WO2021155639A1 - Igy for resisting sars-cov-2 and other coronaviruses, small molecule antibody thereof and use thereof - Google Patents
Igy for resisting sars-cov-2 and other coronaviruses, small molecule antibody thereof and use thereof Download PDFInfo
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- WO2021155639A1 WO2021155639A1 PCT/CN2020/084751 CN2020084751W WO2021155639A1 WO 2021155639 A1 WO2021155639 A1 WO 2021155639A1 CN 2020084751 W CN2020084751 W CN 2020084751W WO 2021155639 A1 WO2021155639 A1 WO 2021155639A1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1002—Coronaviridae
- C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
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- A—HUMAN NECESSITIES
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- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- A—HUMAN NECESSITIES
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- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/50—Isolated enzymes; Isolated proteins
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- A—HUMAN NECESSITIES
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- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
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- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
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- A—HUMAN NECESSITIES
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- A61L9/00—Disinfection, sterilisation or deodorisation of air
- A61L9/14—Disinfection, sterilisation or deodorisation of air using sprayed or atomised substances including air-liquid contact processes
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- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/10—Washing or bathing preparations
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/02—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from eggs
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
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- A—HUMAN NECESSITIES
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- A61L2101/00—Chemical composition of materials used in disinfecting, sterilising or deodorising
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- A—HUMAN NECESSITIES
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- A61L2209/00—Aspects relating to disinfection, sterilisation or deodorisation of air
- A61L2209/20—Method-related aspects
- A61L2209/21—Use of chemical compounds for treating air or the like
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Definitions
- the invention relates to the technical field of biological medicines, in particular to an anti-SARS-CoV-2 and other coronavirus IgY and small molecule antibodies as well as preparation methods and applications.
- coronavirus The coronavirus that infects humans was first isolated by scientists in the 1960s. It was named "coronavirus" because the corona structure on the surface of the virus can be observed under an electron microscope.
- Coronavirus is not only one of the main pathogens of the common cold, but also the culprit of several severe epidemics, including the severe acute respiratory syndrome coronavirus that raged in 2003 and the Middle East respiratory syndrome that affected Saudi Arabia, South Korea and other places in the past few years. Disease coronavirus.
- coronavirus People infected with the coronavirus will have fever, cough, shortness of breath, and difficulty breathing, which can lead to viremia and systemic diseases dominated by lung lesions, pneumonia, acute respiratory syndrome, and so on. This virus can also trigger a "cytokine storm” (cytokine storm), attacking normal human organs, causing organs such as lungs and kidneys to fail and even cause death.
- cytokine storm cytokine storm
- the purpose of the present invention is to provide an anti-SARS-CoV-2 and other coronavirus IgY and its small molecule antibodies as well as preparation methods and applications, so as to solve the problem that there is no effective prevention and treatment of SARS-CoV-2 and other coronaviruses in the prior art.
- Virus infection disease drug problem is to provide an anti-SARS-CoV-2 and other coronavirus IgY and its small molecule antibodies as well as preparation methods and applications, so as to solve the problem that there is no effective prevention and treatment of SARS-CoV-2 and other coronaviruses in the prior art.
- an anti-SARS-CoV-2 and other coronavirus specific IgY, an anti-SARS-CoV-2 and other coronavirus small molecule antibodies, the anti-SARS-CoV -2 and other coronavirus-specific IgY and its small molecule antibodies are prepared by the following methods:
- the present invention adopts the following methods to make antigens for immunization:
- Virus inactivation ⁇ -propiolactone, etc. can be used as an inactivator. Under the condition of constantly stirring the virus liquid, slowly add the inactivator, act at 4-10°C, and then bath at about 37°C. Vero cells were passed continuously for 3 generations to detect the inactivation effect, and all operations were performed in the biological safety cabinet in the P3 laboratory.
- the present invention takes the SARS-CoV-2 coronavirus recombinant antigen component as an example to illustrate.
- the preparation of other coronavirus genetic engineering recombinant antigen components can be carried out in the same way with reference to the following methods. The specific operation process will not be repeated here.
- S protein of SARS-CoV-2 and other coronaviruses plays an important role in the process of virus binding to host cell surface receptors and mediating membrane fusion into cells. It is also the main cause of SARS-CoV-2 and other coronaviruses.
- Antigen protein The S protein fixes the entire S protein on the virus coat membrane through the membrane penetrating part of S2, and fuses with the host cell membrane, while S1 is related to the recognition and binding of the virus and the receptor; blocking the binding site of the S protein can prevent the virus from infecting cells; therefore In the present invention, S protein is selected as the main recombinant protein antigen component.
- the C-terminal repeat fragment of the S2 protein of SARS-CoV-2 or other coronaviruses binds to the cell membrane receptor ACE2 and fuses the virus envelope with the cell membrane, thereby allowing the virus to enter the cell for infection; therefore, the S2 protein was selected As a recombinant protein antigen component.
- the receptor binding domain (RBD) of SARS-CoV-2 or other coronaviruses contains the main neutralizing epitope of the S protein, which mediates the interaction between the virus and the target cell, and can affect the binding of the virus to the target cell. It plays a very important role in spreading across species. Therefore, the present invention selects receptor binding domain (RBD) as an important recombinant protein antigen component.
- the N protein of SARS-CoV-2 and other coronaviruses is an important structural protein, located in the core part of the virus particle. It combines with viral RNA to form a complex, which recognizes the characteristic sequence of viral genome RNA and interacts with other structural proteins. It is of great significance and plays an important role in the process of virus assembly; therefore, the present invention selects N protein as an important recombinant protein antigen component.
- the Escherichia coli expressing SARS-CoV-2 or other coronavirus GST-N protein was disrupted by ultrasound, centrifuged to take the supernatant, purified with GST fusion protein purification kit and identified.
- the empty vector expression supernatant is centrifuged and then purified; then, it is coupled with KLH and BSA to obtain purified SARS-CoV-2 or other coronavirus N protein antigen components.
- D. Transform the recombinant plasmid into Escherichia coli BL21 (DE3), and screen the engineered bacteria that efficiently express SARS-CoV-2 or the fusion protein of S protein fragment and N protein fragment of other coronaviruses.
- SARS-CoV-2 or other coronavirus S protein full sequence antigen SARS-CoV-2 or other coronavirus S protein full sequence antigen
- the agent is placed in a high-speed homogenizer and homogenized at a high speed of 30,000 rpm to form a water-in-oil emulsion to obtain SARS-CoV-2 or other coronavirus fusion protein antigens.
- any egg-laying poultry including chickens, ostriches, ducks, and geese
- any egg-laying poultry can be used for immunization to prepare immune eggs. Due to the limitation of space, only hens are used as an example. Other poultry can be performed in the same manner as described below.
- the SARS-CoV-2 or other coronavirus inactivated viral antigens prepared by the previous method and five genetic recombinant SARS-CoV-2 or other coronavirus protein antigens were used to immunize laying hens, and injected once every two weeks for a total of injections. 2-5 times, at least 12 days after the last injection, the immunized eggs from laying hens were collected and coded and labeled to obtain six immune eggs against SARS-CoV-2 or other coronavirus IgY.
- the crude extract of anti-SARS-CoV-2 or other coronavirus IgY is prepared by pure water extraction method, chloroform extraction method, cold ethanol precipitation method or ammonium sulfate precipitation method.
- the present invention is illustrated by taking the pure water extraction method as an example, other preparation methods refer to conventional methods, and the specific operation process is not repeated here.
- any combination of 2-6 of the above six anti-SARS-CoV-2 or other coronavirus IgY can be mixed in a certain proportion to form an anti-SARS-CoV-2 or other coronavirus compound IgY.
- Anti-SARS-CoV-2 or other coronavirus antigen IgY (anti-SARS-CoV-2 or other coronavirus S-IgY) + (anti-SARS-CoV-2 or other coronavirus RBD-IgY)
- the above anti-SARS-CoV-2 or other coronavirus IgY can be used in any combination as needed.
- the anti-SARS-CoV-2 or other coronavirus pure IgY solution can also be freeze-dried or medium-low temperature spray drying or fluidized bed drying and other drying methods that do not affect the activity of the antibody to prepare anti-SARS-CoV-2 or other coronavirus pure IgY dry powder.
- any combination of 2-6 of the above six anti-SARS-CoV-2 or other coronavirus IgY can be mixed in a certain proportion to form an anti-SARS-CoV-2 or other coronavirus complex small molecule antibody Fab.
- Anti-SARS-CoV-2 or other coronavirus antigen-small molecule antibody Fab + (anti-SARS-CoV-2 or other coronavirus RBD-small molecule antibody Fab)
- Anti-SARS-CoV-2 or other coronavirus virus antigen small molecule antibody Fab + (anti-SARS-CoV-2 or other coronavirus S-small molecule antibody Fab) + (anti-SARS-CoV-2 or other coronavirus Virus RBD-Small Molecule Antibody Fab)
- Anti-SARS-CoV-2 or other coronavirus virus S-small molecule antibody Fab (anti-SARS-CoV-2 or other coronavirus RBD-small molecule antibody Fab) + (anti-SARS-CoV-2 or other Coronavirus N-small molecule antibody Fab)
- the above anti-SARS-CoV-2 or other coronavirus small molecule antibody Fab can be used in any combination as needed.
- the prepared one or more composite anti-SARS-CoV-2 or other coronavirus small molecule antibody Fabs are long-actingly modified with polyethylene glycol, dextran or polyamino acid to obtain long-acting anti-SARS- CoV-2 or other coronavirus small molecule antibody Fab.
- the present invention takes the method of using polyethylene glycol for long-acting modification as an example to illustrate as follows, and other long-acting modification methods can refer to the following methods.
- One or more composite anti-SARS-CoV-2 or other coronavirus small molecule antibody Fab solutions are freeze-dried, spray-dried at medium and low temperature, fluidized bed drying, and other drying methods that do not affect antibody activity to prepare one or more A composite anti-SARS-CoV-2 or other coronavirus small molecule antibody Fab dry powder.
- the present invention also provides that the above-mentioned anti-SARS-CoV-2 or other coronavirus IgY and its small molecule antibodies are used in the preparation of drugs, disinfection products, health care products or medical devices for the prevention and treatment of SARS-CoV-2 or other coronavirus infections. Applications.
- the present invention also provides a composition comprising the above-mentioned anti-SARS-CoV-2 or other coronavirus IgY and small molecule antibodies thereof and at least one other pharmaceutically acceptable component.
- the anti-SARS-CoV-2 or other coronavirus IgY and its small molecule antibodies are added with auxiliary materials or base materials or chemical medicines and traditional Chinese medicines to make aerosols or sprays for atomized inhalation therapy , Oral sprays, nasal sprays, nose drops, eye drops, air disinfectants, air fresheners, disinfectants, hand sanitizers, powders, tablets, lozenges, oral liquids, oral agents, capsules At least one.
- the present invention also provides the application of the above-mentioned composition in the preparation of medicines, disinfection products, health products or medical devices for preventing and treating SARS-CoV-2 or other coronavirus infections.
- the prepared anti-SARS-CoV-2 or other coronavirus IgY and its small molecule antibodies can be made into various stable preparations.
- the formulations include but are not limited to these formulations:
- the formulation further includes one or more of excipients, fillers, solvents, co-solvents, surfactants and capsule auxiliary materials.
- the preparation is an atomizer, disinfectant, disinfectant, air freshener, hand sanitizer, etc. for atomized inhalation therapy.
- the preparation is a tablet, spray, powder, liquid or capsule.
- the anti-SARS-CoV-2 or other coronavirus IgY and its small molecule antibodies and compositions and preparation methods and applications of the present invention have the following beneficial effects: the anti-SARS-CoV-2 or other coronavirus IgY of the present invention and its Small molecule antibodies can not only directly inhibit SARS-CoV-2 and other coronaviruses, but also specifically bind to the key protein S protein of SARS-CoV-2 and other coronaviruses and its RBD domains and block them to make them Loss of the function of mediating virus infecting cells and spreading; thus, preventing SARS-CoV-2 and other coronaviruses from infecting respiratory epithelial cells.
- these antibodies can also neutralize the virus excreted by infected cells, eliminate secondary infections and block the spread of disease.
- These unique advantages are particularly useful for the prevention of SARS-CoV-2 and other coronavirus infections and the prevention and control of the spread of epidemics.
- Significant significance Using the anti-SARS-CoV-2 or other coronavirus IgY and its small molecule antibodies and compositions of the present invention, including the series of products made with these antibodies and compositions, can achieve the purpose of some loss, precise prevention and control, and no The toxic and side effects of chemical antiviral drugs and chemical inhibitors and the problem of inducing drug resistance.
- Antibody antigen Antibody binding titer Anti-SARS-CoV-2 virus antigen IgY SARS-CoV-2 virus antigen 1:32768
- the concentration of the anti-SARS-CoV-2 virus antigen IgY antibody solution in the test sample is 1 mg/mL.
- the prepared anti-SARS-CoV-2 virus antigen IgY has a high antibody binding titer to the SARS-CoV-2 virus.
- SARS-CoV-2-S protein full sequence peptide SARS-CoV-2-S1 protein immunodominance determining region polypeptide, SARS-CoV-2-S2 protein immunodominance determining region polypeptide, and SARS-CoV-2 receptor binding Domain (RBD) fragment recombinant polypeptide protein, SARS-CoV-2-N protein antigen, SARS-CoV-2-S protein fragment and N protein fragment fusion protein antigen as the detection antigen, using the "ELISA" method (enzyme-linked immunoassay ) Test the antibody titer of the prepared anti-SARS-CoV-2 coronavirus IgY and its small molecule antibodies. The results are shown in the following table:
- the concentration of the anti-SARS-CoV-2-IgY series antibody solution in the test sample is 1mg/mL.
- SARS-CoV-2-S protein full sequence peptide SARS-CoV-2-S1 protein immunodominance determining region polypeptide, SARS-CoV-2-S2 protein immunodominance determining region polypeptide, and SARS-CoV-2 receptor binding Domain (RBD) fragment recombinant polypeptide protein, SARS-CoV-2-N protein antigen, SARS-CoV-2-S protein fragment and N protein fragment fusion protein antigen as the detection antigen, using the "ELISA" method (enzyme-linked immunoassay ) Detect the antibody titer of the prepared anti-SARS-CoV-2 small molecule antibody Fab, and the results are shown in the following table:
- the concentration of the anti-SARS-CoV-2 small molecule antibody Fab antibody solution in the test sample is 1 mg/mL.
- the prepared anti-SARS-CoV-2 small molecule antibody Fab has a high antibody binding titer to the corresponding SARS-CoV-2 protein antigen.
- Example 1 The neutralization test method is used to determine the potency of anti-SARS-CoV-2 virus antigen IgY in neutralizing SARS-CoV-2 virus
- Anti-SARS-CoV-2 virus antigen IgY protein content 40mg/ml 2 specimens are divided into No. 1 and No. 2
- Negative antibody (IgY antibody of unimmunized eggs): protein content 60mg/ml
- Negative control 2 sets of baby umbilical cord serum, specimen No. 5 and No. 6
- SARS-CoV-2 virus isolate I Isolation and identification from throat test specimens of patients infected with SARS-CoV-2 virus.
- SARS-CoV-2 virus isolate II Isolation and identification from the serum samples of patients infected with SARS-CoV-2 virus.
- Cells Passage cells of African green monkey kidney.
- the neutralization test method was used to determine the anti-SARS-CoV-2 virus antigen IgY (using two isolated SARS-CoV-2 virus strains); the Reed-Muench method was used to calculate the 50% antibody And the end.
- Concentration 4 wells at the same time set up anti-SARS-CoV-2 virus antigen IgY antibody, patient recovery period serum and infant umbilical cord serum control, virus control, normal cell control. Incubate in a 37°C 5% CO 2 incubator for 5 days. Observe the virus (CPE) under an inverted microscope every day. The morphological change of 25% or less is "+”, and the morphological change of 26%-50% is “++", 51% ⁇ 75% of the morphological change is "+++”, 76%-100% of the morphological change is "++++", and the test is terminated when the virus control appears "+++++++”.
- the Reed-Muench method was used to calculate the 50% antibody neutralization endpoint.
- Anti-SARS-CoV-2 virus antigen IgY antibody No. 1 specimen: 1:512 antibody can protect 50% of cells from cytopathic.
- Anti-SARS-CoV-2 virus antigen IgY antibody No. 2 specimen 1:256 antibody can protect 50% of cells from cytopathic.
- Negative antibody Can not neutralize SARS-CoV-2 virus.
- Serum No. 3 specimen of convalescent patients infected with SARS-CoV-2: 1:512 serum can protect 50% of cells from cytopathic changes.
- Example 2 Western blot detection was used to determine the binding of the anti-SARS-CoV-2 virus antigen IgY antibody to the SARS-CoV-2 virus.
- Sample processing Combine SARS-CoV-2 virus N antigen (200gg/ml), inactivated SARS-CoV-2 virus vero cells (CPE+++) and 5X loading buffer (electrophoresis loading buffer: 25mmol Tris-CL (pH6.8), 50mmol DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerol) mix;
- Sample processing 5 ⁇ l SARS-CoV-2 virus N antigen (200 ⁇ g/ml), inactivated SARS-CoV-2 virus vero cell loading buffer (electrophoresis loading buffer: 25mmol Tris-CL (pH6.8) ), 50mmol DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerin) mixed;
- Example 3 Production of anti-SARS-CoV-2-S protein full sequence polypeptide-small molecule antibody Fab nebulizer for various nebulization inhalers and public environment air disinfection machines, air fresheners and household air disinfectors, Air fresheners and mobile spray disinfection equipment are used as medicinal atomized liquids.
- the solution C is divided into cleaned and disinfected spray bottles, and the label is attached to the factory.
- Example 4 Production of anti-SARS-CoV-2-S and N fusion protein-small molecule antibody Fab nasal spray or nose drops or eye drops
- solution A While stirring, add the anti-SARS-CoV-2-S and N fusion protein-small molecule antibody Fab solution or dry powder to solution A, stir at low speed for 60 minutes, until completely mixed uniformly, to obtain solution B;
- the solution C is dispensed into the cleaned and disinfected nasal drops or eye drops and nasal spray bottles, and the labels are attached to the factory.
- Example 5 Production of anti-SARS-CoV-2-S and N fusion protein-small molecule antibody Fab mouth spray or mouthwash
- solution A While stirring, add the anti-SARS-CoV-2-S protein full-sequence peptide-small molecule antibody Fab solution or dry powder to solution A, stir at low speed for 60 minutes, until completely mixed uniformly, to obtain solution B;
- the solution C is divided into a cleaned and disinfected oral spray can or mouthwash bottle, and the label is attached to the factory.
- Sorbitol is passed through a 60-mesh sieve twice for use
- Carboxymethyl cellulose is dispersed in 30% ethanol to make a 1% ethanol solution
- each tablet is 600mg
- Example 8 Production of anti-SARS-CoV-2-S protein complex antigen-IgY powder, and the non-atomized device for powder supply is used as a medicinal powder.
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Abstract
Provided are an IgY for resisting SARS-CoV-2 and other coronaviruses, a small molecule antibody thereof, a preparation method therefor and the use thereof. Also provided are an atomizing agent, spray, oral spray, nasal spray, nasal drops, eye drops, air disinfectant, hand sanitizer, powder, tablet, mouth lozenge, oral liquid and capsule for atomizing inhalation therapy and made from the IgY for resisting SARS-CoV-2 and other coronaviruses and the small molecule antibody thereof, which are used in drugs, disinfection products, health-care products and medical devices for preventing and treating SARS-CoV-2 infections and other coronavirus infections.
Description
本发明涉及生物药品技术领域，具体涉及一种抗SARS-CoV-2和其它冠状病毒IgY及其小分子抗体以及制备方法和应用。The invention relates to the technical field of biological medicines, in particular to an anti-SARS-CoV-2 and other coronavirus IgY and small molecule antibodies as well as preparation methods and applications.
感染人类的冠状病毒在20世纪60年代首次被科学家分离出来，因在电子显微镜下可观察到病毒外表的冠状构造而得名“冠状病毒”。The coronavirus that infects humans was first isolated by scientists in the 1960s. It was named "coronavirus" because the corona structure on the surface of the virus can be observed under an electron microscope.
冠状病毒不仅是普通感冒的主要病原体之一，更是几次严重疫情的罪魁祸首，包括2003年肆虐的严重急性呼吸综合征冠状病毒，以及前几年影响沙特***、韩国等地的中东呼吸综合症冠状病毒。Coronavirus is not only one of the main pathogens of the common cold, but also the culprit of several severe epidemics, including the severe acute respiratory syndrome coronavirus that raged in 2003 and the Middle East respiratory syndrome that affected Saudi Arabia, South Korea and other places in the past few years. Disease coronavirus.
人感染了冠状病毒后会出现发热、咳嗽、气促和呼吸困难，进而引起病毒血症以及肺部病变为主的全身性疾病，出现肺炎、急性呼吸综合症等。这种病毒还会触发“细胞因子风暴”(cytokine storm)，攻击人的正常器官，使肺、肾等器官衰竭甚至造成死亡。People infected with the coronavirus will have fever, cough, shortness of breath, and difficulty breathing, which can lead to viremia and systemic diseases dominated by lung lesions, pneumonia, acute respiratory syndrome, and so on. This virus can also trigger a "cytokine storm" (cytokine storm), attacking normal human organs, causing organs such as lungs and kidneys to fail and even cause death.
世卫组织指出，对于新型冠状病毒没有疫苗，无法提前预防，也没有有效的治疗药物。The WHO pointed out that there is no vaccine for the new coronavirus, it cannot be prevented in advance, and there is no effective treatment.
世卫组织警示，一些已知的冠状病毒在动物中传播，但尚未感染人类；随着全球监测工作的改善，可能会发现更多冠状病毒，人类与冠状病毒之间的斗争任重道远。The WHO warned that some known coronaviruses are spreading in animals but have not yet infected humans. With the improvement of global surveillance, more coronaviruses may be discovered, and the struggle between humans and coronaviruses has a long way to go.
因此，研究开发一种可有效预防和治疗SARS-CoV-2及其它冠状病毒感染疾病的新药，是关系到人类健康和生命的大事，也是摆在国际医学科学界面前的一项重要课题。Therefore, the research and development of a new drug that can effectively prevent and treat SARS-CoV-2 and other coronavirus infections is a major event related to human health and life, and it is also an important topic at the interface of international medical science.
发明内容Summary of the invention
本发明的目的是提供一种抗SARS-CoV-2和其它冠状病毒IgY及其小分子抗体以及制备方法和应用，解决现有技术中尚未有可以有效预防和治疗SARS-CoV-2及其它冠状病毒感染疾病药物的问题。The purpose of the present invention is to provide an anti-SARS-CoV-2 and other coronavirus IgY and its small molecule antibodies as well as preparation methods and applications, so as to solve the problem that there is no effective prevention and treatment of SARS-CoV-2 and other coronaviruses in the prior art. Virus infection disease drug problem.
本发明解决技术问题所采用的技术方案是：一种抗SARS-CoV-2和其它冠状病毒特异性IgY、一种抗SARS-CoV-2和其它冠状病毒小分子抗体，所述抗SARS-CoV-2和其它冠状病毒特异性IgY及其小分子抗体由如下方法制备得到：The technical solution adopted by the present invention to solve the technical problem is: an anti-SARS-CoV-2 and other coronavirus specific IgY, an anti-SARS-CoV-2 and other coronavirus small molecule antibodies, the anti-SARS-CoV -2 and other coronavirus-specific IgY and its small molecule antibodies are prepared by the following methods:
一、制备抗原：1. Preparation of antigen:
本发明采用以下方法制作免疫用抗原：The present invention adopts the following methods to make antigens for immunization:
(一)制作抗原成份(1) Making antigen components
1.灭活SARS-CoV-2或其它冠状病毒抗原成份1. Inactivate SARS-CoV-2 or other coronavirus antigen components
(1)使用SARS-CoV-2或其它冠状病毒感染Vero、Vero-E6和2BS细胞株。(1) Use SARS-CoV-2 or other coronaviruses to infect Vero, Vero-E6 and 2BS cell lines.
(2)优选最佳病毒感染量和最佳培养条件,培养SARS-CoV-2或其它冠状病毒，将收获的病毒置于-20℃冰箱中反复冻融裂解细胞3次备用。(2) Optimizing the optimal virus infection and optimal culture conditions, culturing SARS-CoV-2 or other coronaviruses, and placing the harvested virus in a refrigerator at -20°C for repeated freezing and thawing to lyse the cells 3 times for use.
(3)灭活病毒：可选用β-丙内酯等作灭活剂，在不断搅拌病毒液的条件下,缓慢加入灭活剂，4-10℃作用，再37℃左右水浴。用Vero细胞连续传3代检测灭活效果,所有操作都在P3实验室内的生物安全柜进行。(3) Virus inactivation: β-propiolactone, etc. can be used as an inactivator. Under the condition of constantly stirring the virus liquid, slowly add the inactivator, act at 4-10°C, and then bath at about 37°C. Vero cells were passed continuously for 3 generations to detect the inactivation effect, and all operations were performed in the biological safety cabinet in the P3 laboratory.
2.基因工程重组抗原成份2. Genetically engineered recombinant antigen components
本发明以SARS-CoV-2冠状病毒重组抗原成份为例来说明，其它冠状病毒基因工程重组抗原成份的制作可参照以下方法同样进行，具体操作过程，这里就不赘述。The present invention takes the SARS-CoV-2 coronavirus recombinant antigen component as an example to illustrate. The preparation of other coronavirus genetic engineering recombinant antigen components can be carried out in the same way with reference to the following methods. The specific operation process will not be repeated here.
(1)基因重组SARS-CoV-2或其它冠状病毒的S蛋白抗原成份(1) Genetically recombined SARS-CoV-2 or S protein antigen components of other coronaviruses
研究揭示，SARS-CoV-2及其它冠状病毒的S蛋白在病毒与宿主细胞表面受体结合及介导膜融合进入细胞的过程中起重要作用，也是SARS-CoV-2及其它冠状病毒主要的抗原蛋白。S蛋白通过S2的穿膜部分把整个S蛋白固定在病毒外壳膜，与宿主细胞膜融合，而S1则与病毒和受体的识别结合有关；封闭S蛋白的结合位点可阻止病毒感染细胞；因此，本发明选择S蛋白作为主要的重组蛋白抗原成份。Studies have revealed that the S protein of SARS-CoV-2 and other coronaviruses plays an important role in the process of virus binding to host cell surface receptors and mediating membrane fusion into cells. It is also the main cause of SARS-CoV-2 and other coronaviruses. Antigen protein. The S protein fixes the entire S protein on the virus coat membrane through the membrane penetrating part of S2, and fuses with the host cell membrane, while S1 is related to the recognition and binding of the virus and the receptor; blocking the binding site of the S protein can prevent the virus from infecting cells; therefore In the present invention, S protein is selected as the main recombinant protein antigen component.
以SARS-CoV-2冠状病毒的S蛋白重组抗原成份为例来说明，具体操作如下：Take the S protein recombinant antigen component of SARS-CoV-2 coronavirus as an example to illustrate, the specific operations are as follows:
A.SARS-CoV-2或其它冠状病毒的S蛋白全序列多肽A. S protein full sequence peptide of SARS-CoV-2 or other coronaviruses
a.先从GenBank中获取SARS-CoV-2之S蛋白氨基酸序列如下：a. First obtain the S protein amino acid sequence of SARS-CoV-2 from GenBank as follows:
b.载体蛋白偶联抗原表位多肽b. Carrier protein coupled with epitope polypeptide
多肽合成
切割与脱保护
HPLC纯化
氨基酸组分分析
修饰合成多肽
偶联KLH和BSA获得重组多肽蛋白
Peptide synthesis Cutting and deprotection HPLC purification Amino acid composition analysis Modified synthetic peptide Coupling KLH and BSA to obtain recombinant polypeptide protein
B.SARS-CoV-2或其它冠状病毒的S1蛋白免疫优势决定区多肽B. S1 protein immunodominance determining region polypeptide of SARS-CoV-2 or other coronaviruses
a.从Gen Bank数据库中获取SARS-CoV-2之S1蛋白氨基酸序列,使用DNAStar软件选取Hopp&Woods亲水性参数(Hydrophilicity)和Willing抗原性参数(Antigenicity)进行单参数预测，根据软件分析结果选择S1蛋白免疫优势决定区多肽序列a. Obtain the S1 protein amino acid sequence of SARS-CoV-2 from the GenBank database, use DNAStar software to select Hopp&Woods hydrophilicity parameters (Hydrophilicity) and Willing antigenicity parameters (Antigenicity) for single parameter prediction, and select S1 according to the software analysis results Protein immunodominance determining region polypeptide sequence
b.载体蛋白偶联抗原表位多肽b. Carrier protein coupled with epitope polypeptide
多肽合成
切割与脱保护
HPLC纯化
氨基酸组分分析
修饰合成多肽
偶联KLH和BSA获得重组免疫优势决定区(510-672)多肽蛋白
Peptide synthesis Cutting and deprotection HPLC purification Amino acid composition analysis Modified synthetic peptide Coupling KLH and BSA to obtain recombinant immunodominance determining region (510-672) polypeptide protein
c.用原核或真核表达***扩增表达SARS-CoV-2或其它冠状病毒的S1区多肽蛋白c. Use prokaryotic or eukaryotic expression system to amplify and express the S1 polypeptide protein of SARS-CoV-2 or other coronaviruses
I.构建SARS-CoV-2或其它冠状病毒S1区基因分段重组质粒I. Construction of SARS-CoV-2 or other coronavirus S1 region gene segmented recombinant plasmids
II.用原核或真核表达***进行表达II. Use prokaryotic or eukaryotic expression system for expression
III.运用Fmoc固相法或其它方法合成肽链III. Use Fmoc solid phase method or other methods to synthesize peptide chains
IV.与KLH和BSA偶联成多肽IV. Coupling with KLH and BSA to form a polypeptide
C.SARS-CoV-2或其它冠状病毒的S2蛋白免疫优势决定区多肽C. S2 protein immunodominance determining region polypeptide of SARS-CoV-2 or other coronaviruses
研究揭示，SARS-CoV-2或其它冠状病毒的S2蛋白之C-未端重复片段与细胞膜受体ACE2结合并使病毒包膜和细胞膜融合,从而使病毒进入细胞发生感染；因此，选择S2蛋白作为重组蛋白抗原成份。Studies have revealed that the C-terminal repeat fragment of the S2 protein of SARS-CoV-2 or other coronaviruses binds to the cell membrane receptor ACE2 and fuses the virus envelope with the cell membrane, thereby allowing the virus to enter the cell for infection; therefore, the S2 protein was selected As a recombinant protein antigen component.
以SARS-CoV-2冠状病毒的S2蛋白重组抗原成份为例来说明，具体操作如下：Take the S2 protein recombinant antigen component of SARS-CoV-2 coronavirus as an example to illustrate, the specific operation is as follows:
a.从Gen Bank数据库中获取SARS-CoV-2之S2蛋白氨基酸序列,使用DNAStar软件选取Hopp&Woods亲水性参数(Hydrophilicity)和Willing抗原性参数(Antigenicity)进行单参数预测，根据软件分析结果选择S2蛋白免疫优势决定区多肽序列a. Obtain the S2 protein amino acid sequence of SARS-CoV-2 from the GenBank database, use DNAStar software to select Hopp&Woods hydrophilicity parameters (Hydrophilicity) and Willing antigenicity parameters (Antigenicity) for single parameter prediction, and select S2 according to the software analysis results Protein immunodominance determining region polypeptide sequence
b.载体蛋白偶联抗原表位多肽b. Carrier protein coupled with epitope polypeptide
多肽合成
切割与脱保护
HPLC纯化
氨基酸组分分析
修饰合成多肽
偶联KLH和BSA获得重组S2蛋白免疫优势决定区多肽蛋白
Peptide synthesis Cutting and deprotection HPLC purification Amino acid composition analysis Modified synthetic peptide Coupling KLH and BSA to obtain recombinant S2 protein immunodominance determining region polypeptide protein
c.用原核或真核表达***扩增表达SARS-CoV-2或其它冠状病毒的S2区多肽蛋白c. Use prokaryotic or eukaryotic expression system to amplify and express SARS-CoV-2 or other coronavirus S2 region polypeptide protein
I.构建SARS-CoV-2或其它冠状病毒S2区基因分段重组质粒I. Construction of SARS-CoV-2 or other coronavirus S2 region gene segmented recombinant plasmids
II.用原核或真核表达***进行表达II. Use prokaryotic or eukaryotic expression system for expression
III.运用Fmoc固相法或其它方法合成肽链III. Use Fmoc solid phase method or other methods to synthesize peptide chains
IV.与KLH和BSA偶联成多肽IV. Coupling with KLH and BSA to form a polypeptide
D.SARS-CoV-2或其它冠状病毒的受体结合域(RBD)片段重组多肽蛋白D. Recombinant polypeptide protein of the receptor binding domain (RBD) fragment of SARS-CoV-2 or other coronaviruses
SARS-CoV-2或其它冠状病毒的受体结合域(RBD)包含了S蛋白的主要中和性抗原决定簇,介导了病毒与靶细胞的相互作用,可影响病毒与靶细胞的结合,在跨种属传播起了十分重要的作用。因此，本发明选择受体结合域(RBD)作为重要的重组蛋白抗原成份。The receptor binding domain (RBD) of SARS-CoV-2 or other coronaviruses contains the main neutralizing epitope of the S protein, which mediates the interaction between the virus and the target cell, and can affect the binding of the virus to the target cell. It plays a very important role in spreading across species. Therefore, the present invention selects receptor binding domain (RBD) as an important recombinant protein antigen component.
以SARS-CoV-2冠状病毒的受体结合域(RBD)片段重组多肽蛋白重组抗原成份为例来说明，具体操作如下：Take the SARS-CoV-2 coronavirus receptor binding domain (RBD) fragment recombinant polypeptide protein recombinant antigen component as an example to illustrate, the specific operation is as follows:
先从GenBank中获取SARS-CoV-2之RBD蛋白氨基酸序列如下：First obtain the RBD protein amino acid sequence of SARS-CoV-2 from GenBank as follows:
然后，按以下步骤制备COVID-19新冠病毒的受体结合域(RBD)片段Then, follow the steps below to prepare the receptor binding domain (RBD) fragment of the COVID-19 new coronavirus
重组多肽蛋白：Recombinant peptide protein:
a.构建PET32(a)-RBD重组质粒a. Construction of PET32(a)-RBD recombinant plasmid
b.用原核或真核表达***进行表达b. Use prokaryotic or eukaryotic expression system for expression
c.运用Fmoc固相法或其它方法合成肽链c. Use Fmoc solid phase method or other methods to synthesize peptide chains
d.与KLH和BSA偶联成多肽d. Coupling with KLH and BSA to form a polypeptide
(2)基因重组SARS-CoV-2或其它冠状病毒N蛋白抗原成份(2) Genetically recombined SARS-CoV-2 or other coronavirus N protein antigen components
SARS-CoV-2及其它冠状病毒N蛋白是一种重要的结构蛋白,位于病毒颗粒的核心部分，与病毒RNA结合形成复合体,对于病毒基因组RNA特征序列的识别、与其他结构蛋白的相互作用有重要意义,在病毒组装等过程中起重要作用；因此，本发明选择N蛋白作为重要的重组蛋白抗原成份。The N protein of SARS-CoV-2 and other coronaviruses is an important structural protein, located in the core part of the virus particle. It combines with viral RNA to form a complex, which recognizes the characteristic sequence of viral genome RNA and interacts with other structural proteins. It is of great significance and plays an important role in the process of virus assembly; therefore, the present invention selects N protein as an important recombinant protein antigen component.
以SARS-CoV-2冠状病毒的N蛋白重组抗原成份为例来说明，具体操作如下：Take the N protein recombinant antigen component of SARS-CoV-2 coronavirus as an example to illustrate, the specific operation is as follows:
先从GenBank中获取SARS-CoV-2之N蛋白氨基酸序列如下：First obtain the N protein amino acid sequence of SARS-CoV-2 from GenBank as follows:
将表达SARS-CoV-2或其它冠状病毒GST-N蛋白的大肠杆菌经超声破碎,离心取上清,用GST融合蛋白纯化试剂盒纯化后鉴定。用空载体表达上清离心后，再纯化；然后，与KLH和BSA偶联，即得到纯化的SARS-CoV-2或其它冠状病毒N蛋白抗原成份。The Escherichia coli expressing SARS-CoV-2 or other coronavirus GST-N protein was disrupted by ultrasound, centrifuged to take the supernatant, purified with GST fusion protein purification kit and identified. The empty vector expression supernatant is centrifuged and then purified; then, it is coupled with KLH and BSA to obtain purified SARS-CoV-2 or other coronavirus N protein antigen components.
(3)基因重组SARS-CoV-2或其它冠状病毒S蛋白片段和N蛋白片段的融合蛋白抗原成份(3) Genetically recombinant SARS-CoV-2 or other coronavirus S protein fragments and N protein fragments of the fusion protein antigen component
以SARS-CoV-2冠状病毒的S蛋白片段和N蛋白片段的融合蛋白抗原成份为例来说明，具体操作如下：Take the fusion protein antigen component of SARS-CoV-2 coronavirus S protein fragment and N protein fragment as an example to illustrate, the specific operations are as follows:
A.通过计算机分析SARS-CoV-2或其它冠状病毒S蛋白和N蛋白的氨基酸序列,确定含强抗原表位的S蛋白片段和N蛋白片段。A. Analyze the amino acid sequence of the S protein and N protein of SARS-CoV-2 or other coronaviruses by computer to determine the S protein fragment and N protein fragment containing strong epitopes.
B.化学合成S蛋白片段和N蛋白片段的基因序列B. Gene sequence of chemically synthesized S protein fragment and N protein fragment
C.将两个基因片段串联,克隆至质粒pET28a(+)内的Nco Ⅰ/EcoR Ⅰ位点,表达S蛋白片段和N蛋白片段的融合蛋白。C. Connect the two gene fragments in series and clone them into the Nco Ⅰ/EcoR Ⅰ site in plasmid pET28a(+), and express the fusion protein of S protein fragment and N protein fragment.
D.将重组质粒转化大肠杆菌BL21(DE3),筛选获得高效表达SARS-CoV-2或其它冠状病毒S蛋白片段和N蛋白片段融合蛋白的工程菌。D. Transform the recombinant plasmid into Escherichia coli BL21 (DE3), and screen the engineered bacteria that efficiently express SARS-CoV-2 or the fusion protein of S protein fragment and N protein fragment of other coronaviruses.
E.将诱导表达融合蛋白的工程菌离心收集上清,过滤后采用S-Sepearse FF阳离子柱纯化。E. Centrifuge the engineered bacteria that induced the expression of the fusion protein to collect the supernatant, and use S-Sepearse FF cation column to purify after filtration.
F.先将纯化的S蛋白和N蛋之融合蛋白透析,然后再用反相高压液相纯化。F. Dialysis the purified fusion protein of S protein and N egg, and then use reversed-phase high pressure liquid purification.
G.与KLH和BSA偶联，获得SARS-CoV-2或其它冠状病毒S蛋白片段和N蛋白片段的融合蛋白抗原成份。G. Coupling with KLH and BSA to obtain the antigen component of the fusion protein of SARS-CoV-2 or other coronavirus S protein fragments and N protein fragments.
(二)制备免疫用抗原(2) Preparation of antigens for immunization
1.制备灭活SARS-CoV-2或其它冠状病毒抗原1. Preparation of inactivated SARS-CoV-2 or other coronavirus antigens
将所制得的经灭活的SARS-CoV-2或其它冠状病毒液以1-10:1-10的比例(通常为1:1)加入福氏佐剂或其它佐剂，置入高速匀浆器以30,000rpm高速匀化，形成油包水乳液，即制得病毒蛋白抗原。Add the prepared inactivated SARS-CoV-2 or other coronavirus liquids in a ratio of 1-10:1-10 (usually 1:1) with Freund’s adjuvant or other adjuvants, and place them in a high-speed homogenizer. The pulper is homogenized at a high speed of 30,000 rpm to form a water-in-oil emulsion to obtain viral protein antigens.
2.基因工程表达蛋白抗原2. Gene engineering express protein antigen
具体实施方法如下：The specific implementation method is as follows:
(1)SARS-CoV-2或其它冠状病毒S蛋白全序列抗原(1) SARS-CoV-2 or other coronavirus S protein full sequence antigen
将所制得的基因工程表达SARS-CoV-2或其它冠状病毒S蛋白全序列抗原成份按1-10:1-10比例(通常为1:1)加入福氏佐剂或其它佐剂，置入高速匀浆器以30,000rpm高速匀化，形成油包水乳液，即制得SARS-CoV-2或其它冠状病毒S蛋白全序列抗原。Add Freund’s adjuvant or other adjuvants in a ratio of 1-10:1-10 (usually 1:1) to the genetically engineered expression of SARS-CoV-2 or other coronavirus S protein full sequence antigen components. Enter a high-speed homogenizer and homogenize at a high speed of 30,000 rpm to form a water-in-oil emulsion to obtain SARS-CoV-2 or other coronavirus S protein full sequence antigens.
(2)SARS-CoV-2或其它冠状病毒S1蛋白和S2蛋白复合抗原(2) SARS-CoV-2 or other coronavirus S1 protein and S2 protein composite antigen
将所制得的以下B、C两种基因工程表达蛋白抗原成份按1-10:1-10的比例混合均匀，制成SARS-CoV-2或其它冠状病毒S蛋白复合抗原成份混合液；然后再按1-10:1-10比例(通常为1:1)加入福氏佐剂或其它佐剂，置入高速匀浆器以30,000rpm高速匀化，形成油包水乳液，即制得SARS-CoV-2或其它冠状病毒S蛋白复合抗原。Mix the prepared antigen components of the following two genetically engineered proteins B and C at a ratio of 1-10:1-10 to make a mixture of SARS-CoV-2 or other coronavirus S protein complex antigen components; then Then add Freund’s adjuvant or other adjuvants in a ratio of 1-10:1-10 (usually 1:1), put in a high-speed homogenizer and homogenize at a high speed of 30,000 rpm to form a water-in-oil emulsion, that is, SARS is prepared -CoV-2 or other coronavirus S protein complex antigens.
B.SARS-CoV-2或其它冠状病毒的S1蛋白免疫优势决定区多肽B. S1 protein immunodominance determining region polypeptide of SARS-CoV-2 or other coronaviruses
C.SARS-CoV-2或其它冠状病毒的S2蛋白免疫优势决定区多肽C. S2 protein immunodominance determining region polypeptide of SARS-CoV-2 or other coronaviruses
(3)SARS-CoV-2或其它冠状病毒的受体结合域(RBD)片段重组多肽蛋白抗原(3) Recombinant polypeptide protein antigen of the receptor binding domain (RBD) fragment of SARS-CoV-2 or other coronaviruses
将所制得的SARS-CoV-2或其它冠状病毒的受体结合域(RBD)片段重组多肽蛋白抗原成份按1-10:1-10比例(通常为1:1)加入福氏佐剂或其它佐剂，置入高速匀浆器以30,000rpm高速匀化，形成油包水乳液， 即制得SARS-CoV-2或其它冠状病毒的受体结合域(RBD)片段重组多肽蛋白抗原。Add the prepared SARS-CoV-2 or other coronavirus receptor binding domain (RBD) fragments of recombinant polypeptide protein antigen components in a ratio of 1-10:1-10 (usually 1:1) with Freund's adjuvant or Other adjuvants are placed in a high-speed homogenizer and homogenized at a high speed of 30,000 rpm to form a water-in-oil emulsion to prepare a recombinant polypeptide protein antigen of the receptor binding domain (RBD) fragment of SARS-CoV-2 or other coronaviruses.
(4)基因重组SARS-CoV-2或其它冠状病毒N蛋白抗原(4) Genetically recombined SARS-CoV-2 or other coronavirus N protein antigens
将所制得的SARS-CoV-2或其它冠状病毒N蛋白抗原成份按1-10:1-10比例(通常为1:1)加入福氏佐剂或其它佐剂，置入高速匀浆器以30,000rpm高速匀化，形成油包水乳液，即制得SARS-CoV-2或其它冠状病毒N蛋白抗原。Add the prepared SARS-CoV-2 or other coronavirus N protein antigen components in a ratio of 1-10:1-10 (usually 1:1) with Freund’s adjuvant or other adjuvants, and place in a high-speed homogenizer Homogenize at a high speed of 30,000 rpm to form a water-in-oil emulsion to obtain SARS-CoV-2 or other coronavirus N protein antigens.
(5)基因重组SARS-CoV-2或其它冠状病毒S蛋白片段和N蛋白片段的融合蛋白抗原(5) Genetically recombinant SARS-CoV-2 or other coronavirus S protein fragments and N protein fragments of fusion protein antigens
将所制得的SARS-CoV-2或其它冠状病毒S蛋白片段和N蛋白片段的融合蛋白抗原成份按1-10:1-10比例(通常为1:1)加入福氏佐剂或其它佐剂，置入高速匀浆器以30,000rpm高速匀化，形成油包水乳液，即制得SARS-CoV-2或其它冠状病毒融合蛋白抗原。Add Freund’s adjuvant or other adjuvant to the prepared SARS-CoV-2 or other coronavirus S protein fragments and N protein fragments of the fusion protein antigen components in a ratio of 1-10:1-10 (usually 1:1) The agent is placed in a high-speed homogenizer and homogenized at a high speed of 30,000 rpm to form a water-in-oil emulsion to obtain SARS-CoV-2 or other coronavirus fusion protein antigens.
二、制备含高活性抗体的免疫蛋2. Preparation of immune eggs containing highly active antibodies
本发明可选用任何产蛋禽类(包括鸡、鸵鸟、鸭、鹅)免疫制备免疫蛋，限于篇幅仅以母鸡为例说明，其它禽类完全可依下文所述方法同样进行。In the present invention, any egg-laying poultry (including chickens, ostriches, ducks, and geese) can be used for immunization to prepare immune eggs. Due to the limitation of space, only hens are used as an example. Other poultry can be performed in the same manner as described below.
分别采用前面方法制备的SARS-CoV-2或其它冠状病毒灭活的病毒抗原和五种基因重组SARS-CoV-2或其它冠状病毒蛋白抗原免疫产蛋鸡，每隔两周注射一次，共注射2-5次，最后一次注射后的至少第12天后检取产蛋鸡所产免疫蛋，并进行编码标记，得到六种抗SARS-CoV-2或其它冠状病毒IgY免疫蛋。The SARS-CoV-2 or other coronavirus inactivated viral antigens prepared by the previous method and five genetic recombinant SARS-CoV-2 or other coronavirus protein antigens were used to immunize laying hens, and injected once every two weeks for a total of injections. 2-5 times, at least 12 days after the last injection, the immunized eggs from laying hens were collected and coded and labeled to obtain six immune eggs against SARS-CoV-2 or other coronavirus IgY.
三、制备抗SARS-CoV-2或其它冠状病毒IgY粗提物3. Preparation of crude extract of anti-SARS-CoV-2 or other coronavirus IgY
采用纯水提取法、氯仿萃取法、冷乙醇沉淀法或硫酸铵沉淀法制备抗SARS-CoV-2或其它冠状病毒IgY粗提物。本发明以纯水提取法为例说明，其他制备方法参照常规方法操作，具体操作过程，这里就不赘述。The crude extract of anti-SARS-CoV-2 or other coronavirus IgY is prepared by pure water extraction method, chloroform extraction method, cold ethanol precipitation method or ammonium sulfate precipitation method. The present invention is illustrated by taking the pure water extraction method as an example, other preparation methods refer to conventional methods, and the specific operation process is not repeated here.
纯水提取法制备抗SARS-CoV-2或其它冠状病毒IgY粗提物的具体操作方法如下：The specific operation method of preparing anti-SARS-CoV-2 or other coronavirus IgY crude extracts by pure water extraction method is as follows:
把所制备的抗SARS-CoV-2或其它冠状病毒IgY免疫蛋用流动水洗净，酒精擦洗消毒，再用打蛋机打碎抗SARS-CoV-2或其它冠状病毒IgY免疫蛋，采用蛋黄筛筛滤去蛋清，留下蛋黄，搅拌均匀；再按蛋黄液体积的3-8倍加入蒸馏水，进行稀释并混合均匀，用1.0N HCI溶液调pH至5.5-6.5；将调好pH值的稀释液进一步充分搅拌均匀，然后将其冷却至2-6℃，静置 12小时-24小时；将稀释液于高速离心；取分离所得的上清液置超滤器中进行超滤浓缩10-20倍；继而加入浓度为1.0-3.0％的海藻酸钠液，缓慢添加海藻酸钠液至终浓度为0.1-0.2％，搅拌至出现浑浊；再加入1.0-3.0％CaCl
2液，至终浓度为0.1-0.2％，搅拌均匀，3-4℃静置8-12小时；高速离心并取上清液，得到抗SARS-CoV-2或其它冠状病毒IgY粗提物。
Wash the prepared anti-SARS-CoV-2 or other coronavirus IgY immune eggs with running water, scrub and disinfect with alcohol, and then break the anti-SARS-CoV-2 or other coronavirus IgY immune eggs with egg yolk Sieve and filter out the egg white, leave the egg yolk, stir well; then add distilled water at 3-8 times the volume of the egg yolk, dilute and mix well, adjust the pH to 5.5-6.5 with 1.0N HCI solution; adjust the pH to the The diluent is further stirred thoroughly, and then cooled to 2-6°C and allowed to stand for 12-24 hours; centrifuge the diluent at a high speed; take the supernatant obtained from the separation and place it in an ultrafilter for ultrafiltration and concentration 10- 20 times; then add 1.0-3.0% sodium alginate solution, slowly add sodium alginate solution to a final concentration of 0.1-0.2%, stir until turbidity appears; then add 1.0-3.0% CaCl 2 solution to the final concentration It is 0.1-0.2%, stir evenly, and stand at 3-4°C for 8-12 hours; centrifuge at high speed and take the supernatant to obtain the crude extract of anti-SARS-CoV-2 or other coronavirus IgY.
分别是：They are:
1.抗SARS-CoV-2或其它冠状病毒病毒抗原IgY1. Anti-SARS-CoV-2 or other coronavirus antigen IgY
2.抗SARS-CoV-2或其它冠状病毒S-IgY2. Anti-SARS-CoV-2 or other coronavirus S-IgY
3.抗SARS-CoV-2或其它冠状病毒(S1+S2)-IgY3. Anti-SARS-CoV-2 or other coronaviruses (S1+S2)-IgY
4.抗SARS-CoV-2或其它冠状病毒RBD-IgY4. Anti-SARS-CoV-2 or other coronavirus RBD-IgY
5.抗SARS-CoV-2或其它冠状病毒N蛋白抗原IgY5. Anti-SARS-CoV-2 or other coronavirus N protein antigen IgY
6.抗SARS-CoV-2或其它冠状病毒(S+N)融合蛋白IgY6. Anti-SARS-CoV-2 or other coronavirus (S+N) fusion protein IgY
可以将以上六种抗SARS-CoV-2或其它冠状病毒IgY中2-6种任意组合按一定比例混合成抗SARS-CoV-2或其它冠状病毒复合IgY。Any combination of 2-6 of the above six anti-SARS-CoV-2 or other coronavirus IgY can be mixed in a certain proportion to form an anti-SARS-CoV-2 or other coronavirus compound IgY.
以下是两种组合的例子：The following are examples of two combinations:
1.(抗SARS-CoV-2或其它冠状病毒病毒抗原IgY)+(抗SARS-CoV-2或其它冠状病毒S-IgY)1. (Anti-SARS-CoV-2 or other coronavirus virus antigen IgY) + (anti-SARS-CoV-2 or other coronavirus virus S-IgY)
2.(抗SARS-CoV-2或其它冠状病毒病毒抗原-IgY)+(抗SARS-CoV-2或其它冠状病毒RBD-IgY)2. (Anti-SARS-CoV-2 or other coronavirus antigen-IgY) + (anti-SARS-CoV-2 or other coronavirus RBD-IgY)
3.(抗SARS-CoV-2或其它冠状病毒S-IgY)+(抗SARS-CoV-2或其它冠状病毒RBD-IgY)3. (Anti-SARS-CoV-2 or other coronavirus S-IgY) + (anti-SARS-CoV-2 or other coronavirus RBD-IgY)
4.【抗SARS-CoV-2或其它冠状病毒(S1+S2)-IgY】+【抗SARS-CoV-2或其它冠状病毒RBD-IgY】4. [Anti-SARS-CoV-2 or other coronaviruses (S1+S2)-IgY] + [Anti-SARS-CoV-2 or other coronaviruses RBD-IgY]
5.(抗SARS-CoV-2或其它冠状病毒S-IgY)+(抗SARS-CoV-2或其它冠状病毒N-IgY)5. (Anti-SARS-CoV-2 or other coronavirus S-IgY) + (anti-SARS-CoV-2 or other coronavirus N-IgY)
6.【抗SARS-CoV-2或其它冠状病毒(S+N)融合蛋白IgY】+(抗SARS-CoV-2或其它冠状病毒RBD-IgY)6. [Anti-SARS-CoV-2 or other coronavirus (S+N) fusion protein IgY] + (anti-SARS-CoV-2 or other coronavirus RBD-IgY)
以下是3种组合的例子：The following are examples of 3 combinations:
1.(抗SARS-CoV-2或其它冠状病毒病毒抗原IgY)+(抗SARS-CoV-2或其它冠状病毒S-IgY)+(抗SARS-CoV-2或其它冠状病毒RBD-IgY)1. (Anti-SARS-CoV-2 or other coronavirus antigen IgY) + (anti-SARS-CoV-2 or other coronavirus S-IgY) + (anti-SARS-CoV-2 or other coronavirus RBD-IgY)
2.(抗SARS-CoV-2或其它冠状病毒病毒S-IgY)+(抗SARS-CoV-2或其它冠状病毒RBD-IgY)+(抗SARS-CoV-2或其它冠状病毒N-IgY)2.(Anti-SARS-CoV-2 or other coronavirus virus S-IgY)+(Anti-SARS-CoV-2 or other coronavirus RBD-IgY)+(Anti-SARS-CoV-2 or other coronavirus N-IgY)
在实际应用中，可根据需要将以上抗SARS-CoV-2或其它冠状病毒IgY任意组合使用。In practical applications, the above anti-SARS-CoV-2 or other coronavirus IgY can be used in any combination as needed.
3.【抗SARS-CoV-2或其它冠状病毒(S1+S2)-IgY】+【抗SARS-CoV-2或其它冠状病毒RBD-IgY】+(抗SARS-CoV-2或其它冠状病毒N-IgY)3.【Anti-SARS-CoV-2 or other coronavirus (S1+S2)-IgY】+【Anti-SARS-CoV-2 or other coronavirus RBD-IgY】+(Anti-SARS-CoV-2 or other coronavirus N -IgY)
四、制备抗SARS-CoV-2或其它冠状病毒纯IgY溶液或干粉4. Preparation of pure IgY solution or dry powder against SARS-CoV-2 or other coronaviruses
将制得一种或多种复合抗SARS-CoV-2或其它冠状病毒IgY粗提物溶解于pH7.0、0.01mol/L的M PB磷酸盐缓冲液中，再先后分别过离子交换柱和凝胶层析柱或者亲和层析柱层析，得到抗SARS-CoV-2或其它冠状病毒纯IgY溶液。抗SARS-CoV-2或其它冠状病毒纯IgY溶液还可以经过冷冻干燥或中低温喷雾干燥或者流化床干燥以及其他不影响抗体活性的干燥方式制得抗SARS-CoV-2或其它冠状病毒纯IgY干粉。Dissolve the prepared one or more composite anti-SARS-CoV-2 or other coronavirus IgY crude extracts in pH 7.0, 0.01 mol/L M PB phosphate buffer, and then pass through the ion exchange column and Gel chromatography column or affinity chromatography column chromatography to obtain anti-SARS-CoV-2 or other coronavirus pure IgY solution. The anti-SARS-CoV-2 or other coronavirus pure IgY solution can also be freeze-dried or medium-low temperature spray drying or fluidized bed drying and other drying methods that do not affect the activity of the antibody to prepare anti-SARS-CoV-2 or other coronavirus pure IgY dry powder.
五、制备一种或多种复合抗SARS-CoV-2或其它冠状病毒小分子抗体Fab5. Preparation of one or more compound anti-SARS-CoV-2 or other coronavirus small molecule antibody Fab
将一种或多种复合抗SARS-CoV-2或其它冠状病毒纯IgY溶液调节pH至3.0-5.0，按0.01％-0.1％质量比的比例为加入催化蛋白质酶；充分搅拌溶解，产生酶促反应，然后低温高速旋转离心，弃去沉淀得上清液，对上清液进行超滤，得到浓缩液；再将超滤后所获浓缩液进行凝胶层析，将层析收集物透析浓缩，得到一种或多种复合抗SARS-CoV-2或其它冠状病毒小分子抗体Fab。Adjust the pH of one or more composite anti-SARS-CoV-2 or other coronavirus pure IgY solutions to 3.0-5.0, and add the catalytic protein enzyme in the proportion of 0.01%-0.1% by mass; fully stir and dissolve to produce enzymatic The reaction is followed by low-temperature high-speed rotation and centrifugation, the supernatant obtained by the precipitation is discarded, and the supernatant is subjected to ultrafiltration to obtain a concentrated solution; then the concentrated solution obtained after ultrafiltration is subjected to gel chromatography, and the chromatographic collection is dialyzed and concentrated , Get one or more compound anti-SARS-CoV-2 or other coronavirus small molecule antibody Fab.
分别是：They are:
1.抗SARS-CoV-2或其它冠状病毒病毒抗原小分子抗体Fab1. Anti-SARS-CoV-2 or other coronavirus antigen small molecule antibody Fab
2.抗SARS-CoV-2或其它冠状病毒S-小分子抗体Fab2. Anti-SARS-CoV-2 or other coronavirus S-small molecule antibody Fab
3.抗SARS-CoV-2或其它冠状病毒(S1+S2)-小分子抗体Fab3. Anti-SARS-CoV-2 or other coronaviruses (S1+S2)-small molecule antibody Fab
4.抗SARS-CoV-2或其它冠状病毒RBD-小分子抗体Fab4. Anti-SARS-CoV-2 or other coronavirus RBD-small molecule antibody Fab
5.抗SARS-CoV-2或其它冠状病毒N蛋白抗原小分子抗体Fab5. Anti-SARS-CoV-2 or other coronavirus N protein antigen small molecule antibody Fab
6.抗SARS-CoV-2或其它冠状病毒(S+N)融合蛋白小分子抗体Fab6. Anti-SARS-CoV-2 or other coronavirus (S+N) fusion protein small molecule antibody Fab
可以将以上六种抗SARS-CoV-2或其它冠状病毒IgY中2-6种任意组合按一定比例混合成抗SARS-CoV-2或其它冠状病毒复合小分子抗体Fab。Any combination of 2-6 of the above six anti-SARS-CoV-2 or other coronavirus IgY can be mixed in a certain proportion to form an anti-SARS-CoV-2 or other coronavirus complex small molecule antibody Fab.
以下是两种组合的例子：The following are examples of two combinations:
2.(抗SARS-CoV-2或其它冠状病毒病毒抗原小分子抗体Fab)+(抗SARS-CoV-2或其它冠状病毒S-小分子抗体Fab)2. (Anti-SARS-CoV-2 or other coronavirus antigen small molecule antibody Fab) + (anti-SARS-CoV-2 or other coronavirus S-small molecule antibody Fab)
2.(抗SARS-CoV-2或其它冠状病毒病毒抗原-小分子抗体Fab)+(抗SARS-CoV-2或其它冠状病毒RBD-小分子抗体Fab)2. (Anti-SARS-CoV-2 or other coronavirus antigen-small molecule antibody Fab) + (anti-SARS-CoV-2 or other coronavirus RBD-small molecule antibody Fab)
3.(抗SARS-CoV-2或其它冠状病毒S-小分子抗体Fab)+(抗SARS-CoV-2或其它冠状病毒RBD-小分子抗体Fab)3. (Anti-SARS-CoV-2 or other coronavirus S-small molecule antibody Fab) + (anti-SARS-CoV-2 or other coronavirus RBD-small molecule antibody Fab)
4.【抗SARS-CoV-2或其它冠状病毒(S1+S2)-小分子抗体Fab】+【抗SARS-CoV-2或其它冠状病毒RBD-小分子抗体Fab】4. [Anti-SARS-CoV-2 or other coronavirus (S1+S2)-small molecule antibody Fab] + [anti-SARS-CoV-2 or other coronavirus RBD-small molecule antibody Fab]
5.(抗SARS-CoV-2或其它冠状病毒S-小分子抗体Fab)+(抗SARS-CoV-2或其它冠状病毒N-小分子抗体Fab)5. (Anti-SARS-CoV-2 or other coronavirus S-small molecule antibody Fab) + (anti-SARS-CoV-2 or other coronavirus N-small molecule antibody Fab)
6.【抗SARS-CoV-2或其它冠状病毒(S+N)融合蛋白小分子抗体Fab】+(抗SARS-CoV-2或其它冠状病毒RBD-小分子抗体Fab)6. [Anti-SARS-CoV-2 or other coronavirus (S+N) fusion protein small molecule antibody Fab] + (anti-SARS-CoV-2 or other coronavirus RBD-small molecule antibody Fab)
以下是3种组合的例子：The following are examples of 3 combinations:
1.(抗SARS-CoV-2或其它冠状病毒病毒抗原小分子抗体Fab)+(抗SARS-CoV-2或其它冠状病毒S-小分子抗体Fab)+(抗SARS-CoV-2或其它冠状病毒RBD-小分子抗体Fab)1. (Anti-SARS-CoV-2 or other coronavirus virus antigen small molecule antibody Fab) + (anti-SARS-CoV-2 or other coronavirus S-small molecule antibody Fab) + (anti-SARS-CoV-2 or other coronavirus Virus RBD-Small Molecule Antibody Fab)
2.(抗SARS-CoV-2或其它冠状病毒病毒S-小分子抗体Fab)+(抗SARS-CoV-2或其它冠状病毒RBD-小分子抗体Fab)+(抗SARS-CoV-2或其它冠状病毒N-小分子抗体Fab)2. (Anti-SARS-CoV-2 or other coronavirus virus S-small molecule antibody Fab) + (anti-SARS-CoV-2 or other coronavirus RBD-small molecule antibody Fab) + (anti-SARS-CoV-2 or other Coronavirus N-small molecule antibody Fab)
3.【抗SARS-CoV-2或其它冠状病毒(S1+S2)-小分子抗体Fab】+【抗SARS-CoV-2或其它冠状病毒RBD-小分子抗体Fab】+(抗SARS-CoV-2或其它冠状病毒N-小分子抗体Fab)3. [Anti-SARS-CoV-2 or other coronavirus (S1+S2)-small molecule antibody Fab] + [anti-SARS-CoV-2 or other coronavirus RBD- small molecule antibody Fab] + (anti-SARS-CoV- 2 or other coronavirus N-small molecule antibody Fab)
在实际应用中，可根据需要将以上抗SARS-CoV-2或其它冠状病毒小分子抗体Fab任意组合使用。In practical applications, the above anti-SARS-CoV-2 or other coronavirus small molecule antibody Fab can be used in any combination as needed.
六、制备长效化抗SARS-CoV-2或其它冠状病毒小分子抗体Fab6. Preparation of long-acting anti-SARS-CoV-2 or other coronavirus small molecule antibody Fab
将所制得的一种或多种复合抗SARS-CoV-2或其它冠状病毒小分子抗体Fab采用聚乙二醇、右旋糖苷或聚氨基酸长效化修饰，得到长效化的抗SARS-CoV-2或其它冠状病毒小分子抗体Fab。The prepared one or more composite anti-SARS-CoV-2 or other coronavirus small molecule antibody Fabs are long-actingly modified with polyethylene glycol, dextran or polyamino acid to obtain long-acting anti-SARS- CoV-2 or other coronavirus small molecule antibody Fab.
本发明以采用聚乙二醇进行长效化修饰的方法为例说明如下，其它长效化修饰的方法可参照以下方法。The present invention takes the method of using polyethylene glycol for long-acting modification as an example to illustrate as follows, and other long-acting modification methods can refer to the following methods.
调节硼酸缓冲液pH值至6.5-10.0，在抗SARS-CoV-2或其它冠状病毒小分子抗体Fab中加入调节pH后的硼酸缓冲液，使抗人***瘤病毒小分子抗体Fab的浓度为0.1-0.5mg/mL，加入聚乙二醇进行反应，且抗SARS-CoV-2或其它冠状病毒小分子抗体Fab与聚乙二醇的摩尔比的比值为1/10～1/30，反应温度为15℃～30℃，反应时间控制在1h～3h。Adjust the pH of the boric acid buffer to 6.5-10.0, and add the pH-adjusted boric acid buffer to the anti-SARS-CoV-2 or other coronavirus small molecule antibody Fab to make the concentration of the anti-human papillomavirus small molecule antibody Fab 0.1 -0.5mg/mL, add polyethylene glycol for reaction, and the molar ratio of anti-SARS-CoV-2 or other coronavirus small molecule antibody Fab to polyethylene glycol is 1/10～1/30, reaction temperature The temperature is 15℃～30℃, and the reaction time is controlled within 1h～3h.
七、制备抗SARS-CoV-2或其它冠状病毒小分子抗体Fab干粉7. Preparation of anti-SARS-CoV-2 or other coronavirus small molecule antibody Fab dry powder
将一种或多种复合抗SARS-CoV-2或其它冠状病毒小分子抗体Fab溶液经过冷冻干燥或中低温喷雾干燥或者流化床干燥以及其他不影响抗体活性的干燥方式制得一种或多种复合抗SARS-CoV-2或其它冠状病毒小分子抗体Fab干粉。One or more composite anti-SARS-CoV-2 or other coronavirus small molecule antibody Fab solutions are freeze-dried, spray-dried at medium and low temperature, fluidized bed drying, and other drying methods that do not affect antibody activity to prepare one or more A composite anti-SARS-CoV-2 or other coronavirus small molecule antibody Fab dry powder.
本发明还提供了上述的抗SARS-CoV-2或其它冠状病毒IgY及其小分子抗体在制备用于防治SARS-CoV-2或其它冠状病毒感染的药物、消毒产品、保健品或医疗器械中的应用。The present invention also provides that the above-mentioned anti-SARS-CoV-2 or other coronavirus IgY and its small molecule antibodies are used in the preparation of drugs, disinfection products, health care products or medical devices for the prevention and treatment of SARS-CoV-2 or other coronavirus infections. Applications.
本发明还提供了一种组合物，包括了上述的抗SARS-CoV-2或其它冠状病毒IgY及其小分子抗体以及至少一种其它药学上可接受的组分。The present invention also provides a composition comprising the above-mentioned anti-SARS-CoV-2 or other coronavirus IgY and small molecule antibodies thereof and at least one other pharmaceutically acceptable component.
在本发明的组合物中，所述抗SARS-CoV-2或其它冠状病毒IgY及其小分子抗体添加辅料或基料或者化学药、中药，制成雾化吸入疗法的雾化剂、喷雾剂、口喷剂、鼻喷剂、滴鼻剂、滴眼剂、空气消毒剂、空气清新剂、消毒剂、洗手液、粉剂、片剂、***片、口服液、口服剂、胶囊剂中的至少一种。In the composition of the present invention, the anti-SARS-CoV-2 or other coronavirus IgY and its small molecule antibodies are added with auxiliary materials or base materials or chemical medicines and traditional Chinese medicines to make aerosols or sprays for atomized inhalation therapy , Oral sprays, nasal sprays, nose drops, eye drops, air disinfectants, air fresheners, disinfectants, hand sanitizers, powders, tablets, lozenges, oral liquids, oral agents, capsules At least one.
本发明还提供了上述的组合物在制备用于防治SARS-CoV-2或其它冠状病毒感染的药物、消毒产品、保健品或医疗器械中的应用。The present invention also provides the application of the above-mentioned composition in the preparation of medicines, disinfection products, health products or medical devices for preventing and treating SARS-CoV-2 or other coronavirus infections.
如上所叙，可将所制备的抗SARS-CoV-2或其它冠状病毒IgY及其小分子抗体制成各种稳定制剂。所述制剂包括但不限于这些制剂：As mentioned above, the prepared anti-SARS-CoV-2 or other coronavirus IgY and its small molecule antibodies can be made into various stable preparations. The formulations include but are not limited to these formulations:
优选地，该制剂还包括赋形剂、填充剂、溶剂、助溶剂、表面活性剂和胶囊辅料中一种或多种。Preferably, the formulation further includes one or more of excipients, fillers, solvents, co-solvents, surfactants and capsule auxiliary materials.
优选地，该制剂为雾化吸入疗法的雾化剂、消毒剂、消毒液、空气清新剂、洗手液等。Preferably, the preparation is an atomizer, disinfectant, disinfectant, air freshener, hand sanitizer, etc. for atomized inhalation therapy.
优选地，该制剂为片剂、喷雾剂、粉剂、液剂或胶囊。Preferably, the preparation is a tablet, spray, powder, liquid or capsule.
实施本发明的抗SARS-CoV-2或其它冠状病毒IgY及其小分子抗体和组合物以及制备方法和应用，具有以下有益效果：本发明的抗SARS-CoV-2或其它冠状病毒IgY及其小分子抗体不但可直接抑灭SARS-CoV-2和其它 冠状病毒，还可特异性与SARS-CoV-2和其它冠状病毒的关键蛋白S蛋白及其RBD结构域结合并将其封闭，使其失去介导病毒侵染细胞和扩散的功能；从而，阻止SARS-CoV-2和其它冠状病毒感染呼吸道上皮细胞。同时，这些抗体还可中和受感染细胞排出的病毒，消除二次感染和阻断散发传播致病，这些独特优点对于全民预防SARS-CoV-2和其它冠状病毒感染以及防控疫情蔓延具有特别重大的意义。使用本发明的抗SARS-CoV-2或其它冠状病毒IgY及其小分子抗体和组合物包括应用这些抗体和组合物制成的系列产品既可达到有的放失、精准防治的目的，又没有化学抗病毒药和化学抑制剂的毒副作用和诱发耐药性的问题。The anti-SARS-CoV-2 or other coronavirus IgY and its small molecule antibodies and compositions and preparation methods and applications of the present invention have the following beneficial effects: the anti-SARS-CoV-2 or other coronavirus IgY of the present invention and its Small molecule antibodies can not only directly inhibit SARS-CoV-2 and other coronaviruses, but also specifically bind to the key protein S protein of SARS-CoV-2 and other coronaviruses and its RBD domains and block them to make them Loss of the function of mediating virus infecting cells and spreading; thus, preventing SARS-CoV-2 and other coronaviruses from infecting respiratory epithelial cells. At the same time, these antibodies can also neutralize the virus excreted by infected cells, eliminate secondary infections and block the spread of disease. These unique advantages are particularly useful for the prevention of SARS-CoV-2 and other coronavirus infections and the prevention and control of the spread of epidemics. Significant significance. Using the anti-SARS-CoV-2 or other coronavirus IgY and its small molecule antibodies and compositions of the present invention, including the series of products made with these antibodies and compositions, can achieve the purpose of some loss, precise prevention and control, and no The toxic and side effects of chemical antiviral drugs and chemical inhibitors and the problem of inducing drug resistance.
下面结合试验例和实施例，对本发明的抗SARS-CoV-2或其它冠状病毒IgY及其小分子抗体及其组合物以及制备方法和应用作进一步说明：In the following, in combination with test examples and examples, the anti-SARS-CoV-2 or other coronavirus IgY and its small molecule antibodies and their compositions, preparation methods and applications of the present invention will be further explained:
试验例1：Test example 1:
抗SARS-CoV-2病毒抗原IgY对相对应的灭活SARS-CoV-2病毒的抗体结合效价检测。Detection of antibody binding titer of anti-SARS-CoV-2 virus antigen IgY to corresponding inactivated SARS-CoV-2 virus.
选择灭活SARS-CoV-2病毒作为检测抗原，用“ELISA”方法(酶联免疫法)检测所制得的抗SARS-CoV-2病毒抗原IgY的抗体效价，结果如下表所示：Select the inactivated SARS-CoV-2 virus as the detection antigen, and use the "ELISA" method (enzyme-linked immunoassay) to detect the antibody titer of the prepared anti-SARS-CoV-2 virus antigen IgY. The results are shown in the following table:
| 抗体Antibody | 抗原antigen | 抗体结合效价Antibody binding titer |
| 抗SARS-CoV-2病毒抗原IgYAnti-SARS-CoV-2 virus antigen IgY | SARS-CoV-2病毒抗原SARS-CoV-2 virus antigen | 1:327681:32768 |
注：测试样本中的抗SARS-CoV-2病毒抗原IgY抗体溶液浓度均为1mg/mL。Note: The concentration of the anti-SARS-CoV-2 virus antigen IgY antibody solution in the test sample is 1 mg/mL.
从以上检测结果可看出，所制备的抗SARS-CoV-2病毒抗原IgY对SARS-CoV-2病毒有很高的抗体结合效价。It can be seen from the above test results that the prepared anti-SARS-CoV-2 virus antigen IgY has a high antibody binding titer to the SARS-CoV-2 virus.
试验例2：Test Example 2:
抗SARS-CoV-2IgY抗体对相对应的SARS-CoV-2冠状病毒有代表性重组抗原成份的抗体结合效价检测。Anti-SARS-CoV-2IgY antibody to the corresponding SARS-CoV-2 coronavirus representative recombinant antigen component antibody binding titer detection.
分别选择SARS-CoV-2-S蛋白全序列多肽、SARS-CoV-2-S1蛋白免疫优势决定区多肽、SARS-CoV-2-S2蛋白免疫优势决定区多肽、SARS-CoV-2受体结合域(RBD)片段重组多肽蛋白、SARS-CoV-2-N蛋白抗原、SARS-CoV-2-S蛋白片段和N蛋白片段的融合蛋白抗原作为检测抗原，用 “ELISA”方法(酶联免疫法)检测所制得的抗SARS-CoV-2冠状病毒IgY及其小分子抗体的抗体效价，结果如下表所示：Select SARS-CoV-2-S protein full sequence peptide, SARS-CoV-2-S1 protein immunodominance determining region polypeptide, SARS-CoV-2-S2 protein immunodominance determining region polypeptide, and SARS-CoV-2 receptor binding Domain (RBD) fragment recombinant polypeptide protein, SARS-CoV-2-N protein antigen, SARS-CoV-2-S protein fragment and N protein fragment fusion protein antigen as the detection antigen, using the "ELISA" method (enzyme-linked immunoassay ) Test the antibody titer of the prepared anti-SARS-CoV-2 coronavirus IgY and its small molecule antibodies. The results are shown in the following table:
注：测试样本中的抗SARS-CoV-2-IgY系列抗体溶液浓度均为1mg/mL。Note: The concentration of the anti-SARS-CoV-2-IgY series antibody solution in the test sample is 1mg/mL.
从以上检测结果可看出，所制备的抗SARS-CoV-2-IgY系列抗体对相对应的SARS-CoV-2蛋白抗原都有很高的抗体结合效价。It can be seen from the above test results that the prepared anti-SARS-CoV-2-IgY series antibodies have high antibody binding titers to the corresponding SARS-CoV-2 protein antigen.
试验例3：Test example 3:
抗SARS-CoV-2小分子抗体Fab对相对应的SARS-CoV-2蛋白抗原的抗体结合效价检测。Detection of the antibody binding titer of the anti-SARS-CoV-2 small molecule antibody Fab to the corresponding SARS-CoV-2 protein antigen.
分别选择SARS-CoV-2-S蛋白全序列多肽、SARS-CoV-2-S1蛋白免疫优势决定区多肽、SARS-CoV-2-S2蛋白免疫优势决定区多肽、SARS-CoV-2受体结合域(RBD)片段重组多肽蛋白、SARS-CoV-2-N蛋白抗原、SARS-CoV-2-S蛋白片段和N蛋白片段的融合蛋白抗原作为检测抗原，用“ELISA”方法(酶联免疫法)检测所制得的抗SARS-CoV-2小分子抗体Fab的抗体效价，结果如下表所示：Select SARS-CoV-2-S protein full sequence peptide, SARS-CoV-2-S1 protein immunodominance determining region polypeptide, SARS-CoV-2-S2 protein immunodominance determining region polypeptide, and SARS-CoV-2 receptor binding Domain (RBD) fragment recombinant polypeptide protein, SARS-CoV-2-N protein antigen, SARS-CoV-2-S protein fragment and N protein fragment fusion protein antigen as the detection antigen, using the "ELISA" method (enzyme-linked immunoassay ) Detect the antibody titer of the prepared anti-SARS-CoV-2 small molecule antibody Fab, and the results are shown in the following table:
注：测试样本中的抗SARS-CoV-2小分子抗体Fab抗体溶液浓度均为1mg/mL。Note: The concentration of the anti-SARS-CoV-2 small molecule antibody Fab antibody solution in the test sample is 1 mg/mL.
从以上检测结果可看出，所制备的抗SARS-CoV-2小分子抗体Fab对相对应的SARS-CoV-2蛋白抗原都有很高的抗体结合效价。It can be seen from the above test results that the prepared anti-SARS-CoV-2 small molecule antibody Fab has a high antibody binding titer to the corresponding SARS-CoV-2 protein antigen.
实施例1：釆用中和试验法测定抗SARS-CoV-2病毒抗原IgY中和SARS-CoV-2病毒的效价Example 1: The neutralization test method is used to determine the potency of anti-SARS-CoV-2 virus antigen IgY in neutralizing SARS-CoV-2 virus
1.试验材料：1. Test materials:
(1)抗SARS-CoV-2病毒抗原IgY：蛋白含量40mg/ml 2份标本分1号和2号(1) Anti-SARS-CoV-2 virus antigen IgY: protein content 40mg/ml 2 specimens are divided into No. 1 and No. 2
(2)HRP酶标兔抗鸡IgG(2) HRP enzyme-labeled rabbit anti-chicken IgG
(3)阴性抗体(未免疫鸡蛋的IgY抗体)：蛋白含量60mg/ml(3) Negative antibody (IgY antibody of unimmunized eggs): protein content 60mg/ml
(4)阳性对照：SARS-CoV-2病毒感染病人恢复期血清2份，标本3号4号(4) Positive control: 2 samples of convalescent serum from patients infected with SARS-CoV-2, specimen No. 3 and No. 4
(5)阴性对照：婴儿脐带血清2份，标本5号6号(5) Negative control: 2 sets of baby umbilical cord serum, specimen No. 5 and No. 6
(6)病毒：(6) Virus:
a.SARS-CoV-2病毒分离株I：从SARS-CoV-2病毒感染病人咽试子标本分离鉴定。a. SARS-CoV-2 virus isolate I: Isolation and identification from throat test specimens of patients infected with SARS-CoV-2 virus.
b.SARS-CoV-2病毒分离株II：从SARS-CoV-2病毒感染病人血清标本分离鉴定。b. SARS-CoV-2 virus isolate II: Isolation and identification from the serum samples of patients infected with SARS-CoV-2 virus.
(7)细胞：非洲绿猴肾传代细胞。(7) Cells: Passage cells of African green monkey kidney.
2.正式试验：2. Formal test:
在VERO E6细胞培养检测抗SARS-CoV-2病毒抗原IgY抗体试验Test for detecting anti-SARS-CoV-2 virus antigen IgY antibody in VERO E6 cell culture
实验目的：Purpose:
在VERO E6细胞培养内，釆用中和试验法，测定抗SARS-CoV-2病毒抗原IgY(用分离的2株SARS-CoV-2病毒株)；用Reed-Muench法，计算50％抗体中和终点。In VERO E6 cell culture, the neutralization test method was used to determine the anti-SARS-CoV-2 virus antigen IgY (using two isolated SARS-CoV-2 virus strains); the Reed-Muench method was used to calculate the 50% antibody And the end.
固定病毒-稀释抗体法Fixed virus-diluted antibody method
将上述抗SARS-CoV-2病毒抗原IgY抗体、SARS-CoV-2病毒感染病人恢复期血清及婴儿脐带血清，分别在56℃灭活1小时后，分别用Egale’S液2倍稀释9个浓度，既L 8—1：1024与100TCID50 SARS-CoV-2病毒分离株I和SARS-CoV-2病毒分离株II之病毒悬液混合37℃水浴1小时后，接种VERO E6细胞96孔培养板，每浓度4孔，同时设抗SARS-CoV-2病毒抗原IgY抗体、病人恢复期血清及婴儿脐带血清对照，病毒对照、正常细胞对照。37℃ 5％CO
2孵箱培养5天，每天在倒置显微镜下观察病毒(CPE),以25％以下形态变化为“+”，26％～50％形态变化为“++”，51％～75％形态变化为“+++”，76％～100％形态变化为“++++”，以病毒对照出现“+++++++”时结束试验。
The above-mentioned anti-SARS-CoV-2 virus antigen IgY antibody, SARS-CoV-2 virus-infected patient's convalescent serum and baby umbilical cord serum were inactivated at 56°C for 1 hour, and then diluted with Egale's solution to 9 concentrations. Both L 8-1: 1024 and 100TCID50 SARS-CoV-2 virus isolate I and SARS-CoV-2 virus isolate II were mixed with the virus suspension in a 37℃ water bath for 1 hour, and then inoculated in a 96-well culture plate of VERO E6 cells. Concentration 4 wells, at the same time set up anti-SARS-CoV-2 virus antigen IgY antibody, patient recovery period serum and infant umbilical cord serum control, virus control, normal cell control. Incubate in a 37°C 5% CO 2 incubator for 5 days. Observe the virus (CPE) under an inverted microscope every day. The morphological change of 25% or less is "+", and the morphological change of 26%-50% is "++", 51%～ 75% of the morphological change is "+++", 76%-100% of the morphological change is "++++", and the test is terminated when the virus control appears "+++++++".
用Reed-Muench法，计算50％抗体中和终点。The Reed-Muench method was used to calculate the 50% antibody neutralization endpoint.
3.抗SARS-CoV-2病毒抗原IgY抗体中和试验检测结果3. Anti-SARS-CoV-2 virus antigen IgY antibody neutralization test results
(1)抗SARS-CoV-2病毒抗原IgY抗体1号标本：1:512的抗体可保护50％细胞不产生细胞病变。(1) Anti-SARS-CoV-2 virus antigen IgY antibody No. 1 specimen: 1:512 antibody can protect 50% of cells from cytopathic.
(2)抗SARS-CoV-2病毒抗原IgY抗体2号标本：1:256的抗体可保护50％细胞不产生细胞病变。(2) Anti-SARS-CoV-2 virus antigen IgY antibody No. 2 specimen: 1:256 antibody can protect 50% of cells from cytopathic.
阴性抗体:不能中和SARS-CoV-2病毒。Negative antibody: Can not neutralize SARS-CoV-2 virus.
阳性对照：Positive control:
(1)SARS-CoV-2病毒感染病人恢复期血清3号标本：1:512的血清可保护50％细胞不产生细胞病变。(1) Serum No. 3 specimen of convalescent patients infected with SARS-CoV-2: 1:512 serum can protect 50% of cells from cytopathic changes.
(2)SARS-CoV-2病毒感染病人恢复期血清4号标本：1:512的血清可保护50％细胞不产生细胞病变。(2) Convalescent serum specimens of patients infected with SARS-CoV-2 virus: 1:512 serum can protect 50% of cells from cytopathic changes.
阴性对照：Negative control:
(1)婴儿脐带血清5号标本：不能中和SARS-CoV-2病毒。(1) Infant umbilical cord serum No. 5 specimen: it cannot neutralize the SARS-CoV-2 virus.
(2)婴儿脐带血清6号标本：不能中和SARS-CoV-2病毒。(2) Infant umbilical cord serum No. 6 specimen: it cannot neutralize the SARS-CoV-2 virus.
实施例2：采用免疫印迹(Western blot)检测，确定抗SARS-CoV-2病毒抗原IgY抗体与SARS-CoV-2病毒的结合。Example 2: Western blot detection was used to determine the binding of the anti-SARS-CoV-2 virus antigen IgY antibody to the SARS-CoV-2 virus.
具体操作方法如下：The specific operation method is as follows:
1.电泳：1. Electrophoresis:
(1)样品处理：将SARS-CoV-2病毒N抗原(200gg/ml),已灭活的SARS-CoV-2病毒vero细胞(CPE+++)与5X加样缓冲液(电泳加样缓冲液：25mmol Tris-CL(pH6.8),50mmol DTT,2％SDS,0.1％漠酚蓝，10％甘油)混合；(1) Sample processing: Combine SARS-CoV-2 virus N antigen (200gg/ml), inactivated SARS-CoV-2 virus vero cells (CPE+++) and 5X loading buffer (electrophoresis loading buffer: 25mmol Tris-CL (pH6.8), 50mmol DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerol) mix;
样品处理：将SARS-CoV-2病毒N抗原(200μg/ml)5μl,已灭活的SARS-CoV-2病毒vero细胞加样缓冲液(电泳加样缓冲液：25mmol Tris-CL(pH6.8),50mmol DTT,2％SDS,0.1％漠酚蓝，10％甘油)混合；Sample processing: 5μl SARS-CoV-2 virus N antigen (200μg/ml), inactivated SARS-CoV-2 virus vero cell loading buffer (electrophoresis loading buffer: 25mmol Tris-CL (pH6.8) ), 50mmol DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerin) mixed;
(2)沸水中加热10min；(2) Heating in boiling water for 10 minutes;
(3)离心3000rpm,2min；(3) Centrifuge at 3000rpm for 2min;
(4)分别取经以上处理的N抗原(200μg/ml)5μl,已灭活的SARS-CoV-2病毒vero细胞10μl加样，15％SDS-PAGE电泳(15％分离胶：11.5ml水，12.5ml 1.5mmol Tris-CL(pH8.8),25ml 30％SDS-PAGE胶贮液，10％SDS 0.5ml,临用前加10％过硫酸胺10μl TEMED(10μl/ml)。5％成层胶：6.8ml水,1.7ml 30％SDS-PAGE胶贮液，1.25ml 1.5mmolTris-CL(pH6.8)，10％SDS0.1ml,临用前加10％过硫酸胺10μl TEMED(1μl/ml)；(4) Take 5μl of the above-treated N antigen (200μg/ml), 10μl of inactivated SARS-CoV-2 virus vero cells, and apply 15% SDS-PAGE electrophoresis (15% separation gel: 11.5ml water, 12.5 ml 1.5mmol Tris-CL (pH 8.8), 25ml 30% SDS-PAGE gel stock solution, 10% SDS 0.5ml, add 10% ammonium persulfate 10μl TEMED (10μl/ml) immediately before use. 5% layered gel ：6.8ml water, 1.7ml 30% SDS-PAGE gel storage solution, 1.25ml 1.5mmolTris-CL (pH6.8), 10% SDS 0.1ml, add 10% ammonium persulfate 10μl TEMED(1μl/ml) ；
(5)电压80V(成层胶)，156V(分离胶)电泳，待指示剂出凝胶底部10min关闭电源。(5) Voltage 80V (layered gel), 156V (separating gel) for electrophoresis, turn off the power supply after the indicator reaches the bottom of the gel for 10 minutes.
2.转膜：2. Transfer film:
(1)小心将凝胶放置于转移液(39mmol甘氨酸,48mmolTris-CL,0.037％SDS,20％甲醇)中浸泡过三层滤纸之上，小心将硝酸纤维素膜(浸泡5min)放在凝胶之上，覆盖浸泡过的三层滤纸，准确对齐各层，赶尽气泡；(1) Carefully place the gel in the transfer solution (39mmol glycine, 48mmolTris-CL, 0.037% SDS, 20% methanol) soaked on three layers of filter paper, carefully place the nitrocellulose membrane (soaked for 5 minutes) on the gel On top, cover the soaked three layers of filter paper, accurately align the layers, and drive out the bubbles;
(2)60mA转膜1h(2) 60mA transfer film 1h
3.免疫学检测：3. Immunological testing:
(1)转膜完毕，揭去滤纸，小心将膜取出，PBST(0.05％Tween-20/PBS)漂洗2min；(1) After transferring the membrane, remove the filter paper, carefully take out the membrane, and rinse with PBST (0.05% Tween-20/PBS) for 2 minutes;
(2)5％脱脂奶(脱脂奶/PBS)封闭过夜；(2) 5% skimmed milk (skimmed milk/PBS) blocked overnight;
(3)将抗SARS-CoV-2病毒抗原IgY悬液1:2、1:4稀释(PBS),37℃与膜反应2h；(3) Dilute the anti-SARS-CoV-2 virus antigen IgY suspension 1:2, 1:4 (PBS), and react with the membrane at 37°C for 2h;
(4)PBST洗膜3次，10min/次；(4) Wash the membrane with PBST 3 times, 10min/time;
(5)辣根过氧化物酶标记的抗鸡IgG(l:250)稀释，37℃与膜反应2h；(5) Dilute horseradish peroxidase-labeled anti-chicken IgG (1:250) and react with the membrane at 37°C for 2h;
(6)PBST洗膜3次，10min/次；(6) Wash the membrane with PBST 3 times, 10min/time;
(7)0.05mol/L DAB(3,3-二氨基联苯胺50mg,50mmolTris-CL(pH8.0),100ml,0.01％H
20
2)显色；
(7) 0.05mol/L DAB (3,3-diaminobenzidine 50mg, 50mmolTris-CL (pH8.0), 100ml, 0.01% H 2 O 2 ) color development;
(8)待出现明显条带即用蒸馏水冲洗终止反应。(8) When there are obvious bands, rinse with distilled water to stop the reaction.
4.结果：4. Results:
(1)与SARS病毒裂解后不同组分(M,N抗原)反应(lane1)；(1) React with different components (M, N antigen) after SARS virus lysis (lane1);
(2)与表达的N抗原(40KD处)反应(lane2)
o
(2) React with the expressed N antigen (at 40KD) (lane2) o
实施例3：生产抗SARS-CoV-2-S蛋白全序列多肽-小分子抗体Fab雾化剂，供各种雾化吸入机和公共环境空气消毒机、空气清新器和家庭用空气消毒器、空气清新器以及移动喷雾消毒器械作为药用雾化液使用。Example 3: Production of anti-SARS-CoV-2-S protein full sequence polypeptide-small molecule antibody Fab nebulizer for various nebulization inhalers and public environment air disinfection machines, air fresheners and household air disinfectors, Air fresheners and mobile spray disinfection equipment are used as medicinal atomized liquids.
配方：formula:
工艺：Process:
(1)将配方量K-30、S-40、吐温-80、薄荷油、香精用紫外光照射灭菌消毒24小时，无菌密封备用；(1) Sterilize the formula quantity K-30, S-40, Tween-80, peppermint oil, and essence with ultraviolet light for 24 hours, and seal aseptically for later use;
(2)按配方量蒸馏水加热至至90℃，再分别加入S-40和K-30分散剂，搅拌30分钟以上，溶解均匀；再冷却至60℃，分别缓慢加入吐温(滴加)和薄荷油以及甘油(滴加)，低速揽拌60分钟至充分溶解；然后，冷却至室温，形成溶液A；(2) Heat distilled water according to the formula to 90°C, then add S-40 and K-30 dispersants separately, stir for more than 30 minutes, and dissolve uniformly; then cool to 60°C, and slowly add Tween (dropwise) and Peppermint oil and glycerin (dropped), stir at low speed for 60 minutes until fully dissolved; then, cool to room temperature to form solution A;
(3)一边搅拌一边将抗SARS-CoV-2-S蛋白全序列多肽-小分子抗体Fab纳米脂质体溶液或干粉加入溶液A中，低速搅拌60min，直至完全混合均匀，得溶液B；(3) While stirring, add the anti-SARS-CoV-2-S protein full-sequence peptide-small molecule antibody Fab nanoliposome solution or dry powder into solution A, stir at low speed for 60 minutes, until it is completely mixed and uniform, and solution B is obtained;
(4)一边搅拌一边将香精加入溶液B中，低速搅拌60min，直至完全溶解，得溶液C；(4) Add the essence to solution B while stirring, and stir at low speed for 60 minutes until it is completely dissolved to obtain solution C;
(5)用pH计测量溶液C的pH值，用柠檬酸调节pH值至6.8±0.1；(5) Measure the pH value of solution C with a pH meter, and adjust the pH value to 6.8±0.1 with citric acid;
(6)静置至上部泡沫全部消溶后，再将溶液C分装在清洗消毒过的喷雾瓶中，贴上标签出厂。(6) After the upper foam is completely dissolved, the solution C is divided into cleaned and disinfected spray bottles, and the label is attached to the factory.
实施例4：生产抗SARS-CoV-2-S和N融合蛋白-小分子抗体Fab鼻喷剂或滴鼻剂或滴眼剂Example 4: Production of anti-SARS-CoV-2-S and N fusion protein-small molecule antibody Fab nasal spray or nose drops or eye drops
配方：formula:
工艺：Process:
(1)将配方量K-30、S-40、吐温-80、薄荷油、香精用紫外光照射灭菌消毒24小时，无菌密封备用；(1) Sterilize the formula quantity K-30, S-40, Tween-80, peppermint oil, and essence with ultraviolet light for 24 hours, and seal aseptically for later use;
(2)按配方量蒸馏水加热至至90℃，再分别加入S-40和K-30分散剂，搅拌30分钟以上，溶解均匀；再冷却至60℃，分别缓慢加入吐温(滴加)和薄荷油以及甘油(滴加)，低速揽拌60分钟至充分溶解；然后，冷却至室温，形成溶液A；(2) Heat distilled water according to the formula to 90°C, then add S-40 and K-30 dispersants separately, stir for more than 30 minutes, and dissolve uniformly; then cool to 60°C, and slowly add Tween (dropwise) and Peppermint oil and glycerin (dropped), stir at low speed for 60 minutes until fully dissolved; then, cool to room temperature to form solution A;
(3)一边搅拌一边将抗SARS-CoV-2-S和N融合蛋白-小分子抗体Fab溶液或干粉加入溶液A中，低速搅拌60min，直至完全混合均匀，得溶液B；(3) While stirring, add the anti-SARS-CoV-2-S and N fusion protein-small molecule antibody Fab solution or dry powder to solution A, stir at low speed for 60 minutes, until completely mixed uniformly, to obtain solution B;
(4)一边搅拌一边将香精加入溶液B中，低速搅拌60min，直至完全溶解，得溶液C；(4) Add the essence to solution B while stirring, and stir at low speed for 60 minutes until it is completely dissolved to obtain solution C;
(5)用pH计测量溶液C的pH值，用柠檬酸调节pH值至6.8±0.1；(5) Measure the pH value of solution C with a pH meter, and adjust the pH value to 6.8±0.1 with citric acid;
(6)静置至上部泡沫全部消溶后，再将溶液C分装在清洗消毒过的滴鼻器或滴眼器以及鼻用喷雾瓶中，贴上标签出厂。(6) After the upper foam is completely dissolved, the solution C is dispensed into the cleaned and disinfected nasal drops or eye drops and nasal spray bottles, and the labels are attached to the factory.
实施例5：生产抗SARS-CoV-2-S和N融合蛋白-小分子抗体Fab口喷剂或漱口水Example 5: Production of anti-SARS-CoV-2-S and N fusion protein-small molecule antibody Fab mouth spray or mouthwash
配方：formula:
工艺：Process:
(1)将配方量K-30、S-40、吐温-80、薄荷油、香精用紫外光照射灭菌消毒24小时，无菌密封备用；(1) Sterilize the formula quantity K-30, S-40, Tween-80, peppermint oil, and essence with ultraviolet light for 24 hours, and seal aseptically for later use;
(2)按配方量蒸馏水加热至至90℃，再分别加入S-40和K-30分散剂，搅拌30分钟以上，溶解均匀；再冷却至60℃，分别缓慢加入吐温(滴加)和薄荷油以及甘油(滴加)，低速揽拌60分钟至充分溶解；然后，冷却至室温，形成溶液A；(2) Heat distilled water according to the formula to 90°C, then add S-40 and K-30 dispersants separately, stir for more than 30 minutes, and dissolve uniformly; then cool to 60°C, and slowly add Tween (dropwise) and Peppermint oil and glycerin (dropped), stir at low speed for 60 minutes until fully dissolved; then, cool to room temperature to form solution A;
(3)一边搅拌一边将抗SARS-CoV-2-S蛋白全序列多肽-小分子抗体Fab溶液或干粉加入溶液A中，低速搅拌60min，直至完全混合均匀，得溶液B；(3) While stirring, add the anti-SARS-CoV-2-S protein full-sequence peptide-small molecule antibody Fab solution or dry powder to solution A, stir at low speed for 60 minutes, until completely mixed uniformly, to obtain solution B;
(4)一边搅拌一边将香精加入溶液B中，低速搅拌60min，直至完全溶解，得溶液C；(4) Add the essence to solution B while stirring, and stir at low speed for 60 minutes until it is completely dissolved to obtain solution C;
(5)用pH计测量溶液C的pH值，用柠檬酸调节pH值至6.8±0.1；(5) Measure the pH value of solution C with a pH meter, and adjust the pH value to 6.8±0.1 with citric acid;
(6)静置至上部泡沫全部消溶后，再将溶液C分装在清洗消毒过的口腔用喷雾罐或漱口瓶中，贴上标签出厂。(6) After standing until the upper foam is completely dissolved, the solution C is divided into a cleaned and disinfected oral spray can or mouthwash bottle, and the label is attached to the factory.
实施例6：生产抗SARS-CoV-2受体结合域(RBD)-IgY洗手液Example 6: Production of anti-SARS-CoV-2 receptor binding domain (RBD)-IgY hand sanitizer
配方：formula:
| 原料raw material | 含量(％)content(%) |
| 抗SARS-CoV-2受体结合域(RBD)-IgYAnti-SARS-CoV-2 receptor binding domain (RBD)-IgY | 5.005.00 |
| 403(脂肪醇聚氧乙烯醚磺基琥珀酸单酯二钠)403 (fatty alcohol polyoxyethylene ether sulfosuccinic acid monoester disodium) | 33.4833.48 |
| 503(脂肪醇聚氧乙烯醚硫酸铵，浓度70％)503 (fatty alcohol polyoxyethylene ether ammonium sulfate, concentration 70%) | 20.5120.51 |
| 医药级甘油Pharmaceutical grade glycerin | 2.002.00 |
| 海藻精华Seaweed Extract | 0.100.10 |
| 香精(玫瑰花香精)Flavor (rose flavor) | 0.050.05 |
| 薄荷油peppermint | 0.05ml0.05ml |
| 蒸馏水Distilled water | 38.8138.81 |
工艺：Process:
(1)将配方量海藻精华、医药级甘油、薄荷油、香精以及403、503发泡剂用紫外光照射灭菌消毒24小时，无菌密封备用；(1) Sterilize the formula amount of seaweed extract, pharmaceutical grade glycerin, peppermint oil, flavor, and 403 and 503 foaming agents with ultraviolet light for 24 hours, and seal them aseptically for later use;
(2)将配方量蒸馏水加热至90℃，停留15分钟；再冷却至60℃，一边搅拌一边先后加入海藻精华和医药级甘油，低速揽拌30分钟，直至完全溶解，冷却至室温，得溶液A；(2) Heat the formula amount of distilled water to 90°C, stay for 15 minutes; then cool to 60°C, add seaweed extract and pharmaceutical grade glycerin one after another while stirring, stir at low speed for 30 minutes, until completely dissolved, cool to room temperature to obtain a solution A;
(3)一边搅拌一边将香精(玫瑰花香精)加入溶液A中，低速搅拌60min，直至完全溶解，得溶液B；(3) Add the flavor (rose flavor) to solution A while stirring, and stir at low speed for 60 minutes until it is completely dissolved to obtain solution B;
(4)将403加热至80℃，然后一边搅拌一边将503徐徐滴加入到403中，低速搅拌30min，得到溶液C；(4) Heat 403 to 80°C, then slowly add 503 to 403 dropwise while stirring, and stir at low speed for 30 minutes to obtain solution C;
(5)保持溶液C温度在80℃，一边搅拌一边将薄荷油徐徐滴加入到溶液C中，低速搅拌60min，得到溶液D；(5) Keep the temperature of solution C at 80°C, slowly add peppermint oil to solution C while stirring, and stir at low speed for 60 minutes to obtain solution D;
(6)将溶液D加热至90℃高温灭菌5min，然后冷却至室温；(6) Heat solution D to 90°C and sterilize for 5 minutes, and then cool to room temperature;
(7)一边搅拌一边将溶液B缓慢地加到溶液D中，低速搅拌60min；如未形成均质稳定的乳状溶液，则需延长搅拌时间，制得溶液E；(7) While stirring, slowly add solution B to solution D, and stir at low speed for 60 minutes; if a homogeneous and stable milky solution is not formed, the stirring time needs to be extended to obtain solution E;
(8)一边搅拌一边将抗SARS-CoV-2受体结合域(RBD)-IgY缓慢地加到溶液E中，低速搅拌60min；如未形成均质稳定的乳状溶液，则需延长搅拌时间，制得溶液F；(8) While stirring, slowly add the anti-SARS-CoV-2 receptor binding domain (RBD)-IgY to solution E, and stir at low speed for 60 minutes; if a homogeneous and stable emulsion solution is not formed, the stirring time needs to be extended. Prepare solution F;
(9)用pH计测量溶液F的pH值，采用柠檬酸或pH4.5磷酸氢二钠–柠檬酸缓冲液调节pH值至4.5±0.1；(9) Measure the pH value of solution F with a pH meter, and adjust the pH value to 4.5±0.1 with citric acid or pH4.5 disodium hydrogen phosphate-citric acid buffer;
(10)静置一夜，直至上部泡沫全部消溶后，再将溶液F液分装在清洗消毒过的洗手液容器中，贴上标签出厂。(10) Let it stand overnight until all the foam on the upper part is dissolved, then disperse the solution F into the cleaned and disinfected hand sanitizer container, label it and leave the factory.
实施例7：生产抗SARS-CoV-2-S蛋白复合抗原-IgY***片Example 7: Production of anti-SARS-CoV-2-S protein complex antigen-IgY lozenges
配方：formula:
工艺Craft
(1)山梨糖醇过60目筛两次备用；(1) Sorbitol is passed through a 60-mesh sieve twice for use;
(2)羧甲基纤维素分散于30％乙醇中制成1％乙醇溶液；(2) Carboxymethyl cellulose is dispersed in 30% ethanol to make a 1% ethanol solution;
(3)将(1)项用适量(2)项制软材，14目筛网制粒，60℃通风干燥，18目筛整粒；用40目筛筛出适量细粉与抗SARS-CoV-2-S蛋白复合抗原-IgY充分混匀；(3) Use an appropriate amount of (1) to make soft materials with a 14-mesh sieve for granulation, ventilate and dry at 60°C, and 18-mesh sieve for granulation; use a 40-mesh sieve to sieve out an appropriate amount of fine powder and anti-SARS-CoV -2-S protein complex antigen-IgY fully mixed;
(4)再拌入硬脂酸镁，一起与整批颗粒混合均匀，密闭4小时以上；(4) Stir in magnesium stearate again, mix it with the whole batch of granules together, and seal it for more than 4 hours;
(5)压片机压片，每片600mg；(5) Tablet pressing machine, each tablet is 600mg;
(6)检验合格后，包装，全验出厂。(6) After passing the inspection, the packaging will be fully inspected and shipped out of the factory.
实施例8：生产抗SARS-CoV-2-S蛋白复合抗原-IgY粉剂，供粉未雾化装置作药用粉剂。Example 8: Production of anti-SARS-CoV-2-S protein complex antigen-IgY powder, and the non-atomized device for powder supply is used as a medicinal powder.
配方：formula:
工艺：Process:
(1)将配方量抗SARS-CoV-2-S蛋白复合抗原-IgY干粉或抗SARS-CoV-2S蛋白复合抗原-IgY干粉和配方量药用葡萄糖混合，充分搅拌均匀；(1) Mix the formula amount of anti-SARS-CoV-2-S protein complex antigen-IgY dry powder or anti-SARS-CoV-2S protein complex antigen-IgY dry powder and formula amount of medicinal glucose, and stir well;
(2)将粉剂分装，检验出厂。(2) Divide the powder and inspect and leave the factory.
本发明所包含的信息，在未脱离上述权利要求的精神和保护范围下，本发明各种偏离精确的描述，对于与本发明相关的本领域技术人员来说是显而易见的。本发明并不认为限制在所定义的程序、性质或组成的范围内，因为优选的实施例和其他描述只用于说明目前提供发明的特定方面。对于在化学、生物化学或相关领域的技术人员来说，实现本发明于各种修改的描述模式，都应属于本发明所附权利要求的保护范围内。The information contained in the present invention, without departing from the spirit and protection scope of the above claims, the precise description of various deviations of the present invention will be obvious to those skilled in the art related to the present invention. The present invention is not considered to be limited to the scope of the defined procedures, properties or compositions, because the preferred embodiments and other descriptions are only used to illustrate specific aspects of the present invention. For those skilled in chemistry, biochemistry or related fields, the description modes for implementing the present invention in various modifications should fall within the protection scope of the appended claims of the present invention.
应当理解的是，对本领域普通技术人员来说，可以根据上述说明加以改进或变换，所有这些改进或变换都应属于本发明所附权利要求的保护范围之内。It should be understood that those of ordinary skill in the art can make improvements or changes based on the above description, and all these improvements or changes should fall within the protection scope of the appended claims of the present invention.
Claims (19)
- 一种抗冠状病毒的抗体制备方法，其特征在于，包括如下步骤：An anti-coronavirus antibody preparation method is characterized in that it comprises the following steps:S1、制备抗原：将目标冠状病毒的灭活的冠状病毒或冠状病毒蛋白成分加入佐剂，制得病毒抗原或蛋白抗原；S1. Preparation of antigen: adding the inactivated coronavirus or coronavirus protein component of the target coronavirus to an adjuvant to prepare a virus antigen or protein antigen;S2、制备免疫蛋：将所述病毒抗原或蛋白抗原免疫产蛋禽类(包括鸡、鸵鸟、鸭、鹅)，得到抗冠状病毒的免疫蛋；S2. Preparation of immune eggs: immunizing egg-laying poultry (including chickens, ostriches, ducks, and geese) with the virus antigen or protein antigen to obtain immune eggs against coronavirus;S3、分离抗体：从所述免疫蛋中分离抗所述目标冠状病毒的IgY抗体。S3. Isolation of antibodies: IgY antibodies against the target coronavirus are separated from the immunized eggs.
- 根据权利要求1所述的抗体制备方法，其特征在于，所述步骤S1中，所述抗原成分为如下中的至少一种：灭活的冠状病毒液、冠状病毒重组S蛋白、冠状病毒重组S1蛋白、冠状病毒重组S2蛋白、冠状病毒的受体结合域RBD重组蛋白、冠状病毒重组N蛋白、冠状病毒重组S蛋白片段和N蛋白片段的融合蛋白。The antibody preparation method according to claim 1, wherein in the step S1, the antigen component is at least one of the following: inactivated coronavirus liquid, coronavirus recombinant S protein, coronavirus recombinant S1 Protein, coronavirus recombinant S2 protein, coronavirus receptor binding domain RBD recombinant protein, coronavirus recombinant N protein, coronavirus recombinant S protein fragment and fusion protein of N protein fragment.
- 根据权利要求2所述的抗体制备方法，其特征在于，所述灭活的冠状病毒液使用如下方法制备：The antibody preparation method according to claim 2, wherein the inactivated coronavirus liquid is prepared by the following method:(1)使用目标冠状病毒感染体外培养用的细胞株；(1) Use the target coronavirus to infect cell lines for in vitro culture;(2)选择病毒感染量和培养条件培养目标冠状病毒，将收获的病毒反复冻融裂解备用；(2) Select the virus infection and culture conditions to cultivate the target coronavirus, and freeze the harvested virus repeatedly for use;(3)灭活病毒：在不断搅拌病毒液的条件下,缓慢加入灭活剂来灭活病毒。(3) Inactivate the virus: Under the condition of constantly stirring the virus liquid, slowly add an inactivating agent to inactivate the virus.
- 根据权利要求2所述的抗体制备方法，其特征在于，所述冠状病毒S蛋白为基因重组表达的冠状病毒S蛋白全序列多肽，偶联KLH和BSA获得的重组多肽蛋白。The antibody preparation method according to claim 2, wherein the coronavirus S protein is a full-sequence polypeptide of the coronavirus S protein expressed by genetic recombination, which is a recombinant polypeptide protein obtained by coupling KLH and BSA.
- 根据权利要求2所述的抗体制备方法，其特征在于，所述冠状病毒重组S1蛋白、冠状病毒重组S2蛋白使用如下方法制备：The antibody preparation method according to claim 2, wherein the coronavirus recombinant S1 protein and coronavirus recombinant S2 protein are prepared using the following method:(1)分别表达冠状病毒S1蛋白免疫优势决定区多肽和冠状病毒S2蛋白免疫优势决定区多肽；(1) Express the immunodominance determining region polypeptide of the coronavirus S1 protein and the immunodominance determining region polypeptide of the coronavirus S2 protein respectively;(2)分别与KLH和BSA偶联成多肽，获得冠状病毒重组S1蛋白重组冠状病毒重组S1蛋白。(2) Coupling with KLH and BSA to form polypeptides to obtain recombinant coronavirus S1 protein.
- 根据权利要求2所述的抗体制备方法，其特征在于，所述冠状病毒的受体结合域RBD重组蛋白使用如下方法制备：The antibody preparation method according to claim 2, wherein the RBD recombinant protein of the coronavirus receptor binding domain is prepared using the following method:(1)构建PET32(a)-RBD重组质粒；(1) Construction of PET32(a)-RBD recombinant plasmid;(2)用原核或真核表达***进行表达；(2) Use prokaryotic or eukaryotic expression system for expression;(3)运用Fmoc固相法或其它方法合成肽链；(3) Use Fmoc solid-phase method or other methods to synthesize peptide chains;(4)与KLH和BSA偶联成多肽。(4) Coupling with KLH and BSA to form a polypeptide.
- 根据权利要求2所述的抗体制备方法，其特征在于，所述冠状病毒N蛋白为基因重组表达的冠状病毒N蛋白全序列多肽，偶联KLH和BSA获得的重组N蛋白。The antibody preparation method according to claim 2, wherein the coronavirus N protein is a full-sequence polypeptide of the coronavirus N protein expressed by genetic recombination, and is a recombinant N protein obtained by coupling KLH and BSA.
- 根据权利要求2所述的抗体制备方法，其特征在于，所述冠状病毒S蛋白片段和N蛋白片段的融合蛋白使用如下方法制备：The antibody preparation method according to claim 2, wherein the fusion protein of the coronavirus S protein fragment and N protein fragment is prepared using the following method:(1)化学合成S蛋白片段和N蛋白片段的基因序列；(1) The gene sequence of chemically synthesized S protein fragment and N protein fragment;(2)将两个基因片段串联,克隆至质粒的表达位点；(2) Connect the two gene fragments in series and clone them to the expression site of the plasmid;(3)将重组质粒转化原核或真核表达***，筛选获得高效表达的工程菌，表达和纯化冠状病毒S蛋白片段和N蛋白片段融合蛋白。(3) Transform the recombinant plasmid into a prokaryotic or eukaryotic expression system, screen and obtain highly expressed engineering bacteria, and express and purify the fusion protein of the coronavirus S protein fragment and N protein fragment.(4)与KLH和BSA偶联，获得冠状病毒S蛋白片段和N蛋白片段的融合蛋白抗原成份。(4) Coupling with KLH and BSA to obtain the fusion protein antigen component of the coronavirus S protein fragment and N protein fragment.
- 根据权利要求1所述的抗体制备方法，其特征在于，所述步骤S1中，所述佐剂为福氏佐剂或其它佐剂，所述灭活的冠状病毒液或重组蛋白成分与所述福氏佐剂经过高速匀浆后形成所述抗原，所述抗原为油包水乳液。The antibody preparation method according to claim 1, wherein in the step S1, the adjuvant is Freund’s adjuvant or other adjuvants, and the inactivated coronavirus liquid or recombinant protein component is combined with the Freund's adjuvant is homogenized at a high speed to form the antigen, and the antigen is a water-in-oil emulsion.
- 根据权利要求1所述的抗体制备方法，其特征在于，所述步骤S2中，每隔两周免疫注射一次产蛋禽类，共注射2-5次，最后一次注射后的至少第12天后检取产蛋禽类所产免疫蛋。The antibody preparation method according to claim 1, characterized in that, in the step S2, the laying poultry is immunized every two weeks for a total of 2-5 injections, and at least the 12th day after the last injection is taken. Immune eggs produced by laying poultry.
- 根据权利要求1所述的抗体制备方法，其特征在于，所述步骤S3中，采用纯水提取法、氯仿萃取法、冷乙醇沉淀法或硫酸铵沉淀法分离抗所述目标冠状病毒的IgY粗提物。The antibody preparation method according to claim 1, wherein in step S3, pure water extraction, chloroform extraction, cold ethanol precipitation, or ammonium sulfate precipitation is used to separate crude IgY against the target coronavirus. Extract.
- 根据权利要求1所述的抗体制备方法，其特征在于，所述纯水提取法包括如下步骤：The antibody preparation method according to claim 1, wherein the pure water extraction method comprises the following steps:(1)取所述免疫蛋的蛋黄，搅拌均匀；(1) Take the yolk of the immune egg and stir it evenly;(2)再按蛋黄液体积的3-8倍加入蒸馏水，进行稀释并混合均匀，调pH至5.5-6.5；(2) Add distilled water at 3-8 times the volume of the egg yolk, dilute and mix well, and adjust the pH to 5.5-6.5;(3)冷却至2-6℃，静置12-24小时后高速离心；取离心分离所得的上清液置超滤器中进行超滤浓缩10-20倍；(3) Cool to 2-6°C, stand for 12-24 hours and then centrifuge at high speed; take the supernatant obtained from centrifugal separation and place it in an ultrafilter for ultrafiltration and concentration 10-20 times;(4)加入浓度为1.0-3.0％的海藻酸钠液，缓慢添加海藻酸钠液至终浓度为0.1-0.2％，搅拌至出现浑浊；(4) Add sodium alginate solution with a concentration of 1.0-3.0%, slowly add sodium alginate solution to a final concentration of 0.1-0.2%, and stir until turbidity appears;(5)再加入1.0-3.0％CaCl 2液，至终浓度为0.1-0.2％，搅拌均匀，3-4℃静置8-12小时； (5) Add 1.0-3.0% CaCl 2 solution to the final concentration of 0.1-0.2%, stir evenly, and let stand at 3-4°C for 8-12 hours;(6)高速离心并取上清液，得到IgY粗提物。(6) Centrifuge at high speed and take the supernatant to obtain the crude IgY extract.
- 根据权利要求12所述的抗体制备方法，其特征在于，所述IgY粗提物先后分别过离子交换柱和凝胶层析柱或者亲和层析柱层析，得到抗冠状病毒纯IgY溶液。The antibody preparation method according to claim 12, characterized in that the crude IgY extract is passed through an ion exchange column and a gel chromatography column or an affinity chromatography column respectively to obtain an anti-coronavirus pure IgY solution.
- 根据权利要求13所述的抗体制备方法，其特征在于，还包括把抗冠状病毒IgY酶解制备一种或多种复合抗SARS-CoV-2或其它冠状病毒小分子抗体Fab。The antibody preparation method according to claim 13, characterized in that it further comprises enzymatically hydrolyzing the anti-coronavirus IgY to prepare one or more composite anti-SARS-CoV-2 or other coronavirus small molecule antibody Fabs.
- 根据权利要求13和14所述的抗体制备方法，其特征在于，还包括如下步骤：The antibody preparation method according to claims 13 and 14, characterized in that it further comprises the following steps:将所述纯IgY溶液和小分子抗体Fab制备成IgY干粉、长效化小分子抗体Fab、小分子抗体Fab干粉中的至少一种。The pure IgY solution and the small molecule antibody Fab are prepared into at least one of IgY dry powder, long-acting small molecule antibody Fab, and small molecule antibody Fab dry powder.
- 含有权利要求1-15中所制备抗体的组合物。A composition containing the antibody prepared in claims 1-15.
- 根据权利要求16所述的组合物，其特征在于，还包括赋形剂、填充剂、溶剂、助溶剂、表面活性剂、胶囊辅料、化学药、中药中一种或多种。The composition according to claim 16, characterized in that it further comprises one or more of excipients, fillers, solvents, co-solvents, surfactants, capsule excipients, chemical medicines, and traditional Chinese medicines.
- 根据权利要求16所述的组合物，其特征在于，所述组合物制成雾化吸入疗法的雾化剂、喷雾剂、口喷剂、鼻喷剂、滴鼻剂、滴眼剂、空气消毒剂、空气清新剂、消毒剂、洗手液、粉剂、片剂、***片、口服液、口服剂、胶囊剂中的至少一种。The composition according to claim 16, wherein the composition is made into an atomizer, spray, mouth spray, nasal spray, nasal drops, eye drops, air disinfection for nebulized inhalation therapy At least one of medicines, air fresheners, disinfectants, hand sanitizers, powders, tablets, buccal tablets, oral liquids, oral preparations, and capsules.
- 权利要求1-17中制备的抗体和组合物，在制备用于防治冠状病毒感染的药物、消毒产品、保健品或医疗器械中的应用。The antibody and composition prepared in claims 1-17 are used in the preparation of drugs, disinfection products, health products or medical devices for preventing and treating coronavirus infection.
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| WO2023019724A1 (en) * | 2021-08-17 | 2023-02-23 | 上海浙江大学高等研究院 | Monoclonal antibody 35b5, preparation method therefor, and use thereof |
| CN116162156A (en) * | 2022-11-21 | 2023-05-26 | 中国科学院微生物研究所 | Preparation and application of influenza and new crown bigeminal polyclonal antibody |
| US11732030B2 (en) | 2020-04-02 | 2023-08-22 | Regeneron Pharmaceuticals, Inc. | Anti-SARS-CoV-2-spike glycoprotein antibodies and antigen-binding fragments |
| US11999777B2 (en) | 2021-06-02 | 2024-06-04 | Regeneron Pharmaceuticals, Inc. | Methods for treating or preventing SARS-CoV-2 infections and COVID-19 with anti-SARS-CoV-2 spike glycoprotein antibodies |
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| CN1450086A (en) * | 2003-05-13 | 2003-10-22 | 雅臣药业集团（远东）有限公司 | Anti SARS specificity IgY and combination preparation thereof |
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