CN113201068A - Gene recombination anti 2019-nCoV and other coronavirus IgY and small molecule antibody and application thereof - Google Patents
Gene recombination anti 2019-nCoV and other coronavirus IgY and small molecule antibody and application thereof Download PDFInfo
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- 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]
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- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
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Abstract
The invention relates to a gene recombinant anti-2019-nCoV and other coronavirus IgY and a micromolecule antibody thereof, and a preparation method and application thereof. The anti-2019-nCoV and other coronavirus IgY and the micromolecular antibody thereof can directly inhibit the '2019-nCoV' and other coronaviruses, and block a key protein S protein and an RBD structure domain thereof which act on the coronaviruses and host cells, so that the key protein S protein and the RBD structure domain thereof cannot be combined with a cell membrane receptor ACE 2; thus, infection of respiratory epithelial cells by "2019-nCoV" and other coronaviruses was prevented. At the same time, these antibodies can also neutralize coronaviruses excreted by infected cells, blocking secondary infection and spread. The invention also provides an atomizing agent, a spraying agent, an oral spraying agent, a nasal drop, an eye drop, an air disinfectant, a hand sanitizer, powder, a tablet, a buccal tablet, oral liquid and a capsule which are prepared from the anti-2019-nCoV and other coronavirus IgY and micromolecule antibodies thereof, and the atomizing agent, the spraying agent, the nasal drop, the eye drop, the air disinfectant, the hand sanitizer, the powder, the tablet, the buccal tablet, the oral liquid and the capsule are applied to medicines, disinfection products, health care products and medical devices for preventing and treating the infection of the 2019-nCoV and other coronavirus.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a gene recombinant anti-2019-nCoV and other coronavirus IgY and a small molecule antibody thereof, and a preparation method and application thereof.
Background
Human infecting coronavirus was first isolated by scientists in the 60's 20 th century, and is named "coronavirus" because the corona structure of the virus' exterior can be observed under an electron microscope.
Coronavirus is not only one of the main pathogens of the common cold, but also the chief culprits of several serious epidemics, including severe acute respiratory syndrome coronavirus abused in 2003, and middle east respiratory syndrome coronavirus affecting saudi arabia, korea, etc. in the last few years.
After a person is infected with coronavirus, fever, cough, shortness of breath and dyspnea occur, so that viremia, pulmonary diseases, pneumonia, acute respiratory syndrome and the like are caused. The virus can also trigger 'cytokine storm' (cytokine storm), attack normal organs of human, and cause organ failure such as lung, kidney and the like and even death.
The world health organization indicates that there is no vaccine for the novel coronavirus, which cannot be prevented in advance, nor is there an effective therapeutic agent.
The world health organization warns that some known coronaviruses are transmitted in animals, but have not yet infected humans; as global monitoring efforts improve, more coronaviruses may be discovered and the struggle between humans and coronaviruses is far reaching.
Therefore, research and development of a new drug capable of effectively preventing and treating the '2019-nCoV' and other coronavirus infection diseases are great matters related to human health and life, and an important subject before the international medical science and science interface.
Disclosure of Invention
The invention aims to provide a gene recombinant anti-2019-nCoV and other coronavirus IgY and a small molecule antibody thereof, a preparation method and application thereof, and solves the problem that no medicine capable of effectively preventing and treating the 2019-nCoV and other coronavirus infection diseases exists in the prior art.
The technical scheme adopted by the invention for solving the technical problem is as follows: a gene recombination anti-2019-nCoV and other coronavirus specific IgY, a gene recombination anti-2019-nCoV and other coronavirus small molecule antibody, wherein the gene recombination anti-2019-nCoV and other coronavirus specific IgY and small molecule antibody thereof are prepared by the following method:
firstly, preparing an antigen:
the invention adopts the following method to prepare the antigen for immunization:
(one) preparation of antigen component
1. Inactivating "2019-nCoV" or other coronavirus antigen components
(1) Vero, Vero-E6 and 2BS cell lines were infected with "2019-nCoV" or other coronaviruses.
(2) Preferably, the optimal virus infection amount and the optimal culture conditions are selected, the '2019-nCoV' or other coronaviruses are cultured, and the harvested viruses are placed in a refrigerator at the temperature of-20 ℃ for repeated freeze thawing and cell lysis for 3 times for standby.
(3) Inactivating the virus: the beta-propiolactone and the like can be used as an inactivator, the inactivator is slowly added under the condition of continuously stirring the virus liquid, the effect is realized at 4-10 ℃, and then water bath is carried out at about 37 ℃. The inactivation effect was tested by passage 3 in succession using Vero cells, all performed in a biosafety cabinet in the P3 laboratory.
2. Genetically engineered recombinant antigen components
The invention takes the '2019-nCoV' coronavirus recombinant antigen component as an example for illustration, and the preparation of other coronavirus genetic engineering recombinant antigen components can be carried out by the same method as follows, and the specific operation process is not repeated here.
(1) Gene recombination "2019-nCoV" or other coronavirus S protein antigen components
Studies reveal that the S protein of '2019-nCoV' and other coronaviruses plays an important role in the process of binding the viruses with host cell surface receptors and mediating membrane fusion into cells, and is also the main antigen protein of '2019-nCoV' and other coronaviruses. The S protein fixes the whole S protein on the virus outer shell membrane through the membrane penetrating part of S2, and is fused with the host cell membrane, and S1 is related to the recognition and combination of virus and receptor; blocking the binding site of the S protein prevents the virus from infecting the cell; thus, the present invention selects the S protein as the major recombinant protein antigenic component.
The specific operation is as follows:
polypeptide of the complete sequence of the S protein of "2019-nCoV" or other coronaviruses
a. The SARS-CoVS protein sequence was obtained from Gen Bank with a total of 1256 amino acids. Selecting Hopp & Woods Hydrophilicity parameter (hydrophicity) and Willing Antigenicity parameter (Antigenicity) by using DNAStar software to perform single-parameter prediction, and selecting an antigenic polypeptide sequence with Hydrophilicity and strong Antigenicity according to the analysis result of software
b. Carrier protein coupled antigen epitope polypeptide
Polypeptide synthesis
Cleavage and deprotection
HPLC purification
Analysis of amino acid composition
Modified synthetic polypeptides
Coupling KLH and BSA to obtain recombinant polypeptide protein
S1 protein immunodominant determinant polypeptide of "2019-nCoV" or other coronaviruses
a. The SARS-CoVS protein sequence was obtained from Gen Bank with a total of 1256 amino acids. Selecting Hopp & Woods Hydrophilicity parameter (hydrophicity) and Willing Antigenicity parameter (Antigenicity) by using DNAStar software to perform single-parameter prediction, and selecting S1 protein immunodominance decision region polypeptide sequence according to software analysis result
b. Carrier protein coupled antigen epitope polypeptide
Polypeptide synthesis
Cleavage and deprotection
HPLC purification
Analysis of amino acid composition
Modified synthetic polypeptides
Coupling KLH and BSA to obtain recombinant immunodominance decision region (510-672) polypeptide protein
c. Amplification expression of polypeptide protein of S1 region of '2019-nCoV' or other coronavirus by using prokaryotic or eukaryotic expression system
I. Construction of a Gene-segmented recombinant plasmid of the S1 region of "2019-nCoV" or other coronaviruses
Expression with prokaryotic or eukaryotic expression systems
Peptide chain synthesis using Fmoc solid phase method or other methods
Conjugation to KLH and BSA to Polypeptides
Polypeptide of immunodominant determinant of protein S2 of "2019-nCoV" or other coronaviruses
Studies have revealed that the C-terminal repeat of the S2 protein of "2019-nCoV" or other coronaviruses binds to the cell membrane receptor ACE2 and fuses the viral envelope and the cell membrane, thereby allowing the virus to enter the cell and become infected; therefore, the S2 protein was selected as the recombinant protein antigenic component. The specific operation is as follows:
a. the SARS-CoVS protein sequence was obtained from Gen Bank with a total of 1256 amino acids. Selecting Hopp & Woods Hydrophilicity parameter (hydrophicity) and Willing Antigenicity parameter (Antigenicity) by using DNAStar software to perform single-parameter prediction, and selecting S2 protein immunodominance decision region polypeptide sequence according to the analysis result of the software
b. Carrier protein coupled antigen epitope polypeptide
Polypeptide synthesis
Cleavage and deprotection
HPLC purification
Analysis of amino acid composition
Modified synthetic polypeptides
Coupling KLH and BSA to obtain recombinant S2 protein immunodominance decision region polypeptide protein
c. Amplification expression of polypeptide protein of S2 region of '2019-nCoV' or other coronavirus by using prokaryotic or eukaryotic expression system
I. Construction of a Gene-segmented recombinant plasmid of the S2 region of "2019-nCoV" or other coronaviruses
Expression with prokaryotic or eukaryotic expression systems
Peptide chain synthesis using Fmoc solid phase method or other methods
Conjugation to KLH and BSA to Polypeptides
Recombinant poly-R-binding domain (RBD) fragments of "2019-nCoV" or other coronaviruses
Peptide proteins
The Receptor Binding Domain (RBD) of the '2019-nCoV' or other coronaviruses contains the main neutralizing antigenic determinant of the S protein, mediates the interaction of the viruses and target cells, can influence the binding of the viruses and the target cells, and plays an important role in the spread of the virus across species. Thus, the present invention selects the Receptor Binding Domain (RBD) as an important antigenic component of a recombinant protein.
a. Construction of PET32(a) -RBD recombinant plasmid
b. Expression using prokaryotic or eukaryotic expression systems
c. Peptide chain synthesis by Fmoc solid phase method or other methods
d. Coupling with KLH and BSA to Polypeptides
(2) Gene recombination "2019-nCoV" or other coronavirus N protein antigen components
The '2019-nCoV' and other coronavirus N proteins are important structural proteins, are positioned in the core part of virus particles, are combined with virus RNA to form a complex, have important significance for the recognition of virus genome RNA characteristic sequences and the interaction with other structural proteins, and play an important role in the processes of virus assembly and the like; therefore, the present invention selects the N protein as an important recombinant protein antigen component.
The specific operation is as follows:
escherichia coli expressing '2019-nCoV' or other coronavirus GST-N proteins is subjected to ultrasonic disruption, centrifuged to take supernatant, and purified by a GST fusion protein purification kit for identification. Expressing the supernatant with an empty vector, centrifuging, and purifying; then, coupling with KLH and BSA to obtain purified "2019-nCoV" or other coronavirus N protein antigen components.
(3) Gene recombination "2019-nCoV" or fusion protein antigen component of S protein segment and N protein segment of other coronavirus
The specific operation is as follows:
A. the amino acid sequences of the S protein and the N protein of the '2019-nCoV' or other coronaviruses are analyzed by a computer to determine the S protein fragment and the N protein fragment containing strong antigen epitopes.
B. Gene sequences for chemical synthesis of S protein fragments and N protein fragments
C. The two gene fragments were connected in series and cloned into the Nco I/EcoRI site in the plasmid pET28a (+) to express a fusion protein of the S protein fragment and the N protein fragment.
D. And transforming the recombinant plasmid into escherichia coli BL21(DE3), and screening to obtain engineering bacteria for efficiently expressing '2019-nCoV' or other coronavirus S protein fragment and N protein fragment fusion proteins.
E. And (3) centrifuging the engineering bacteria for inducing expression of the fusion protein, collecting supernatant, filtering, and purifying by adopting an S-Sepearse FF cation column.
F. The purified fusion protein of S protein and N protein is dialyzed and then purified by reversed phase high pressure liquid phase.
G. Coupled with KLH and BSA, the fusion protein antigen component of the S protein segment and the N protein segment of the '2019-nCoV' or other coronaviruses is obtained.
(II) preparation of antigen for immunization
1. Preparation of inactivated "2019-nCoV" or other coronavirus antigens
Adding the prepared inactivated "2019-nCoV" or other coronavirus solution into Freund's adjuvant or other adjuvants at a ratio of 1-10:1-10 (usually 1:1), and homogenizing at high speed at 30,000rpm in a high-speed homogenizer to form water-in-oil emulsion, to obtain virus protein antigen.
2. Genetically engineered expression of protein antigens
The specific implementation method comprises the following steps:
(1) "2019-nCoV" or other coronavirus S protein complete sequence antigen
Adding Freund' S adjuvant or other adjuvants into the prepared genetic engineering expression "2019-nCoV" or other coronavirus S protein complete sequence antigen component according to the ratio of 1-10:1-10 (usually 1:1), and placing into a high-speed homogenizer to homogenize at high speed of 30,000rpm to form water-in-oil emulsion, thus obtaining the "2019-nCoV" or other coronavirus S protein complete sequence antigen.
(2) "2019-nCoV" or other coronavirus S1 protein and S2 protein composite antigen
Mixing the prepared B, C following two genetic engineering expression protein antigen components uniformly according to the proportion of 1-10:1-10, and preparing 2019-nCoV or other coronavirus S protein composite antigen component mixed liquor; then adding Freund' S adjuvant or other adjuvants at a ratio of 1-10:1-10 (usually 1:1), and homogenizing at high speed at 30,000rpm in a high speed homogenizer to form water-in-oil emulsion, to obtain "2019-nCoV" or other coronavirus S protein complex antigen.
S1 protein immunodominant determinant polypeptide of "2019-nCoV" or other coronaviruses
Polypeptide of immunodominant determinant of protein S2 of "2019-nCoV" or other coronaviruses
(3) Recombinant polypeptide protein antigen of Receptor Binding Domain (RBD) fragment of '2019-nCoV' or other coronaviruses
Adding Freund's adjuvant or other adjuvants into the prepared recombinant polypeptide protein antigen component of the Receptor Binding Domain (RBD) fragment of the ' 2019-nCoV ' or other coronaviruses according to the ratio of 1-10:1-10 (usually 1:1), and placing the mixture into a high-speed homogenizer to homogenize at high speed at 30,000rpm to form water-in-oil emulsion, namely preparing the recombinant polypeptide protein antigen of the Receptor Binding Domain (RBD) fragment of the ' 2019-nCoV ' or other coronaviruses.
(4) Gene recombination "2019-nCoV" or other coronavirus N protein antigen
Adding Freund's adjuvant or other adjuvants into the prepared "2019-nCoV" or other coronavirus N protein antigen components at a ratio of 1-10:1-10 (usually 1:1), and homogenizing at high speed at 30,000rpm in a high-speed homogenizer to form water-in-oil emulsion, thereby preparing "2019-nCoV" or other coronavirus N protein antigens.
(5) Fusion protein antigen of gene recombination '2019-nCoV' or other coronavirus S protein fragment and N protein fragment
Adding Freund ' S adjuvant or other adjuvants into the prepared fusion protein antigen components of the ' 2019-nCoV ' or other coronavirus S protein fragment and N protein fragment in a ratio of 1-10:1-10 (usually 1:1), and homogenizing at high speed of 30,000rpm in a high-speed homogenizer to form water-in-oil emulsion, thus obtaining the ' 2019-nCoV ' or other coronavirus fusion protein antigen.
Secondly, preparing immune eggs containing high-activity antibodies
The invention can be used for preparing immune eggs by immunizing any laying poultry, is limited by the space and only takes hen as an example, and other poultry can be completely carried out by the same method as described below.
The '2019-nCoV' or other coronavirus inactivated virus antigen and the five gene recombination '2019-nCoV' or other coronavirus protein antigen prepared by the method are respectively adopted to immunize laying hens, the injection is performed once every two weeks for 2-5 times, the immune eggs laid by the laying hens are detected at least 12 days after the last injection, and the coding and marking are performed to obtain six anti- '2019-nCoV' or other coronavirus IgY immune eggs.
Thirdly, preparing crude IgY extract resisting '2019-nCoV' or other coronaviruses
Preparing the crude extract of the IgY resisting the 2019-nCoV or other coronaviruses by a pure water extraction method, a chloroform extraction method, a cold ethanol precipitation method or an ammonium sulfate precipitation method. The invention is illustrated by taking a pure water extraction method as an example, and other preparation methods refer to conventional methods for operation, and the specific operation process is not repeated here.
The specific operation method for preparing the crude extract of the anti-2019-nCoV or other coronavirus IgY by the pure water extraction method is as follows:
washing the prepared anti-2019-nCoV or other coronavirus IgY immune eggs with flowing water, scrubbing and sterilizing with alcohol, breaking the anti-2019-nCoV or other coronavirus IgY immune eggs with a eggbeater, filtering out egg white by using a yolk sieve, leaving yolk, and stirring uniformly; according to the volume of egg yolk liquidAdding distilled water 3-8 times the amount of the filtrate, diluting, mixing, and adjusting pH to 5.5-6.5 with 1.0N HCI solution; further fully and uniformly stirring the diluent with the adjusted pH value, then cooling the diluent to 2-6 ℃, and standing for 12-24 hours; centrifuging the diluted solution at high speed; taking supernatant obtained by separation, and placing the supernatant into an ultrafilter for ultrafiltration and concentration by 10-20 times; then adding sodium alginate solution with the concentration of 1.0-3.0%, slowly adding the sodium alginate solution until the final concentration is 0.1-0.2%, and stirring until turbidity appears; then adding 1.0-3.0% of CaCl2Mixing the solution until the final concentration is 0.1-0.2%, stirring, and standing at 3-4 deg.C for 8-12 hr; high-speed centrifugation is carried out, and supernatant is taken, thus obtaining the crude anti-2019-nCoV or other coronavirus IgY extract.
The method comprises the following steps:
1. anti-2019-nCoV or other coronavirus antigen IgY
2. anti-2019-nCoV or other coronavirus S-IgY
3. Anti "2019-nCoV" or other coronaviruses (S1+ S2) -IgY
4. anti-2019-nCoV or other coronavirus RBD-IgY
5. anti-2019-nCoV or other coronavirus N protein antigen IgY
6. anti-2019-nCoV or other coronavirus (S + N) fusion protein IgY
2-6 arbitrary combinations of the six anti-2019-nCoV or other coronavirus IgY can be mixed into the anti-2019-nCoV or other coronavirus composite IgY according to a certain proportion.
The following are examples of two combinations:
(anti-2019-nCoV or other coronavirus antigen IgY) + (anti-2019-nCoV or other coronavirus S-IgY)
(anti-2019-nCoV or other coronavirus antigen-IgY) + (anti-2019-nCoV or other coronavirus RBD-IgY)
(anti-2019-nCoV or other coronavirus S-IgY) + (anti-2019-nCoV or other coronavirus RBD-IgY)
4. [ ANTI-2019-nCoV OR OTHER CORONAVIRUS (S1+ S2) -IgY ] + [ ANTI-2019-nCoV OR OTHER CORONAVIRUS RBD-IgY ]
(anti-2019-nCoV or other coronavirus S-IgY) + (anti-2019-nCoV or other coronavirus N-IgY)
6. [ ANTI-2019-nCoV OR OTHER CORONAVIRUS (S + N) FUSION PROTEIN IgY ] + (ANTI-2019-nCoV OR OTHER CORONAVIRUS RBD-IgY)
The following are examples of 3 combinations:
(anti-2019-nCoV or other coronavirus antigen IgY) + (anti-2019-nCoV or other coronavirus S-IgY) + (anti-2019-nCoV or other coronavirus RBD-IgY)
(anti-2019-nCoV or other coronavirus S-IgY) + (anti-2019-nCoV or other coronavirus RBD-IgY) + (anti-2019-nCoV or other coronavirus N-IgY)
In practical applications, the above anti-2019-nCoV or other coronavirus IgY can be used in any combination according to requirements.
3. [ ANTI-2019-nCoV OR OTHER CORONAVIRUS (S1+ S2) -IgY ] + [ ANTI-2019-nCoV OR OTHER CORONAVIRUS RBD-IgY ] + (ANTI-2019-nCoV OR OTHER CORONAVIRUS N-IgY)
Fourthly, preparing anti-2019-nCoV or other coronavirus pure IgY solution or dry powder
The prepared crude IgY extract of one or more compound anti-2019-nCoV or other coronaviruses is dissolved in M PB phosphate buffer solution with the pH value of 7.0 and 0.01mol/L, and then the crude IgY extract is respectively subjected to ion exchange column chromatography and gel chromatography column chromatography or affinity chromatography column chromatography to obtain the pure IgY solution of the anti-2019-nCoV or other coronaviruses. The anti-2019-nCoV or other coronavirus pure IgY solution can be further subjected to freeze drying or spray drying at medium or low temperature or fluidized bed drying and other drying modes without influencing the activity of the antibody to prepare anti-2019-nCoV or other coronavirus pure IgY dry powder.
Fifthly, preparing anti-2019-nCoV or other coronavirus pure IgY nano dry powder
There are various methods for preparing the IgY nano dry powder, and the present invention is illustrated as follows by one of the methods:
the prepared one or more composite anti-2019-nCoV or other coronavirus pure IgY nano dry powder is added into a superfine pulverizer to be ground and pulverized, and the obtained mixture is processed into superfine particles with the size of 1-100nm and the particle size of more than or equal to 15000 meshes, so that the corresponding nano anti-2019-nCoV or other coronavirus pure IgY nano dry powder is prepared.
Sixthly, preparing nano liposome anti 2019-nCoV or other coronavirus pure IgY dry powder
There are various methods for preparing nanoliposome IgY, and the present invention is illustrated by one of the methods as follows:
1. mixing lecithin and cholesterol 50% according to the weight ratio of 1:5, dissolving in diethyl ether;
2. adding 4mmol/L Phosphate Buffer Solution (PBS) into the prepared anti-2019-nCoV or other coronavirus pure IgY nano dry powder according to the proportion of 1:100 or other proper proportions to prepare an IgY solution with the concentration of 1%;
3. lecithin and cholesterol in ether solution and IgY in PBS solution were mixed as 3: 1 proportion or other proper proportions are mixed evenly;
4. ultrasonic treatment for 2min (0.5 min per treatment, intermittent. 0.5 min);
5. performing rotary evaporation in water bath under reduced pressure until the gel is in gel state, performing vortex oscillation to allow the gel to phase-convert, and continuously evaporating to remove ether;
6. ultracentrifugation (35000r/min, 30min) is carried out to separate and remove the unincorporated IgY;
7. washing the precipitate twice with water, and centrifuging in a high-speed centrifuge to obtain precipitate;
8. diluting the obtained precipitate with 10mmol/L PBS to obtain nanometer liposome anti-2019-nCoV or other coronavirus pure IgY solution; the solution is dried by adopting freeze drying, medium-low temperature spray drying or fluidized bed drying and other drying modes which do not influence the activity of the antibody, and then the nanoliposome anti-2019-nCoV or other coronavirus pure IgY dry powder is obtained.
Seventhly, preparing one or more compound anti-2019-nCoV or other coronavirus small molecule antibody Fab
Adjusting the pH of one or more composite anti-2019-nCoV or other coronavirus pure IgY solutions to 3.0-5.0, and adding catalytic protease according to the mass ratio of 0.01-0.1%; fully stirring and dissolving to generate enzymatic reaction, then carrying out low-temperature high-speed rotation and centrifugation, removing precipitate to obtain supernatant, and carrying out ultrafiltration on the supernatant to obtain concentrated solution; and performing gel chromatography on the concentrated solution obtained after ultrafiltration, and dialyzing and concentrating the chromatography collection to obtain one or more compound anti-2019-nCoV or other coronavirus small molecule antibody Fab.
The method comprises the following steps:
1. anti-2019-nCoV or other coronavirus virus antigen small-molecule antibody Fab
2. anti-2019-nCoV or other coronavirus S-small molecule antibody Fab
3. anti-2019-nCoV or other coronavirus (S1+ S2) -small molecule antibody Fab
4. anti-2019-nCoV or other coronavirus RBD-small molecule antibody Fab
5. Small-molecule antibody Fab against N protein antigen of '2019-nCoV' or other coronaviruses
6. Small antibody Fab against "2019-nCoV" or other coronavirus (S + N) fusion proteins
2-6 arbitrary combinations of the six anti-2019-nCoV or other coronavirus IgY can be mixed into the anti-2019-nCoV or other coronavirus composite small molecule antibody Fab according to a certain proportion.
The following are examples of two combinations:
(anti-2019-nCoV or other coronavirus antigen small-molecule antibody Fab) + (anti-2019-nCoV or other coronavirus S-small-molecule antibody Fab)
(anti-2019-nCoV or other coronavirus antigen-small antibody Fab) + (anti-2019-nCoV or other coronavirus RBD-small antibody Fab)
(anti-N2019-nCoV or other coronavirus S-small antibody Fab) + (anti-N2019-nCoV or other coronavirus RBD-small antibody Fab)
4. [ ANTI-2019-nCoV OR OTHER CORONAVIRUS (S1+ S2) -SMALL ANTIBODY Fab ] + [ ANTI-2019-nCoV OR OTHER CORONAVIRUS RBD-SMALL ANTIBODY Fab ]
(anti-2019-nCoV or other coronavirus S-small antibody Fab) + (anti-2019-nCoV or other coronavirus N-small antibody Fab)
6. [ anti-2019-nCoV or other coronavirus (S + N) fusion protein small antibody Fab ] + (anti-2019-nCoV or other coronavirus RBD-small antibody Fab)
The following are examples of 3 combinations:
(anti-2019-nCoV or other coronavirus antigen small-molecule antibody Fab) + (anti-2019-nCoV or other coronavirus S-small-molecule antibody Fab) + (anti-2019-nCoV or other coronavirus RBD-small-molecule antibody Fab)
(anti-2019-nCoV or other coronavirus S-small antibody Fab) + (anti-2019-nCoV or other coronavirus RBD-small antibody Fab) + (anti-2019-nCoV or other coronavirus N-small antibody Fab)
3. [ anti- "2019-nCoV" or other coronavirus (S1+ S2) -small molecule antibody Fab ] + [ anti- "2019-nCoV" or other coronavirus RBD-small molecule antibody Fab ] + (anti- "2019-nCoV" or other coronavirus N-small molecule antibody Fab ]
In practical applications, the above anti-2019-nCoV or other coronavirus small molecule antibody Fab can be used in any combination according to requirements.
Eighthly, preparing long-acting anti-2019-nCoV or other coronavirus small molecule antibody Fab
The prepared one or more composite anti-2019-nCoV or other coronavirus micromolecule antibody Fab is subjected to long-acting modification by adopting polyethylene glycol, dextran or polyamino acid to obtain the long-acting anti-2019-nCoV or other coronavirus micromolecule antibody Fab.
The present invention is described below by taking a method of long-acting modification with polyethylene glycol as an example, and other methods of long-acting modification can be referred to the following methods.
Adjusting the pH value of a boric acid buffer solution to 6.5-10.0, adding the boric acid buffer solution after pH adjustment into the anti-2019-nCoV or other coronavirus micromolecule antibody Fab to ensure that the concentration of the anti-HPV micromolecule antibody Fab is 0.1-0.5mg/mL, adding polyethylene glycol for reaction, wherein the molar ratio of the anti-2019-nCoV or other coronavirus micromolecule antibody Fab to the polyethylene glycol is 1/10-1/30, the reaction temperature is 15-30 ℃, and the reaction time is controlled to be 1-3 h.
Preparation of Fab dry powder of anti-2019-nCoV or other coronavirus small molecule antibody
One or more composite anti-2019-nCoV or other coronavirus small molecule antibody Fab solution is subjected to freeze drying or medium-low temperature spray drying or fluidized bed drying and other drying modes which do not influence the activity of the antibody to prepare one or more composite anti-2019-nCoV or other coronavirus small molecule antibody Fab dry powder.
To enhance the ability of such small molecule antibodies to penetrate the respiratory mucosa and further increase the therapeutic index, one or more of the complex anti- "2019-nCoV" or other coronavirus small molecule antibodies Fab can be nanolipolated as described above.
Ten, preparing anti-2019-nCoV or other coronavirus small molecule antibody Fab coupled with cell-penetrating peptide
In order to improve the capability of anti-2019-nCoV or other coronavirus small molecule antibody Fab to permeate cell membranes and enter cells infected by the '2019-nCoV' or other coronaviruses to kill the '2019-nCoV' or other coronaviruses, the invention adopts naturally-derived CPPs or artificially-synthesized CPPs to couple one or more composite anti-2019-nCoV or other coronavirus small molecule antibody Fab. The invention is illustrated by taking one coupling method as an example, and other coupling methods refer to the following methods:
taking 1-3 parts of anti-2019-nCoV or other coronavirus small molecular antibody Fab dry powder, 5-10 parts of CPPs-EGFP1-3 parts and BSA-NS protein microspheres by mass, respectively dissolving the anti-2019-nCoV or other coronavirus small molecular antibody Fab dry powder, the BSA-NS protein microspheres by mass and 5-10 parts in PBS (pH7.5 and 0.01 mol/L) to obtain an anti-2019-nCoV or other coronavirus small molecular antibody Fab solution with the concentration of 0.5-1.5g/L, a CPPs-EGFP solution with the concentration of 0.5-1.5g/L and a protein microsphere solution with the concentration of 2.5-5g/L, respectively slowly adding 10-25mol/L ethanol solution according to the volume ratio of 1: 10-1: 30, stirring and reacting at room temperature for 10-30 minutes, respectively adding the reaction mixture of the anti-2019-nCoV or other coronavirus small molecular antibody Fab and the reaction mixture of the CPEGFP into an ultrafiltration centrifugal tube, respectively taking acetate buffer solution with pH4.5 and 0.01mol/L and PBS with pH7.5 and 0.01mol/L as washing liquid, and centrifugally washing at 4 ℃; adding the BSA-NS protein microsphere reaction mixture into an ultrafiltration centrifugal tube, centrifugally washing the mixture at 4 ℃ by PBS (phosphate buffer solution) with the pH value of 7.5 and 0.01mol/L to remove residual SPDP, respectively obtaining SPDP derivatives of anti-2019-nCoV or other coronavirus micromolecule antibodies Fab-PDP, CPPs-EGFP-PDP and BSA-NS-PDP, and storing the derivatives at 4 ℃ for later use; adding a proper amount of DTT into the obtained anti-2019-nCoV or other coronavirus micromolecule antibody Fab-PDP acetate solution to ensure that the final concentration of the DTT in the reaction system is 50mmol/L, slightly stirring for 30 minutes at room temperature, adding into an ultrafiltration centrifugal tube, centrifugally washing at 4 ℃ by taking PBS (phosphate buffer solution) with pH7.5 and 0.01mol/L as a washing solution, removing residual DTT to obtain an anti-2019-nCoV or other coronavirus micromolecule antibody Fab-PDP-SH solution, immediately mixing with CPPs-EG-FP-PDP and BSA-NS-PDP solutions, stirring for 15-20 hours at 4 ℃, centrifugally washing by PBS, precipitating, and finally obtaining the CPPs-EG-FP-BSA-NS-anti-Fab-2019-nCoV or other coronavirus micromolecule antibody protein conjugates.
The invention also provides application of the anti-2019-nCoV or other coronavirus IgY and the micromolecular antibody thereof in preparation of medicines, disinfection products, health-care products or medical devices for preventing and treating 2019-nCoV or other coronavirus infection.
The invention also provides a composition which comprises the anti-2019-nCoV or other coronavirus IgY and a small molecule antibody thereof and at least one other pharmaceutically acceptable component.
In the composition, the anti-2019-nCoV or other coronavirus IgY and micromolecular antibodies thereof are added with auxiliary materials or base materials or chemical drugs and traditional Chinese medicines to prepare at least one of an atomizing agent, a spraying agent, an oral spraying agent, a nasal drop, an eye drop, an air disinfectant, an air freshener, a disinfectant, hand sanitizer, powder, a tablet, a buccal tablet, oral liquid, an oral agent and a capsule.
The invention also provides application of the composition in preparation of medicines, disinfection products, health care products or medical instruments for preventing and treating 2019-nCoV or other coronavirus infections.
As mentioned above, the prepared anti-2019-nCoV or other coronavirus IgY and small molecule antibodies thereof can be prepared into various stable preparations. Such formulations include, but are not limited to, these:
preferably, the preparation further comprises one or more of excipient, filler, solvent, cosolvent, surfactant and capsule adjuvant.
Preferably, the formulation is a nebulant, disinfectant, air freshener, hand sanitizer, and the like.
Preferably, the formulation is a tablet, spray, powder, liquid or capsule.
The implementation of the anti-2019-nCoV or other coronavirus IgY and the micromolecule antibody and the composition thereof, the preparation method and the application thereof have the following beneficial effects: the anti-2019-nCoV or other coronavirus IgY and the micromolecular antibody thereof can directly inhibit the 2019-nCoV and other coronaviruses, and can be specifically combined with the key protein S protein and RBD structure domain thereof of the 2019-nCoV and other coronaviruses and seal the key protein S protein and RBD structure domain thereof, so that the functions of mediating virus infection and diffusion are lost; thus, infection of respiratory epithelial cells by "2019-nCoV" and other coronaviruses was prevented. Meanwhile, the antibodies can neutralize virus discharged by infected cells, eliminate secondary infection and block sporadic transmission pathogeny, and the unique advantages are particularly significant for preventing '2019-nCoV' and other coronavirus infections and preventing and controlling epidemic spread in the whole population. The anti-2019-nCoV or other coronavirus IgY and the micromolecule antibody and the composition thereof provided by the invention comprise series products prepared by applying the antibodies and the composition, so that the aims of releasing and accurately preventing and treating can be achieved, and the problems of toxic and side effects of chemical antiviral drugs and chemical inhibitors and induction of drug resistance do not exist.
Detailed Description
The following test examples and examples are combined to further illustrate the anti-2019-nCoV or other coronavirus IgY and its small molecule antibody, its composition, preparation method and application:
test example 1:
and (3) detecting the antibody binding titer of the anti-2019-nCoV virus antigen IgY to the corresponding inactivated 2019-nCoV virus.
The inactivated "2019-nCoV" virus is selected as a detection antigen, and the titer of the prepared anti-virus antigen IgY of the "2019-nCoV" virus is detected by an ELISA method (enzyme-linked immunosorbent assay), and the results are shown in the following table:
| antibodies | Antigens | Antibody binding potency |
| anti-2019-nCoV virus antigen IgY | "2019-nCoV" virus antigen | 1:32768 |
Note: the concentrations of the anti-2019-nCoV virus antigen IgY antibody solutions in the test samples are all 1 mg/mL.
From the detection results, the prepared anti-2019-nCoV virus antigen IgY has high antibody binding titer to the 2019-nCoV virus.
Test example 2:
the anti-2019-nCoV IgY antibody has the antibody binding titer detection of a representative recombinant antigen component of the corresponding '2019-nCoV' coronavirus.
The complete sequence polypeptide of the "2019-nCoV" S protein, the polypeptide of the immunodominance determinant of the "2019-nCoV" S1 protein, the polypeptide of the immunodominance determinant of the "2019-nCoV" S2 protein, the recombinant polypeptide protein of the "2019-nCoV" Receptor Binding Domain (RBD) fragment, the antigen of the "2019-nCoV" N protein, the antigen of the "2019-nCoV" S protein fragment and the fusion protein antigen of the N protein fragment are respectively selected as detection antigens, and the antibody titer of the prepared anti-2019-nCoV coronavirus IgY and small molecule antibodies thereof is detected by an ELISA method (enzyme-linked immunosorbent assay), and the results are shown in the following table:
note: the concentrations of the anti-2019-nCoV-IgY series antibody solutions in the test sample are all 1 mg/mL.
From the detection results, the prepared anti-2019-nCoV ' -IgY series antibodies have high antibody binding titer to the corresponding protein antigens of the ' 2019-nCoV '.
Test example 3:
and (3) detecting the antibody binding titer of the anti-2019-nCoV small-molecule antibody Fab on the corresponding 2019-nCoV protein antigen.
The complete sequence polypeptide of the "2019-nCoV" S protein, the polypeptide of the immunodominance determinant of the "2019-nCoV" S1 protein, the polypeptide of the immunodominance determinant of the "2019-nCoV" S2 protein, the recombinant polypeptide protein of the "2019-nCoV" Receptor Binding Domain (RBD) fragment, the antigen of the "2019-nCoV" N protein, the antigen of the "2019-nCoV" S protein fragment and the fusion protein antigen of the N protein fragment are respectively selected as detection antigens, and the antibody titer of the prepared anti-small molecule antibody Fab of the "2019-nCoV" is detected by an ELISA method (enzyme-linked immunosorbent assay), and the results are shown in the following table:
note: the concentrations of the Fab antibody solutions of the anti-2019-nCoV small-molecule antibodies in the test samples are all 1 mg/mL.
From the detection results, the prepared Fab of the anti-2019-nCoV small-molecule antibody has high antibody binding titer to the corresponding protein antigen of the 2019-nCoV.
Example 1: the titer of the virus resisting the virus antigen IgY of '2019-nCoV' and the virus of '2019-nCoV' is determined by a neutralization test method
1. Test materials:
(1) anti-2019-nCoV virus antigen IgY: 2 portions of standard sample with protein content of 40mg/ml are divided into No. 1 and No. 2
(2) HRP enzyme-labeled rabbit anti-chicken IgG
(3) Negative antibodies (IgY antibodies of non-immunized eggs): protein content 60mg/ml
(4) Positive control: 2 portions of serum of a patient infected by the virus of '2019-nCoV' in convalescent period, No. 3 specimen and No. 4 specimen
(5) Negative control: infant umbilical cord serum 2 parts, specimen No. 5 No. 6
(6) Virus:
"2019-nCoV" viral isolate I: separating and identifying from an pharyngeal test sub specimen of a virus infection patient of '2019-nCoV'.
"2019-nCoV" viral isolate II: separating and identifying from a serum specimen of a virus-infected patient of '2019-nCoV'.
(7) Cell: african green monkey kidney passage cells.
2. And (3) formal test:
detection of anti "2019-nCoV" viral antigen IgY antibody in VERO E6 cell culture assay for purposes of the experiments:
the anti-2019-nCoV virus antigen IgY (isolated 2 strains of "2019-nCoV" virus) was determined in VERO E6 cell culture by neutralization assay; the 50% antibody neutralization endpoint was calculated using the Reed-Muench method.
Fixed virus-diluted antibody method
Inactivating the above IgY antibody against the virus antigen of "2019-nCoV", serum of a patient infected with the virus of "2019-nCoV" in convalescent period and infant umbilical cord serum at 56 deg.C for 1 hr, and diluting with Egale' S solution 2 timesRelease 9 concentrations, i.e. L8-1: 1024 and 100TCID50 virus suspension of the "2019-nCoV" virus isolate I and the "2019-nCoV" virus isolate II are mixed in a water bath at 37 ℃ for 1 hour, and then inoculated to a VERO E6 cell 96-well culture plate, wherein each concentration is 4 wells, and an anti-2019-nCoV virus antigen IgY antibody, patient recovery period serum and infant umbilical cord serum control, a virus control and a normal cell control are simultaneously arranged. 5% CO at 37 ℃2The incubation was carried out for 5 days, and the virus (CPE) was observed under an inverted microscope every day with a morphological change of less than 25% to "+", 26% -50% to "+", 51% -75% to "+ + + +", and 76% -100% to "+ + + + + +", and the experiment was terminated with the appearance of a viral control "+ + + + + + + + + + + +".
The 50% antibody neutralization endpoint was calculated using the Reed-Muench method.
3. Neutralization test detection result of anti-2019-nCoV virus antigen IgY antibody
(1) Anti "2019-nCoV" virus antigen IgY antibody No. 1 specimen: the 1:512 antibody protects 50% of cells from cytopathic effects.
(2) Anti "2019-nCoV" virus antigen IgY antibody No. 2 specimen: the 1:256 antibody protects 50% of cells from cytopathic effects.
Negative antibody, which can not neutralize the virus of '2019-nCoV'.
Positive control:
(1) "2019-nCoV" virus infected patient convalescent phase No. 3 specimen: serum at 1:512 protected 50% of cells from cytopathic effects.
(2) "2019-nCoV" virus infected patient convalescent phase No. 4 specimen: serum at 1:512 protected 50% of cells from cytopathic effects.
Negative control:
(1) infant umbilical cord serum sample No. 5: the "2019-nCoV" virus could not be neutralized.
(2) Infant umbilical cord serum sample No. 6: the "2019-nCoV" virus could not be neutralized.
Example 2: and (3) determining the combination of the anti-2019-nCoV virus antigen IgY antibody and the 2019-nCoV virus by adopting immunoblotting (Western blot) detection.
The specific operation method comprises the following steps:
1. electrophoresis:
(1) sample treatment: mixing the virus N antigen (200gg/ml) of the '2019-nCoV', inactivated virus vero cells (CPE + +) of the '2019-nCoV' and 5X sample adding buffer solution (electrophoresis sample adding buffer solution: 25mmol Tris-CL (pH6.8),50mmol DTT, 2% SDS, 0.1% gossypol blue and 10% glycerol);
sample treatment: mixing 5 μ l of "2019-nCoV" virus N antigen (200 μ g/ml), and inactivated "2019-nCoV" virus vero cell loading buffer (electrophoresis loading buffer: 25mmol Tris-CL (pH6.8),50mmol DTT, 2% SDS, 0.1% gossypol blue, 10% glycerol);
(2) heating in boiling water for 10 min;
(3) centrifuging at 3000rpm for 2 min;
(4) mu.l of the N antigen treated above (200. mu.g/ml), 10. mu.l of the inactivated "2019-nCoV" virus vero cells were each loaded, and 15% SDS-PAGE was run (15% separation gel: 11.5ml of water, 12.5ml of 1.5mmol Tris-CL (pH8.8),25ml of 30% SDS-PAGE gel stock, 0.5ml of 10% SDS, 10. mu.l TEMED (10. mu.l/ml), 5% stratified gel: 6.8ml of water, 1.7ml of 30% SDS-PAGE gel stock, 1.25ml of 1.5 mmol-Tris (pH6.8), 0.1ml of 10% SDS, 10. mu.l TEMED (1. mu.l/ml) of ammonium persulfate was added immediately before use;
(5) voltage 80V (layering gel), electrophoresis 156V (separating gel), and turning off the power supply 10min after the indicator is out of the bottom of the gel.
2. Film transfer:
(1) carefully placing the gel on three layers of filter paper soaked in transfer solution (39mmol glycine, 48mmol Tris-CL, 0.037% SDS, 20% methanol), carefully placing a nitrocellulose membrane (soaking for 5min) on the gel, covering the soaked three layers of filter paper, accurately aligning the layers, and removing bubbles;
(2)60mA film rotating for 1h
3. And (3) immunological detection:
(1) after the membrane is transferred, removing the filter paper, carefully taking out the membrane, and rinsing the membrane for 2min by PBST (0.05% Tween-20/PBS);
(2) 5% skim milk (skim milk/PBS) was blocked overnight;
(3) diluting the anti-2019-nCoV virus antigen IgY suspension 1:2 and 1:4 (PBS), and reacting with a membrane for 2h at 37 ℃;
(4) PBST membrane washing for 3 times, 10 min/time;
(5) diluting anti-chicken IgG (l:250) marked by horseradish peroxidase, and reacting with the membrane for 2h at 37 ℃;
(6) PBST membrane washing for 3 times, 10 min/time;
(7)0.05mol/L DAB (3, 3-diaminobenzidine 50mg,50mmol Tris-CL (pH8.0), 100ml, 0.01% H202) Developing color;
(8) the reaction was stopped by rinsing with distilled water until a clear band appeared.
4. As a result:
(1) reacting with different components (M, N antigen) after SARS virus lysis (lane 1);
(2) reaction with expressed N antigen (at 40 KD) (lane2) o
Example 3: the anti-2019-nCoV protein full-sequence polypeptide-small-molecule antibody Fab atomizing agent is produced and used as medicinal atomizing liquid for various atomizing inhalation machines, public environment air disinfectors, air fresheners, household air disinfectors, air fresheners and mobile spray disinfection instruments.
The formula is as follows:
the process comprises the following steps:
(1) sterilizing K-30, S-40, tween-80, oleum Menthae Dementholatum and essence with ultraviolet irradiation for 24 hr, and aseptically sealing;
(2) heating distilled water according to the formula amount to 90 ℃, then respectively adding S-40 and K-30 dispersing agents, stirring for more than 30 minutes, and uniformly dissolving; cooling to 60 deg.C, adding Tween, oleum Menthae Dementholatum and glycerol slowly, stirring at low speed for 60min to dissolve completely; then, cooling to room temperature to form a solution A;
(3) adding the anti-2019-nCoV S protein complete sequence polypeptide-small molecule antibody Fab nano liposome solution or dry powder into the solution A while stirring, and stirring at a low speed for 60min until the mixture is completely and uniformly mixed to obtain a solution B;
(4) adding the essence into the solution B while stirring, and stirring at low speed for 60min until the essence is completely dissolved to obtain a solution C;
(5) measuring the pH value of the solution C by using a pH meter, and adjusting the pH value to 6.8 +/-0.1 by using citric acid;
(6) standing until the upper foam is completely dissolved, subpackaging the solution C in spray bottles which are cleaned and disinfected, and labeling for delivery.
Example 4: production of anti-2019-nCoV S and N fusion protein-small molecule antibody Fab nasal spray or nasal drops or eye drops
The formula is as follows:
the process comprises the following steps:
(1) sterilizing K-30, S-40, tween-80, oleum Menthae Dementholatum and essence with ultraviolet irradiation for 24 hr, and aseptically sealing;
(2) heating distilled water according to the formula amount to 90 ℃, then respectively adding S-40 and K-30 dispersing agents, stirring for more than 30 minutes, and uniformly dissolving; cooling to 60 deg.C, adding Tween, oleum Menthae Dementholatum and glycerol slowly, stirring at low speed for 60min to dissolve completely; then, cooling to room temperature to form a solution A;
(3) adding the anti-2019-nCoV S and N fusion protein-micromolecular antibody Fab nano liposome solution or dry powder into the solution A while stirring, and stirring at a low speed for 60min until the solution B is completely and uniformly mixed to obtain a solution B;
(4) adding the essence into the solution B while stirring, and stirring at low speed for 60min until the essence is completely dissolved to obtain a solution C;
(5) measuring the pH value of the solution C by using a pH meter, and adjusting the pH value to 6.8 +/-0.1 by using citric acid;
(6) standing until the upper foam is completely dissolved, subpackaging the solution C in cleaned and sterilized nose dropping device or eye dropping device and nasal spray bottle, and labeling for delivery.
Example 5: production of anti-2019-nCoV S and N fusion protein-small molecule antibody Fab mouth spray or mouthwash
The formula is as follows:
the process comprises the following steps:
(1) sterilizing K-30, S-40, tween-80, oleum Menthae Dementholatum and essence with ultraviolet irradiation for 24 hr, and aseptically sealing;
(2) heating distilled water according to the formula amount to 90 ℃, then respectively adding S-40 and K-30 dispersing agents, stirring for more than 30 minutes, and uniformly dissolving; cooling to 60 deg.C, adding Tween, oleum Menthae Dementholatum and glycerol slowly, stirring at low speed for 60min to dissolve completely; then, cooling to room temperature to form a solution A;
(3) adding the anti-2019-nCoV S protein complete sequence polypeptide-small molecule antibody Fab nano liposome solution or dry powder into the solution A while stirring, and stirring at a low speed for 60min until the mixture is completely and uniformly mixed to obtain a solution B;
(4) adding the essence into the solution B while stirring, and stirring at low speed for 60min until the essence is completely dissolved to obtain a solution C;
(5) measuring the pH value of the solution C by using a pH meter, and adjusting the pH value to 6.8 +/-0.1 by using citric acid;
(6) standing until the upper foam is completely dissolved, subpackaging the solution C in a spray can or a gargle bottle for oral cavity after cleaning and disinfection, and labeling for delivery.
Example 6: production of anti-2019-nCoV Receptor Binding Domain (RBD) -IgY hand sanitizer
The formula is as follows:
| raw materials | Content (%) |
| Anti "2019-nCoV" Receptor Binding Domain (RBD) -IgY | 5.00 |
| 403 (disodium fatty alcohol polyoxyethylene ether sulfosuccinate) | 33.48 |
| 503 (fatty alcohol polyoxyethylene ether ammonium sulfate, concentration 70%) | 20.51 |
| Pharmaceutical grade glycerin | 2.00 |
| Seaweed essence | 0.10 |
| Essence (Rose essence) | 0.05 |
| Mint oil | 0.05ml |
| Distilled water | 38.81 |
The process comprises the following steps:
(1) sterilizing the formula amount of seaweed essence, pharmaceutical grade glycerin, peppermint oil, essence and 403 and 503 foaming agents by ultraviolet irradiation for 24 hours, and sealing aseptically for later use;
(2) heating the formula amount of distilled water to 90 ℃, and staying for 15 minutes; cooling to 60 ℃, adding the seaweed essence and the pharmaceutical-grade glycerol while stirring, stirring at a low speed for 30 minutes until the seaweed essence and the pharmaceutical-grade glycerol are completely dissolved, and cooling to room temperature to obtain a solution A;
(3) adding essence (flos Rosae Rugosae essence) into the solution A while stirring, and stirring at low speed for 60min until completely dissolved to obtain solution B;
(4) heating 403 to 80 ℃, slowly dripping 503 into 403 while stirring, and stirring at low speed for 30min to obtain solution C;
(5) slowly dripping oleum Menthae Dementholatum into solution C while stirring at 80 deg.C, and stirring at low speed for 60min to obtain solution D;
(6) heating the solution D to 90 ℃, sterilizing at high temperature for 5min, and then cooling to room temperature;
(7) slowly adding the solution B into the solution D while stirring, and stirring at a low speed for 60 min; if homogeneous stable emulsion solution is not formed, the stirring time is prolonged to prepare solution E;
(8) slowly adding the anti-2019-nCoV Receptor Binding Domain (RBD) -IgY into the solution E while stirring, and stirring at low speed for 60 min; if homogeneous stable emulsion solution is not formed, the stirring time is prolonged to prepare solution F;
(9) measuring the pH value of the solution F by using a pH meter, and adjusting the pH value to 4.5 +/-0.1 by using citric acid or a disodium hydrogen phosphate-citric acid buffer solution with the pH value of 4.5;
(10) standing overnight until the upper foam is completely dissolved, packaging the solution F in a cleaned and sterilized hand sanitizer container, and labeling for delivery.
Example 7: production of anti-2019-nCoV S protein composite antigen-IgY buccal tablet
The formula is as follows:
process for the preparation of a coating
(1) Sieving sorbitol with 60 mesh sieve twice;
(2) dispersing carboxymethyl cellulose in 30% ethanol to obtain 1% ethanol solution;
(3) preparing soft materials from item (1) with appropriate amount of item (2), granulating with 14 mesh screen, air drying at 60 deg.C, and grading with 18 mesh screen; sieving with 40 mesh sieve to obtain appropriate amount of fine powder, and mixing with 2019-nCoV S protein complex antigen-IgY;
(4) then adding magnesium stearate, mixing with the whole batch of granules uniformly, and sealing for more than 4 hours;
(5) tabletting with a tabletting machine, wherein each tablet is 600 mg;
(6) and after the inspection is qualified, packaging, and completely inspecting and leaving the factory.
Example 8: producing anti-2019-nCoV S protein composite antigen-IgY powder, and using a powder supply non-atomization device as medicinal powder.
The formula is as follows:
the process comprises the following steps:
(1) mixing the formula amount of the '2019-nCoV' S protein composite antigen-IgY dry powder or the nano liposome anti '2019-nCoV' S protein composite antigen-IgY dry powder with the formula amount of medicinal glucose, and fully and uniformly stirring;
(2) packaging the powder, and inspecting.
It will be apparent to those skilled in the art to which the invention relates that the invention may be varied from the precise details disclosed without departing from the spirit and scope of the appended claims. The present invention is not to be considered as limited in scope by the procedures, properties or compositions defined, since the preferred embodiments and other descriptions are intended only to illustrate specific aspects of the invention presently provided. Various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry, biochemistry or related fields are intended to be within the scope of the following claims.
It will be understood that modifications and variations can be made by those skilled in the art in light of the above teachings, and it is intended to cover all such modifications and variations as fall within the scope of the appended claims.
Claims (18)
1. A method for preparing an anti-coronavirus antibody, comprising the steps of:
s1, preparing antigen: adding adjuvant into inactivated coronavirus or coronavirus protein component of target coronavirus to obtain virus antigen or protein antigen;
s2, preparing immune eggs: immunizing egg-laying poultry with the virus antigen or the protein antigen to obtain an immune egg against coronavirus;
s3, isolated antibody: isolating IgY antibodies against said target coronavirus from said immunized egg.
2. The method for producing an antibody according to claim 1, wherein in step S1, the antigen component is at least one of: inactivated coronavirus liquid, coronavirus recombinant S protein, coronavirus recombinant S1 protein, coronavirus recombinant S2 protein, coronavirus receptor binding domain recombinant protein, coronavirus recombinant N protein, and fusion protein of coronavirus recombinant S protein fragment and N protein fragment.
3. The method of claim 2, wherein the inactivated coronary virus fluid is prepared using the following method:
(1) infecting a cell line for in vitro culture with the target coronavirus;
(2) selecting the virus infection amount and culture conditions to culture the target coronavirus, and repeatedly freezing and thawing the harvested virus for later use;
(3) inactivating the virus: under the condition of continuously stirring virus liquid, slowly adding an inactivator to inactivate the virus.
4. The method for preparing antibody according to claim 2, wherein the coronavirus S protein is a coronavirus S protein complete sequence polypeptide expressed by gene recombination, and a recombinant polypeptide protein obtained by coupling KLH and BSA.
5. The method of claim 2, wherein the recombinant coronavirus S1 protein and the recombinant coronavirus S2 protein are prepared by the following steps:
(1) respectively expressing an immunodominance determinant polypeptide of coronavirus S1 protein and an immunodominance determinant polypeptide of coronavirus S2 protein;
(2) respectively coupled with KLH and BSA to form polypeptide to obtain coronavirus recombinant S1 protein recombinant coronavirus recombinant S1 protein.
6. The method of claim 2, wherein the recombinant protein of the receptor binding domain of coronavirus is prepared by the following method:
(1) constructing a PET32(a) -RBD recombinant plasmid;
(2) expressing by using a prokaryotic or eukaryotic expression system;
(3) synthesizing a peptide chain by using an Fmoc solid phase method or other methods;
(4) coupled to KLH and BSA to form a polypeptide.
7. The method for preparing antibody according to claim 2, wherein the coronavirus N protein is a coronavirus N protein complete sequence polypeptide expressed by gene recombination, and the recombinant N protein is obtained by coupling KLH and BSA.
8. The method for producing an antibody according to claim 2, wherein the fusion protein of the coronavirus S protein fragment and the N protein fragment is produced by the following method:
(1) chemically synthesizing the gene sequences of the S protein fragment and the N protein fragment;
(2) connecting two gene segments in series, and cloning to an expression site of a plasmid;
(3) transforming the recombinant plasmid into a prokaryotic or eukaryotic expression system, screening to obtain high-efficiency expression engineering bacteria, and expressing and purifying coronavirus S protein fragment and N protein fragment fusion protein.
(4) Coupling with KLH and BSA to obtain the fusion protein antigen components of the S protein fragment and the N protein fragment of the coronavirus.
9. The method for preparing antibody according to claim 1, wherein in step S1, the adjuvant is freund 'S adjuvant or other adjuvants, the inactivated coronavirus solution or recombinant protein component and the freund' S adjuvant are subjected to high speed homogenization to form the antigen, and the antigen is water-in-oil emulsion.
10. The method for preparing antibody according to claim 1, wherein in step S2, the egg-laying fowl is immunized every two weeks for 2-5 times, and the immunized egg of the egg-laying fowl is collected at least 12 days after the last injection.
11. The method of claim 1, wherein in step S3, the crude IgY extract against the target coronavirus is isolated by pure water extraction, chloroform extraction, cold ethanol precipitation or ammonium sulfate precipitation.
12. The method for producing an antibody according to claim 1, wherein the pure water extraction method comprises the steps of:
(1) taking the yolk of the immune egg, and uniformly stirring;
(2) adding distilled water 3-8 times the volume of egg yolk liquid, diluting, mixing, and adjusting pH to 5.5-6.5;
(3) cooling to 2-6 deg.C, standing for 12-24 hr, and centrifuging at high speed; taking supernatant obtained by centrifugal separation, and placing the supernatant into an ultrafilter for ultrafiltration and concentration by 10-20 times;
(4) adding 1.0-3.0% sodium alginate solution, slowly adding sodium alginate solution to final concentration of 0.1-0.2%, and stirring to obtain turbid solution;
(5) then adding 1.0-3.0% of CaCl2Mixing the solution until the final concentration is 0.1-0.2%, stirring, and standing at 3-4 deg.C for 8-12 hr;
(6) centrifuging at high speed and collecting supernatant to obtain crude IgY extract.
13. The method for preparing antibody of claim 12, wherein said crude IgY extract is passed through ion exchange column and gel column chromatography or affinity column chromatography, respectively, to obtain anti-coronavirus pure IgY solution.
14. The method for producing an antibody according to claim 13, further comprising the steps of:
and preparing the pure IgY solution into at least one of IgY dry powder, IgY nano dry powder, nanoliposome IgY dry powder, small molecule antibody Fab, long-acting small molecule antibody Fab, small molecule antibody Fab dry powder, nanoliposome small molecule antibody Fab and small molecule antibody Fab coupled with cell-penetrating peptide.
15. A composition comprising an antibody produced according to claims 1-14.
16. The composition of claim 15, further comprising one or more of excipients, fillers, solvents, cosolvents, surfactants, capsule excipients, chemicals, and traditional Chinese medicines.
17. The composition of claim 15, wherein the composition is formulated as at least one of a nebulizer, a spray, a mouth spray, a nasal drop, an eye drop, an air sanitizer, an air freshener, a sanitizer, a hand sanitizer, a powder, a tablet, a buccal tablet, an oral liquid, and a capsule.
18. Use of the antibodies and compositions prepared in claims 1-16 for the preparation of a medicament, a disinfectant product, a health product or a medical device for the prevention and treatment of coronavirus infection.
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| PCT/CN2020/084751 WO2021155639A1 (en) | 2020-02-03 | 2020-04-14 | Igy for resisting sars-cov-2 and other coronaviruses, small molecule antibody thereof and use thereof |
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| US11999777B2 (en) | 2020-06-03 | 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 |
| CN113388031B (en) * | 2021-08-17 | 2021-11-09 | 上海浙江大学高等研究院 | Monoclonal antibody 35B5, and preparation method and application thereof |
| CN114699522A (en) * | 2022-03-23 | 2022-07-05 | 上海亚健医学科技有限公司 | Production process of oral spray based on gene recombination anti-mutation virus bacteria and fungi |
| 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 |
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| CN1556113A (en) * | 2004-01-07 | 2004-12-22 | 珠海百奥生物技术有限公司 | Yolk antibody of anti SARS coronavirus and its preparation method and liquid preparation |
| US20080063664A1 (en) * | 2006-09-05 | 2008-03-13 | Academia Sinica | High-yield transgenic mammalian expression system for generating virus-like particles |
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| CN1318449C (en) * | 2003-05-13 | 2007-05-30 | 雅臣药业集团（远东）有限公司 | Anti SARS specificity IgY and combination preparation thereof |
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| CN1556113A (en) * | 2004-01-07 | 2004-12-22 | 珠海百奥生物技术有限公司 | Yolk antibody of anti SARS coronavirus and its preparation method and liquid preparation |
| US20080063664A1 (en) * | 2006-09-05 | 2008-03-13 | Academia Sinica | High-yield transgenic mammalian expression system for generating virus-like particles |
| CN108840929A (en) * | 2018-06-20 | 2018-11-20 | 深圳市雅臣智能生物工程有限公司 | Anti-human papilloma virus (anti-HPV) small molecular antibody and combinations thereof and preparation method and application |
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