CN111548413B - Antibody for resisting novel coronavirus, preparation method and application thereof - Google Patents
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- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
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Abstract
The invention discloses an antibody for resisting novel coronavirus, which is a high-titer specific antibody extracted from chicken egg yolk, wherein the chicken egg yolk is mainly obtained from eggs laid by immunized hens, and the immunized hens are healthy hens injected with novel coronavirus vaccines. The invention also discloses a prevention and control auxiliary medicament for the novel coronavirus, which takes the anti-novel coronavirus antibody as a main component, the medicament exerts the characteristics of the anti-novel coronavirus antibody, can be specifically combined with the novel coronavirus in the oropharynx and the nasal cavity in a short time, has long retention time, no pain and irritation, good safety performance and no toxic or side effect, can be used for preventing and blocking the infection of the novel coronavirus, can be used for the auxiliary treatment of early infection, and can be used for the prevention and control auxiliary medicament for the group type coronavirus.
Description
Technical Field
The invention relates to the technical field of biological antibody immunity, in particular to an antibody for resisting novel coronavirus, and a preparation method and application thereof.
Background
Coronaviruses belong to the order of the nested viruses (Nidovirales), the family of Coronaviridae (Coronaviridae), and the genus coronaviruses (Coronavirus), are important pathogens of diseases of many domestic animals and pets, including humans, and cause a variety of acute and chronic diseases. The ninth international committee for taxonomic classification of virology reported the classification of the coronaviridae family into three genera, i.e., alpha, beta, and gamma. The novel coronavirus belongs to the subgenus Sarbecovirus of the beta-coronavirus.
The novel coronavirus pneumonia is a new infectious disease, and people have no immunity and are generally susceptible. Pneumonia caused by the novel coronavirus infection can occur in people with low immune function and normal immune function, and has a certain relation with the amount of the viruses. For people with poor immune function, such as the elderly, pregnant and lying-in women, or people with liver and kidney dysfunction and chronic diseases, the disease condition after infection is serious. According to the latest evidence analysis of new coronavirus transmission pathways by WHO, three transmission pathways including symptomatic, presymptomatic and asymptomatic are included.
Since the spread of the novel coronavirus all over the world, the number of cases is rising, and various countries adopt a series of measures such as isolation, personal protection, travel limitation, stopping unnecessary economic activities and the like to control the development of epidemic situations. At present, no specific medicine or vaccine exists for preventing, treating and controlling novel coronavirus, so that epidemic propagation cannot be really controlled, and the disease can be dealt with only by a symptomatic treatment and tight monitoring method. There is an urgent need to find drugs that can be used in therapeutic treatments to combat new coronaviruses; the new coronavirus pneumonia treatment is not specific at present, the disease treatment is limited to symptomatic treatment, the clinical result is unclear after the use of part of antiviral drugs, and the use of the drugs cannot realize specific killing of pathogens, so that the toxic and side effects are great.
Vaccines have been considered as the most effective means of controlling population immunity. At present, no novel coronavirus vaccine is on the market, the most effective mode for preventing and controlling the virus is isolation, and the measure brings great inconvenience and economic loss to production and life of people. Vaccine development is vigorously carried out all over the world, and 115 candidate vaccine research projects aiming at COVID-2019 are available all over the world. Vaccine technologies currently under investigation include: nucleic acids (DNA and RNA), virus-like particles, synthetic peptides, viral vectors (both replicative and non-replicative), recombinant proteins, attenuated viruses, and inactivated viruses. The fastest time to market for the vaccine is more than 18 months. The vaccine development and the marketing time are long, the large-area coverage inoculation is difficult in a short time, and the contradiction is generated between the urgent need of people for group protection.
The main methods for killing new coronaviruses at present are chemical killing methods and physical killing methods. The chemical killing method is to kill viruses by using chemical agents which can kill bacteria and viruses in a broad spectrum, such as 84 disinfectant, medical alcohol and the like, wherein the chemical agents generally have certain toxicity to human bodies, so the chemical agents are mostly used for killing the environment and cannot be directly applied to the surfaces of sensitive organs of human bodies, such as eyes, ears, mouths, noses and the like, and some chemical agents with strong chemical toxicity even cannot be excessively contacted with the skin of the human bodies, so the protective effect on the human bodies is very limited; the physical killing method needs to kill viruses by physical means such as high temperature, high pressure, ultraviolet rays and the like, has high dependence on external environment, cannot be directly applied to the protection of the surface of a human body, cannot specifically kill pathogens by both the chemical killing method and the physical killing method, has large side effect, and has very limited prevention and control auxiliary effect on group viruses. Therefore, there is an urgent need for effective means for preventing and emergently blocking novel coronavirus infection, which can be applied to the surface of the human body, without causing adverse effects on the human body.
Disclosure of Invention
The present invention aims to provide an antibody against a novel coronavirus.
Another object of the present invention is to provide a method for producing the antibody against the novel coronavirus.
The invention also aims to provide a medicine which is prepared by the antibody for resisting the novel coronavirus and is locally applied to nasal cavity and oropharyngeal mucosa, so that the medicine has no limitation and side effect of systemic administration, and has quick response, small dosage and good curative effect of local administration; the antibody is not easy to be absorbed by human body, does not destroy the ecological environment of normal flora in nasal cavity and oropharynx, does not cause the imbalance of local flora, and has no toxic and side effect; and the composition can not activate a complement system, is not easy to generate non-specific combination with rheumatoid factors, is not easy to fix staphylococcus aureus protein A, and is not easy to combine with protein G and human cell FC receptors, so that the composition is not easy to generate allergic reaction, has no limitation and side effect of systemic medication, and can be used for prevention and control adjuvant medication of novel coronavirus.
The invention is realized by the following technical scheme: an antibody against a novel coronavirus, wherein the antibody is a high-titer specific antibody extracted from a chicken yolk obtained mainly from an egg laid by an immunized hen, and the immunized hen is a healthy hen injected with the novel coronavirus vaccine.
Wherein, the novel coronavirus vaccine is mainly prepared from inactivated novel coronavirus or recombinant novel coronavirus antigen.
The spine protein of the new coronavirus invading cells can only be used for site-directed infection of the cells by using ACE 2. Birds are not currently extensively detected as possessing ACE2 protease. This locus is currently predominantly contained in mammalian cells. Thus, fish, birds and reptiles are very unlikely to be intermediate hosts for new coronary pneumonia compared to mammals. Therefore, the antibody against the new coronavirus is prepared by immunizing a hen and then extracting from the yolk of the hen, and has obvious contingency. The inactivated new type coronavirus (SARS-CoV-2) or recombined new type coronavirus (SARS-CoV-2) S antigen is used to prepare vaccine immune hen, and produce high titer specific antibody (specific IgY) in the egg yolk, and the specific egg yolk antibody is separated and purified by biological technique, and the enzyme linked immunosorbent assay is used to detect the titer of the finished egg yolk antibody, the titer is more than or equal to 1:5000, and the vaccine immune hen has unexpected technical effect.
The preparation process of the antibody against the novel coronavirus is as follows:
using recombined expressed new coronavirus (SARS-CoV-2) S antigen or virus inactivated new coronavirus (SARS-CoV-2) antigen, diluting, adding adjuvant and emulsifying to obtain various vaccines for immunization; injecting the vaccine into egg laying hen to obtain immunized egg, sterilizing, separating yolk from egg white, collecting yolk, extracting with water dilution method, centrifuging to obtain water soluble component, and extracting with ammonium sulfate to obtain novel coronavirus antibody crude extract; dissolving the crude extract with buffer solution, inactivating, and ultrafiltering for purification; preparing a novel coronavirus egg yolk antibody according to the antibody titer detection result, sterilizing, filtering, and adding a proper amount of stabilizer to prepare a novel coronavirus egg yolk antibody finished product.
The invention relates to a prevention and control adjuvant drug for novel coronavirus, which is a drug composition prepared by taking an antibody for resisting the novel coronavirus as a main component and adding auxiliary components acceptable in biomedicine. The pharmaceutical composition is in a liquid form, wherein the liquid form is an aqueous solution form, the prevention and control auxiliary medicine can directly act on the oropharynx and the nasal cavity, can be specifically combined with the novel coronavirus in the oropharynx and the nasal cavity in a short time, can stay on an action surface for a long time, effectively neutralizes the novel coronavirus, and has no pain or stimulation to the oropharynx and the nasal mucosa in the whole process of acting pathogens.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) the antibody for resisting the novel coronavirus can be combined with a specific pathogen of the novel coronavirus in a short time, the antibody stays on an acting surface for a long time, the novel coronavirus can be effectively neutralized, the whole process of acting the pathogen is painless and nonirritating to oropharyngeal and nasal mucosa, and the protection range is wider;
(2) the prevention and control auxiliary medicine taking the antibody resisting the novel coronavirus as the main component can be locally applied to mucous membranes of nasal cavities, oral cavities, pharynx, larynx and bronchus, has no limitation and side effect of whole-body medicine, has quick response, small dosage, good curative effect and difficult absorption by human bodies, does not destroy the ecological environment of normal flora in the nasal cavities, the oral cavities, the pharynx, the larynx and the bronchus, and can not cause local flora imbalance;
(3) the prevention and control adjuvant drug taking the antibody against the novel coronavirus as the main component is not easy to activate a complement system, is not easy to generate nonspecific combination with rheumatoid factors, is not easy to fix staphylococcus aureus protein A, is not easy to combine with protein G and human cell FC receptors, and is not easy to generate allergic reaction;
(4) the prevention and control adjuvant drug taking the antibody against the novel coronavirus as the main component does not generate acute toxic reaction, belongs to the actual non-toxic level, can independently play the roles of neutralizing the novel coronavirus and blocking the infection of the virus through nose and oropharynx, and does not need the coordination of complement and other immune cells;
(5) the finished product of the antibody for resisting the novel coronavirus, prepared by the method, has stable biological activity, can be stored at normal temperature for a long time, has the corresponding specific pathogen antibody titer of not less than 1:5000 per milliliter of the antibody for resisting the novel coronavirus, has more antigen binding sites, can effectively neutralize the novel coronavirus, and blocks the transmission of the virus.
Drawings
FIG. 1 is a SDS-PAGE analysis of purified antibodies (chicken egg yolk antibodies IgY) according to the invention;
FIG. 2 is a Western Blot analysis of purified antibodies (chicken egg yolk antibodies IgY) according to the invention;
FIG. 3 is a diagram of the antibody titer of the finished product detected by enzyme-linked immunosorbent assay in the invention.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto, and various substitutions and alterations can be made without departing from the technical idea of the present invention as described above, according to the common technical knowledge and the conventional means in the field.
The present invention will be described in further detail with reference to the following examples for the purpose of making clear the objects, process conditions and advantages of the present invention, which are given by way of illustration only and are not intended to be limiting of the present invention.
Example 1:
the invention particularly provides an antibody for resisting novel coronavirus, which is characterized in that the antibody is a high-titer specific antibody extracted from egg yolk, the egg yolk is mainly obtained from eggs laid by immunized hens, and the immunized hens are healthy hens injected with the novel coronavirus vaccine.
Wherein, the novel coronavirus vaccine is mainly prepared from a recombinant expression novel coronavirus (SARS-CoV-2) S antigen or a virus inactivated novel coronavirus (SARS-CoV-2) antigen.
The specific preparation method comprises the following steps:
1. preparing an antibody:
s1, vaccine preparation:
taking a recombinant expressed novel coronavirus (SARS-CoV-2) S antigen or a virus inactivated novel coronavirus (SARS-CoV-2) antigen, adding a Freund 'S complete adjuvant (priming) or Freund' S incomplete adjuvant (boosting immunity) into the antigen according to the proportion of 1:1, carrying out resonance on the antigen for 3-5 min at 20-25 Hz on a tissue cracking instrument to prepare a vaccine, and after the physical properties and the like are inspected to be qualified, providing the vaccine for immune healthy laying hens.
S2. immunization protocol
Selecting healthy hens of 90-120 days old, and carrying out subcutaneous and intramuscular multi-point injection on the prepared vaccine of 5 mu g/ml and 10 mu g/ml in a primary immunization manner, wherein the injection amount is 0.25-1.0 ml/feather; vaccine boosting immunization is carried out 2-8 weeks after priming, and 0.25-1.0 ml/feather subcutaneous injection and intramuscular injection are carried out at multiple points.
S3, egg collection: after the initial immunization, eggs are periodically taken to detect the increase condition of the egg yolk antibody titer, after the boosting immunization, the egg yolk antibody titer is more than or equal to 1:5000 (detection by an ELISA method), and the eggs are collected.
The antigen immunization dose is preferred according to the egg yolk antibody production of 2 different immunization dose groups.
2. Crude extraction and purification of novel coronavirus antibodies.
S1, cleaning the egg white collected after immunization, breaking the shell, separating egg white and yolk, removing the egg white, collecting the yolk and weighing the yolk.
S2, adding purified water to dilute the homogenate in the step S1 according to the proportion of 1: 6-9, adjusting the pH value to 4.8-6.0 by using 1mol/L hydrochloric acid solution, and standing for 1-24 hours at the temperature of 2-8 ℃.
S3, centrifuging the liquid prepared in the S2 step at 4000-20000 r/min, removing precipitates, and taking supernatant.
S4, adding 10-35% ammonium sulfate into the supernatant obtained in the S3 step for salting out, fully mixing uniformly, and precipitating overnight.
S5, centrifuging and desalting the precipitate prepared in the S4 step at 4000-20000 r/min, taking the precipitate to prepare a yolk antibody crude extract, and removing the ammonium sulfate supernatant.
S6, redissolving the precipitate prepared in the S5 step by using 1/150-1/300 volume weak acid buffer solution, adjusting the pH value to 4.8-6.0, fully dissolving for overnight at 2-8 ℃, and taking the supernatant.
S7, carrying out virus inactivation on the supernatant obtained in the step S6 at 54-60 ℃ for 8-20 hours.
S8, carrying out ultrafiltration on the inactivated supernatant obtained in the S7 step by using an ultrafiltration membrane with the cut-off relative molecular weight of 50-100 kD.
S9, the ultrafiltrate prepared in the step S8 is sterilized and filtered to obtain the IgY stock solution.
S10, adding a stabilizing agent into the IgY stock solution prepared in the step S9 according to the detected antibody titer to prepare a yolk antibody preparation.
3. The yolk antibody extraction effect is shown in fig. 1.
Wherein, 1 and 2 are IgY which is purified by an ultrafiltration tube after ammonium sulfate precipitation.
As shown in FIG. 1, the IgY can be further purified by ultrafiltration after extraction with ammonium sulfate to reduce the amount of impure proteins. The purified IgY has higher purity and less impurity band; SDS-PAGE non-reduction electrophoresis shows that the molecular weight of the extracted anti-neocoronavirus IgY is about 180kD, and the purity of the IgY antibody is over 80 percent by analyzing gel imaging software.
As can be seen from FIG. 2, the molecular weights of the heavy and light chains of IgY were 65kD and 25kD, respectively, according to Western blot analysis.
The finished product yolk antibody titer is detected by an enzyme-linked assay method, as shown in figure 3.
The titer of the anti-novel coronavirus IgY: 64000 is more than or equal to 1.
Example 2:
in this example, compared with the yolk antibody production of 2 groups with different antigen immunization doses, the antigen immunization dose is preferred, and the following table shows the following details:
TABLE 1 yolk antibody titer abrogation at different antigen immunization doses
(NT: no egg laying and no detection after hen immunization.)
As can be seen from the above table, the 5 μ g/feather antigen dose group is preferred, and the yolk antibody titer generated after 6, 8 and 10 weeks of priming is not lower than that generated after 10 μ g/feather antigen dose group.
Example 3:
in this example, to verify the virus-neutralizing property of the yolk antibody against the novel coronavirus, the following experiment was performed:
respectively and uniformly mixing 105-107 TCID50/ml of novel coronavirus solution and 1mg/ml, 2mg/ml and 3mg/ml of novel coronavirus egg yolk antibody samples according to the volume ratio of 1:1, neutralizing at 25-37 ℃ for 1-2 hours, adding into a 96-well plate containing cells, incubating in a carbon dioxide incubator at 33-35 ℃, and observing cytopathic effect day by day under a microscope.
Mixing the novel coronavirus egg yolk antibodies with the concentrations of 2mg/ml and 3mg/ml and the novel coronavirus liquid with the concentration of 105-107 TCID50/ml at the temperature of 25-37 ℃, wherein cells have no pathological changes. Positive control, novel coronary virus fluid, can cause cytopathic effects.
As is clear from the test results, the anti-novel coronavirus IgY prepared in the present invention has an effect of neutralizing the novel coronavirus.
Example 4:
in order to verify that the residence time of the specific egg yolk antibody in different parts of the living organism is long, a drug residence time small animal imaging experiment is carried out on the obtained egg yolk antibody, and the specific process is as follows:
the pH of the buffer system (sodium bicarbonate buffer system) was adjusted to 8.5 to 10.0, and a small amount of a fluorescent dye was added to the yolk antibody solution at 4 ℃ and mixed vertically overnight. Then, the unreacted fluorescent molecules were removed by ultrafiltration centrifugation (5000-.
The test method comprises the following steps: preparing the yolk antibody stock solution containing the fluorescent molecules into oral spray. Three experimental groups were divided: a group: after spraying, water is forbidden for 8 hours, and fasting is avoided; b group: after spraying, fasting is carried out for 8 hours, and water is not forbidden; and c, group: after spraying, water is forbidden and the food is fasted for 8 hours; skin spraying: and after removing part of fur on the back, spraying.
The fluorescence intensity of the spray part is acquired through small animal imaging, the residence time of the yolk antibody stock solution at the spray part is judged through the weakening condition of the fluorescence intensity, after oral cavity spraying, the fluorescence of the yolk antibody stock solutions of different groups is weakened along with the time, after oral cavity spraying, the residence time of different test groups in the oral cavity can reach 8.5 hours, and the effect of the fasting water-forbidden experimental group is good. The yolk antibody stock solution containing the fluorescent molecules can stay at the skin for as long as 70 hours.
Example 5:
in this embodiment, to verify the safety of the antibody against the novel coronavirus on the surface of the living biological eyeball, an acute eye stimulation test is performed on the obtained antibacterial chicken egg yolk complex antibody, and the specific process is as follows:
0.1ml of the test object is dripped into the conjunctival sac of the right eye of the rabbit, the rabbit is passively closed for 4s, 30s later, the rabbit is washed for 30s by enough physiological saline with higher flow rate and without causing the injury of the eyes of the animal, and the left eye is a blank control. Local responses were recorded at 1h, 24h, 48h, 72h, 7d, 14d and 21d after eye-dropping. If the eye irritation response is not completely recovered within 72h, or at 7d and 14d, the test can be terminated early. The test results are shown in Table II.
Results of rabbit eye irritation test using antibodies against novel Epimedium coronavirus
Mean score refers to the sum of the 24h, 48h, 72h scores divided by the number of observation time segments 3.
The test result shows that after the test object is dripped into the conjunctival sac of the right eye of the rabbit, the conjunctiva, the cornea and the iris of 3 eyes of the rabbit are not abnormal. The antibody for resisting the novel coronavirus has no irritation to the result of an acute eye irritation test of rabbits.
Example 6:
in this embodiment, in order to verify the safety of the anti-novel coronavirus antibody on the surface of a living organism, a complete skin irritation test is performed on the obtained antibacterial chicken egg yolk complex antibody for a plurality of times, specifically as follows:
24h before the test, the hair on the two sides of the spine of the back of the rabbit is cut off, and the hair removing range is about 3cm multiplied by 3cm respectively on the left side and the right side. In the test, 0.5ml of the stock solution of the test substance is applied to 2.5cm × 2.5cm of skin on one side, and the skin on the other side is used as a blank control. The test substance was removed 4h after application, applied once daily for 14 days. The result of the complete skin irritation test for rabbits for multiple times is observed after 24 hours of each smearing, and the result is nonirritant.
After the test substance is smeared on the skin of a rabbit, no erythema, edema and inflammatory reaction appear on the skin of the animal, and the average stimulation reaction score of each animal per day is 0, which is shown in the third table.
Results of repeated complete skin irritation test of antibodies against novel coronavirus from Epimetrix on rabbits
As can be seen from the above table, the sample has no irritation to the rabbit after multiple complete skin irritation tests.
While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.
Claims (7)
1. An antibody against a novel coronavirus, wherein the antibody is a specific antibody with high titer extracted from chicken egg yolk, the chicken egg yolk is mainly obtained from eggs laid by immunized hens, the immunized hens are healthy hens injected with the novel coronavirus vaccine, and the antibody is prepared by the following specific steps:
(1) preparing a novel coronavirus vaccine; in the step (1), the specific process for preparing the novel coronavirus vaccine comprises the following steps: taking a recombinant expressed novel coronavirus antigen or a virus inactivated novel coronavirus antigen, adding equivalent volume of Freund's complete adjuvant or Freund's incomplete adjuvant, carrying out emulsification on a tissue cracking instrument at 20-25 Hz for 3-5 min by resonance, and immunizing healthy laying hens after passing safety tests, aseptic tests and physical property tests;
(2) immunizing healthy laying hens with the prepared novel coronavirus vaccine; in the step (2), the specific process of immunizing the hen is as follows: taking healthy laying hens of the right age, and taking the prepared novel coronavirus vaccine to carry out intramuscular and subcutaneous multi-point injection at the position of deltoid muscle or pectoralis muscle of the wing base of the laying hens, wherein the dosage is 0.25-1.0 ml/feather; performing vaccine boosting immunization at intervals of 2-8 weeks after primary immunization, namely performing subcutaneous and intramuscular multipoint injection on the laying hens, wherein the dosage is 0.25-1.0 ml/feather;
(3) collecting the immune eggs laid by the immune hens;
(4) crude extraction and purification of novel coronavirus antibodies from the immunized eggs; in the step (4), the specific process of crude extraction and purification of the novel coronavirus antibody from the immune eggs comprises the following steps:
(4.1) cleaning the egg white collected after immunization, breaking the shell, separating egg white and yolk, removing the egg white, collecting the yolk and weighing the yolk;
(4.2) adding purified water into the yolk according to the ratio of 1: 6-9 to dilute and homogenize the yolk, adjusting the pH value to 4.8-6.0 by using 1mol/L hydrochloric acid solution, and standing for 1-24 hours at the temperature of 2-8 ℃;
(4.3) centrifuging the liquid after standing to remove precipitates and taking a supernatant;
(4.4) adding 10-35% ammonium sulfate into the obtained supernatant for salting out, fully and uniformly mixing, and then precipitating overnight;
(4.5) after the precipitation is finished, carrying out centrifugal desalination to obtain a crude yolk antibody extract, and removing an ammonium sulfate supernatant;
(4.6) re-dissolving the crude yolk antibody extract with a weakly acidic buffer solution with the volume of 1/150-1/300, adjusting the pH to 4.8-6.0, fully dissolving the crude yolk antibody extract, standing overnight at the temperature of 2-8 ℃, and then obtaining a supernatant;
(4.7) performing virus inactivation on the obtained supernatant;
(4.8) ultrafiltering the supernatant after virus inactivation by using an ultrafiltration membrane;
(4.9) taking supernatant of the solution after ultrafiltration to obtain antibody stock solution;
(5) detecting the titer of the antibody stock solution, and adding a stabilizing agent to obtain the antibody preparation for resisting the novel coronavirus.
2. The antibody against a novel coronavirus of claim 1, wherein the novel coronavirus vaccine is prepared mainly from an inactivated novel coronavirus or a recombinant novel coronavirus antigen.
3. The method of producing an antibody against a novel coronavirus according to claim 1 or 2, comprising the steps of:
(1) preparing a novel coronavirus vaccine; in the step (1), the specific process for preparing the novel coronavirus vaccine comprises the following steps: taking a recombinant expressed novel coronavirus antigen or a virus inactivated novel coronavirus antigen, adding equivalent volume of Freund's complete adjuvant or Freund's incomplete adjuvant, carrying out emulsification on a tissue cracking instrument at 20-25 Hz for 3-5 min by resonance, and immunizing healthy laying hens after passing safety tests, aseptic tests and physical property tests;
(2) immunizing healthy laying hens with the prepared novel coronavirus vaccine; in the step (2), the specific process of immunizing the hen is as follows: taking healthy laying hens of the right age, and taking the prepared novel coronavirus vaccine to carry out intramuscular and subcutaneous multi-point injection at the position of deltoid muscle or pectoralis muscle of the wing base of the laying hens, wherein the dosage is 0.25-1.0 ml/feather; performing vaccine boosting immunization at intervals of 2-8 weeks after primary immunization, namely performing subcutaneous and intramuscular multipoint injection on the laying hens, wherein the dosage is 0.25-1.0 ml/feather;
(3) collecting the immune eggs laid by the immune hens;
(4) crude extraction and purification of novel coronavirus antibodies from the immunized eggs; in the step (4), the specific process of crude extraction and purification of the novel coronavirus antibody from the immune eggs comprises the following steps:
(4.1) cleaning the egg white collected after immunization, breaking the shell, separating egg white and yolk, removing the egg white, collecting the yolk and weighing the yolk;
(4.2) adding purified water into the yolk according to the ratio of 1: 6-9 to dilute and homogenize the yolk, adjusting the pH value to 4.8-6.0 by using 1mol/L hydrochloric acid solution, and standing for 1-24 hours at the temperature of 2-8 ℃;
(4.3) centrifuging the liquid after standing to remove precipitates and taking a supernatant;
(4.4) adding 10-35% ammonium sulfate into the obtained supernatant for salting out, fully and uniformly mixing, and then precipitating overnight;
(4.5) after the precipitation is finished, carrying out centrifugal desalination to obtain a crude yolk antibody extract, and removing an ammonium sulfate supernatant;
(4.6) re-dissolving the crude yolk antibody extract with a weakly acidic buffer solution with the volume of 1/150-1/300, adjusting the pH to 4.8-6.0, fully dissolving the crude yolk antibody extract, standing overnight at the temperature of 2-8 ℃, and then obtaining a supernatant;
(4.7) performing virus inactivation on the obtained supernatant;
(4.8) ultrafiltering the supernatant after virus inactivation by using an ultrafiltration membrane;
(4.9) taking supernatant of the solution after ultrafiltration to obtain antibody stock solution;
(5) detecting the titer of the antibody stock solution, and adding a stabilizing agent to obtain the antibody preparation for resisting the novel coronavirus.
4. The method of claim 3, wherein the antibody against the novel coronavirus is prepared by: the centrifugal parameter of the preparation method in the centrifugal process is 4000-20000 r/min.
5. The method of claim 3, wherein the antibody against the novel coronavirus is prepared by: and (4) the interception relative molecular weight of the ultrafiltration membrane used in the step (4.6) is 50-100 kD.
6. A preventive/control adjuvant drug for a novel coronavirus, which is a pharmaceutical composition comprising the antibody of claim 1 or 2 as a main ingredient and a biologically pharmaceutically acceptable adjuvant ingredient.
7. The preventive and controlled adjuvant for a novel coronavirus according to claim 6, wherein the pharmaceutical composition is in the form of a liquid, wherein the liquid is in the form of an aqueous solution, and the preventive and controlled adjuvant is capable of directly acting on the oropharynx and nasal cavity of a living body to neutralize the novel coronavirus.
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| WO2021243417A1 (en) * | 2020-06-05 | 2021-12-09 | Centenary Institute Of Cancer Medicine And Cell Biology | Immunogenic formulations |
| CN112062838B (en) * | 2020-08-25 | 2021-03-23 | 南京医科大学 | Neutralizing single-domain antibody for resisting novel coronavirus SARS-Cov-2 and application thereof |
| CN112175074A (en) * | 2020-10-09 | 2021-01-05 | 梁永明 | Industrial application of gene recombination-based anti-Covid-19 and other coronavirus IgY antibodies and other small molecule antibodies |
| CN114470198B (en) * | 2020-10-26 | 2023-05-05 | 上海博满生物科技有限公司 | IgY spray of SARS-CoV-2 egg yolk neutralization antibody for preventing and treating new coronal pneumonia |
| CN112279912A (en) * | 2020-11-10 | 2021-01-29 | 桂林医学院 | Novel coronavirus poultry egg antibody SARS-CoV-2-IgY preparation and its preparation method and application |
| CN113087791B (en) * | 2021-02-05 | 2024-04-26 | 深圳市雅臣智能生物工程有限公司 | Broad-spectrum anti-variant coronavirus IgY and composite antibody, preparation method and combined preparation |
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Inventor after: Lin Xiaojun Inventor after: Ye Lin Inventor after: Yang Penghui Inventor after: Wu Jie Inventor after: Liao Ming Inventor after: Wang Huan Inventor after: Zhu Ling Inventor after: Lei Yu Inventor after: Hu Fangyan Inventor after: Ye Xiangyang Inventor before: Lin Xiaojun Inventor before: Ye Lin Inventor before: Yang Penghui Inventor before: Wu Jie Inventor before: Liao Ming Inventor before: Wang Huan Inventor before: Zhu Ling Inventor before: Lei Yu Inventor before: Hu Fangyan Inventor before: Ye Xiangyang |
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| GR01 | Patent grant | ||
| GR01 | Patent grant |