preparation of inactivated vaccine:
[0032] The above inactivated antigen (antigen content was 10 7.0 TCID 50 / ml) was mixed with Freund's complete adjuvant at a volume ratio of 1:1. Freund's complete adjuvant was placed in a container and slowly stirred, while the antigen was slowly added, and finally emulsified into a vaccine at high speed, which was the vaccine for the first immunization of laying hens. After antigen inactivation (antigen content was 10 7.0 TCID 50 / ml), it was mixed with Freund's incomplete adjuvant at a volume ratio of 1:1. Freund's incomplete adjuvant was placed in a container and slowly stirred, while the antigen was slowly added, and finally emulsified into a vaccine at high speed, which was the vaccine for subsequent booster immunization of laying hens. The emulsified vaccine was milky white, did not disperse when dropped into water, and passed the sterility inspection.
[0033] III. Immunization of Laying Hens
[0034] 1. Select 10 healthy laying hens at 20 weeks of age and immunize them with the inactivated bovine viral diarrhea vaccine obtained in Step 2. Inject 1 ml (10 7.0 TCID 50 / ml) into the leg muscle of each laying hen. At an interval of 14 days after the first immunization, immunize at least 5 times in the same immunization method. At the same time, select 10 laying hens and inject the same dose of normal saline at the same site at the same time as the control group. Collect the hyperimmunized eggs and make good marks, and store them at 4°C for standby.
[0035] 2. Collect eggs:
[0036] Ten days after the 5th immunization of the laying hens in Step A above, sample and detect the titer of the hyperimmune bovine viral diarrhea virus egg yolk antibody every 2 days. When the neutralization titer is greater than 1:64, collect the hyperimmunized eggs and store them at 4°C.
[0037] 3. Preparation of egg yolk antibody:
[0038] Separate the egg yolks from the eggs collected in Step B above with an egg white-yolk separator. Dilute the separated egg yolks with 10 times the volume of acetic acid-sodium acetate buffer (0.05 mol / L, pH 5.0), stir until the egg yolks are completely dissolved, and let it stand and precipitate at 2-8°C for 12-24 hours.
[0039] Centrifuge the above 10-fold acid-diluted solution using a tubular centrifuge at 6000 r / min for 20 min, and take the supernatant after centrifugation.
[0040] The first ammonium sulfate precipitation: Add solid ammonium sulfate to the supernatant after the above centrifugation to reach a saturation of 33% (add 196 g of solid ammonium sulfate per liter of liquid at 25°C), and perform the first precipitation and purification on it. Stir until the ammonium sulfate is completely dissolved, let it stand and precipitate at room temperature for 2-24 hours. Centrifuge the first ammonium sulfate precipitation solution using a tubular centrifuge at 6000 r / min for 20 min, and take the precipitate after centrifugation.
[0041] The second ammonium sulfate precipitation: Dissolve the above precipitate in physiological saline, with a volume of 1 / 3-1 / 4 of the original volume of the first precipitation. Stir until the precipitate is completely dissolved, add solid ammonium sulfate to reach a saturation of 33% (add 196 g of solid ammonium sulfate per liter of liquid at 25°C), and perform the second precipitation and purification on it. Stir until the ammonium sulfate is completely dissolved, let it stand and precipitate at room temperature for 2-24 hours. Centrifuge the second ammonium sulfate precipitation solution using a tubular centrifuge at 6000 r / min for 20 min, and take the precipitate after centrifugation.
[0042] 3rd ammonium sulfate precipitation: Dissolve the above-mentioned precipitate in physiological saline with the same volume as the second precipitation [Check the volume of the egg yolk, then dilute it with 10 times the volume of the egg yolk, centrifuge and measure the volume of the supernatant (the volume of the first precipitation). After centrifugation, take the precipitate, dissolve it with 1 / 3 - 1 / 4 of the volume of the first precipitation (the volume of the second precipitation), centrifuge and take the precipitate, dissolve it with the volume of the second precipitation (this is the third precipitation, that is, the original volume of physiological saline mentioned in the text)]. Stir until the precipitate is completely dissolved, add solid ammonium sulfate to reach a saturation of 33% (add 196 g of solid ammonium sulfate per liter of liquid at 25 °C), conduct the 3rd precipitation purification on it, stir until the ammonium sulfate is completely dissolved, let it stand at room temperature for precipitation for 12 - 24 hours, centrifuge the 3rd ammonium sulfate precipitation solution using a tubular centrifuge at 6000 r / min for 20 min, and take the precipitate after centrifugation. Dissolve the precipitate in physiological saline with the same volume as the 3rd precipitation, and stir until the precipitate is completely dissolved.
[0043] 4. Ultrafiltration concentration and desalting of egg yolk antibody:
[0044] Mix the egg yolk antibody obtained after ultrafiltration concentration and desalting in step 3 above with the freeze-drying protectant at a ratio of 9:1, dispense it into glass bottles, 2 mL per bottle, and cover it with a rubber stopper loosely. Put the glass bottles containing the egg yolk antibody added with the freeze-drying protectant into a pre-freeze dryer to -70 °C (about 2 - 3 h), turn on the vacuum pump, start the shelf temperature control to -22 °C, work for 18 h, set the shelf temperature to -8 °C, work for 2 - 3 h, press the lid to close the refrigeration and the vacuum pump, and release the vacuum to obtain the freeze-dried product of the egg yolk antibody.
[0045] 5. Determination of the optimal immunization times:
[0046] Collect 20 eggs after each immunization. After separating the egg white and egg yolk, prepare the high-immune egg yolk antibody according to the above method, measure the neutralizing antibody titer of the egg yolk antibody, and determine the immunization times. Method for measuring the neutralizing titer of egg yolk antibody: Dilute each batch of the prepared egg yolk antibody proportionally to 1:2, 1:4, 1:8... 1:512, add 200 TCID 50 / ml bovine viral diarrhea virus to each dilution, place it in a 37 °C 5% CO2 incubator for neutralization for 1 h. Inoculate the well-grown MDBK cells at 1×10 5.0 cells / ml into a 96-well cell culture plate and continue to culture it in a 37 °C 5% CO2 incubator for 5 d. Observe the cytopathic effect of each well of cells, calculate the neutralizing antibody titer, and the results are as Figure 1 , after the first immunization, the antibody titer is 1:8, and after 5 immunizations, the antibody titer can reach 1:512. Therefore, it is determined that the immunization times of the laying hens for preparing the egg yolk antibody is 5 times.
[0047] Example 2. Safety evaluation of egg yolk antibody
[0048] Fifteen 8-week-old SPF-grade BALB / c mice were selected. The yolk antibody prepared above was injected into healthy BALB / c mice at 0.5 ml / mouse and 1 ml / mouse respectively. Five mice were immunized with each dose, and another five mice were set as a control group and injected with an equal amount of normal saline. The mental state and death of the mice were observed daily. The results showed that the mental state of the mice in the 0.5 ml dose group, 1 ml dose group and the control group was good, and all of them were healthy and alive, indicating that the prepared yolk antibody had good safety.
[0049] Example 3. Emergency treatment test on mice
[0050] Ten healthy BALB / c mice were selected and divided into an experimental group and a control group, with 5 mice in each group. On the first day, the experimental group was injected with a virulent strain of bovine viral diarrhea virus at 100 LD50, 1 ml / mouse, and on the second day, yolk antibody was injected as emergency treatment, 1 ml / mouse; the control group was only injected with 100 LD 50 of the virulent strain of bovine viral diarrhea virus, 1 ml / mouse. The mice were continuously observed for 7 days, and the death situation of the mice was recorded. After 3 days of virus challenge, the mice in the control group began to die, and by the 5th day after virus challenge, all the mice in the control group had died; after 6 days of virus challenge, 1 mouse in the experimental group died; the remaining mice were all healthy and alive, and the emergency treatment protection rate of the mice was 80%, as shown in Table 2.
[0051] Table 2
[0052]
[0053] The above-mentioned embodiments only represent several implementation manners of the present invention, which are preferred implementation manners, but should not be construed as limiting the scope of the invention patent. For those of ordinary skill in the art, several improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention.
Claims
1. A method for preparing bovine viral diarrhea virus yolk antibody, characterized in that, The specific steps of the preparation method are as follows: Step 1: Prepare a vaccine against bovine viral diarrhea virus; Step 2: Immunize laying hens with the vaccine obtained in Step 1 to obtain egg yolks; Step 3: Extract antibodies from the egg yolks obtained in Step 2 to obtain yolk antibodies.
2. The preparation method according to claim 1, characterized in that, The vaccine obtained in Step 1 is an inactivated vaccine or a live attenuated vaccine.
3. The preparation method according to claim 2, wherein, The preparation method of the inactivated vaccine is as follows: bovine viral diarrhea virus is mixed with BEI inactivator to obtain the inactivated antigen, and the inactivated antigen is mixed with Freund's incomplete adjuvant in a volume ratio of 1:1 to obtain the inactivated vaccine.
4. The preparation method according to claim 1, wherein The method for immunizing laying hens in Step 2 is intramuscular injection in the legs of the laying hens.
5. The preparation method according to claim 1, characterized in that, The method for obtaining yolk antibodies in Step 3: (1) Dilute the separated egg yolks with 0.05 mol / L acetic acid-sodium acetate buffer solution at 10 times the volume of the egg yolks, stir until the egg yolks are completely dissolved, and let stand at 2-8 °C for precipitation for 12-24 hours; (2) Centrifuge the above 10-fold acid-diluted solution using a tubular centrifuge at 6000 r / min for 20 min, and take the supernatant after centrifugation; (3) Perform ammonium sulfate precipitation three times: add solid ammonium sulfate to the supernatant after centrifugation above to reach a saturation of 33%, perform the first precipitation and purification, stir until the ammonium sulfate is completely dissolved, let stand at room temperature for precipitation for 2-24 hours, centrifuge the ammonium sulfate precipitation solution using a tubular centrifuge at 6000 r / min for 20 min, take the precipitate after centrifugation, and after repeating three times, dissolve the obtained precipitate in physiological saline and stir until the precipitate is completely dissolved to obtain yolk antibodies.
6. The preparation method according to claim 1, characterized in that The obtained yolk antibodies are lyophilized and stored.
7. The preparation method according to claim 6, characterized in that, The method of lyophilization: Pre-freeze the lyophilizer to -70 °C, start the vacuum pump after 2-3 h, start the shelf temperature control to -22 °C, work for 18 h, set the shelf temperature to -8 °C, work for 2-3 h, and then close the refrigeration and the vacuum pump after capping.
8. The preparation method according to claim 1, wherein, The bovine viral diarrhea virus is the CP type of bovine viral diarrhea virus.
9. Yolk antibodies against bovine viral diarrhea virus obtained by the preparation method described in any one of claims 1-8.
10. Use of the yolk antibodies against bovine viral diarrhea virus described in claim 9 in the preparation of a kit for detecting bovine viral diarrhea virus or in the preparation of a drug for treating bovine viral diarrhea disease.