CN112175074A - Industrial application of gene recombination-based anti-Covid-19 and other coronavirus IgY antibodies and other small molecule antibodies - Google Patents
Industrial application of gene recombination-based anti-Covid-19 and other coronavirus IgY antibodies and other small molecule antibodies Download PDFInfo
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- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
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- C07K2317/11—Immunoglobulins specific features characterized by their source of isolation or production isolated from eggs
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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
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Abstract
The invention discloses an anti-Covid-19 and other coronavirus IgY antibody and other micromolecule antibody industrial application based on gene recombination, which comprises the following steps: s1: cloning and expression of the novel coronavirus S protein gene: s2: preparing an antigen, namely performing Baculoviral-Insect Cell expression and His-tag (polyhistidine tag) purification of Insect cells through a bioreactor to ensure that the purity of the antigen is kept at 93%; s3: selecting animals, namely selecting high-yield hens, wherein the age of the hens is the best age of the hens which just lay eggs; s4: selected animals were immunized with oil adjuvant, immunopotentiator. The invention obtains high-purity antigen protein immune hen by constructing and expressing S protein and purifying, so that specific yolk antibody aiming at the S protein is continuously generated in eggs, and the spraying agent is prepared by extracting the specific IgY antibody aiming at SARS-CoV through purification and is used for spraying and disinfecting human oral cavity.
Description
Technical Field
The invention relates to the antibody industrialization application, in particular to the gene recombination-based anti-Covid-19 and other coronavirus IgY antibodies and other micromolecule antibodies industrialization application, and belongs to the technical field of biology.
Background
Antibodies are proteins that are produced by the body as a result of stimulation by antigens and have a protective effect. It (immunoglobulins are not just antibodies) is a large Y-shaped protein secreted by plasma cells (effector B-cells), used by the immune system to identify and neutralize foreign substances such as bacteria, viruses, etc., found only in body fluids such as blood of vertebrates, and the cell membrane surface of B-cells, antibodies recognize a unique feature of specific foreign substances, which are called antigens.
In COVID-19, the S protein on the surface of the virus mediates receptor recognition and membrane fusion, the trimeric S protein is cleaved into S1 and S2 subunits during viral infection, the Receptor Binding Domain (RBD) of the S1 protein, which directly binds to the Peptidase Domain (PD) of the target cell surface receptor ACE2, whereas S2 is responsible for membrane fusion, but the production of IgY antibodies specific for SARS-CoV is currently not achieved by constructing and expressing the S protein.
Disclosure of Invention
The invention aims to provide the industrial application of anti-Covid-19 and other coronavirus IgY antibodies and other small molecule antibodies based on gene recombination so as to solve the problem that the production of SARS-CoV specific IgY antibodies cannot be realized by constructing and expressing S protein at present, which is proposed in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme: the method comprises the following steps:
s1: cloning and expressing a new coronavirus S protein gene;
s2: preparing an antigen, namely performing Baculoviral-Insect Cell expression and His-tag (polyhistidine tag) purification of Insect cells through a bioreactor to ensure that the purity of the antigen is kept at 93%;
s3: selecting animals, namely selecting high-yield hens, wherein the age of the hens is the best age of the hens which just lay eggs;
s4: immunizing selected animals by using an oil adjuvant and an immunopotentiator, wherein the immunization period is about 2 months, the immunization frequency is 3 times, and after the third immunization is finished, eggs produced by immunized hens are collected at the time interval of 2 weeks;
s5: detecting the antibody of the hen, and detecting whether the antibody is produced or not and the titer of each hen by using an ELISA (enzyme-linked immunosorbent assay) method, wherein the ELISA detects the OD (optical density) value, and the formula is as follows: judging the sample OD value/negative control OD value is greater than 2.1 to be positive, taking the reciprocal of the dilution multiple of the hole corresponding to the minimum positive value as the highest titer, and making an antibody titer change table in the immune cycle;
s6: preparing an antibody;
s7: measuring the content of the antibody and the purity of the antibody;
s8: various additives, including preservatives and stabilizers, are added. Adjusting the content of IgY to be more than 3mg/ml, filtering and sterilizing by a 0.2um filter membrane, inactivating viruses and then filling.
In a preferred embodiment of the present invention, the cloning and expression of the new coronavirus S protein gene in step S1 is performed by cloning the new coronavirus S protein gene according to a known probe, wherein the probe is labeled with radioactivity or non-radioactivity, and then hybridized with genomic DNA treated with different endonucleases, and finally the identified fragment is excised from the gel and cloned into a specific vector.
As a preferred embodiment of the present invention, the step S6 includes a primary centrifugation and a secondary centrifugation, and the primary centrifugation includes the following steps:
a 1: weighing the collected eggs; a 2: PBS (phosphate buffered saline); a 3: beating the eggs into a glass ware, and separating egg yolk from egg white; a 4: stirring the separated yolk, wherein the stirring time is controlled to be 0.5 hour; a 5: precipitating the stirred egg yolk, wherein the precipitation time is controlled to be 0.5 hour; a 6: centrifuging the precipitated egg yolk by a centrifuge; a, 7: filtering the centrifuged egg yolk; a 8: adding an additive, adjusting the content of IgY (egg yolk immunoglobulin), carrying out constant volume for 12 hours, a 9: adding 0.45um and 0.22um membranes into the constant-volume egg yolk, and filtering and sterilizing; a 10: performing pasteurization for 0.5 hr, and packaging.
As a preferred technical solution of the present invention, the secondary centrifugation comprises the following steps: b1 weighing the collected eggs; b 2: PBS (phosphate buffered saline); b 3: beating the eggs into a glass ware, and separating egg yolk from egg white; b 4: stirring the separated yolk, wherein the stirring time is controlled to be 0.5 hour; b 5: precipitating the stirred egg yolk, wherein the precipitation time is controlled to be 0.5 hour; b 6: centrifuging the precipitated egg yolk by a centrifuge, and adding a 0.45um membrane for filtration during centrifugation; b 7: adding a precipitator into the supernatant, stirring, performing secondary centrifugation through a centrifuge, and dissolving and precipitating the supernatant after the secondary centrifugation for at least 12 hours; b 8: adding additives to regulate the content of IgY (chicken yolk immunoglobulin); b 9: adding 0.22um membrane for filtration and sterilization; b10 sterilizing by Pasteur for 0.5 hr, filling and packaging.
As a preferable technical scheme of the present invention, the additive is added in the step a8, the content of IgY (chicken egg yolk immunoglobulin) is adjusted, the sodium oxide reagent is added to the diluted egg yolk filtered in the step a7, the sodium oxide reagent is added and the precipitation is carried out, the volume is determined after the precipitation, and the content of IgY (chicken egg yolk immunoglobulin) is ensured to be about 90%.
As a preferred technical scheme of the invention, the IgY detection method comprises electrophoresis, SEC, binding activity, pseudovirus neutralization activity, ACE2 competition activity and HPLC.
Compared with the prior art, the invention has the beneficial effects that:
based on the industrial application of gene recombinant anti-Covid-19 and other coronavirus IgY antibodies and other small molecule antibodies, the invention obtains high-purity antigen protein immune hen by constructing and expressing S protein and purifying, so that specific yolk antibody aiming at the S protein is continuously generated in eggs, and the specific IgY antibody aiming at SARS-CoV is extracted by purification to prepare a spraying agent for spraying and disinfecting human oral cavities.
Drawings
FIG. 1 is a process flow diagram of the present invention;
FIG. 2 is a flow chart of the purification process of IgY antibodies of the present invention;
FIG. 3 is a diagram of the purity detection of IgY protein by HPLC method according to the present invention;
FIG. 4 is a graph showing the detection of SARS-COV2-RBD protein and antibody binding activity by ELISA method according to the present invention;
FIG. 5 is a graph of IgY and SEC activity of the present invention
FIG. 6 is a graph showing the binding activity of IgY to RBD S1h according to the present invention;
FIG. 7 is a graph showing the activity of the IgY pseudovirus of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to FIGS. 1-7, the present invention provides a technical solution for industrial application of anti-Covid-19 and other coronavirus IgY antibodies and other small molecule antibodies based on gene recombination:
as shown in fig. 1-7, the method comprises the following steps:
s1: cloning and expressing a new coronavirus S protein gene;
s2: preparing an antigen, namely performing Baculoviral-Insect Cell expression and His-tag (polyhistidine tag) purification of Insect cells through a bioreactor to ensure that the purity of the antigen is kept at 93%;
s3: selecting animals, namely selecting high-yield hens, wherein the age of the hens is the best age of the hens which just lay eggs;
s4: immunizing selected animals by using an oil adjuvant and an immunopotentiator, wherein the immunization period is about 2 months, the immunization frequency is 3 times, and after the third immunization is finished, eggs produced by immunized hens are collected at the time interval of 2 weeks;
s5: detecting the antibody of the hen, and detecting whether the antibody is produced or not and the titer of each hen by using an ELISA (enzyme-linked immunosorbent assay) method, wherein the ELISA detects the OD (optical density) value, and the formula is as follows: judging the sample OD value/negative control OD value is greater than 2.1 to be positive, taking the reciprocal of the dilution multiple of the hole corresponding to the minimum positive value as the highest titer, and making an antibody titer change table in the immune cycle;
s6: preparing an antibody;
s7: measuring the content of the antibody and the purity of the antibody;
s8: various additives, including preservatives and stabilizers, are added. Adjusting the content of IgY to be more than 3mg/ml, filtering and sterilizing by a 0.2um filter membrane, inactivating viruses and then filling.
Cloning and expressing the new coronavirus S protein gene in step S1, cloning the new coronavirus S protein gene according to a known probe, labeling the probe with radioactivity or nonradioactive label, hybridizing the probe with genomic DNA treated by different endonucleases, cutting the identified fragment from the gel, and cloning to a specific vector.
Step S6 includes primary centrifugation and secondary centrifugation, the primary centrifugation including the steps of: a 1: weighing the collected eggs; a 2: PBS (phosphate buffered saline); a 3: beating the eggs into a glass ware, and separating egg yolk from egg white; a 4: stirring the separated yolk, wherein the stirring time is controlled to be 0.5 hour; a 5: precipitating the stirred egg yolk, wherein the precipitation time is controlled to be 0.5 hour; a 6: centrifuging the precipitated egg yolk by a centrifuge; a, 7: filtering the centrifuged egg yolk; a 8: adding an additive, adjusting the content of IgY (egg yolk immunoglobulin), carrying out constant volume for 12 hours, a 9: adding 0.45um and 0.22um membranes into the constant-volume egg yolk, and filtering and sterilizing; a 10: performing pasteurization for 0.5 hr, and packaging.
The secondary centrifugation comprises the following steps:
b1 weighing the collected eggs; b 2: PBS (phosphate buffered saline); b 3: beating the eggs into a glass ware, and separating egg yolk from egg white; b 4: stirring the separated yolk, wherein the stirring time is controlled to be 0.5 hour; b 5: precipitating the stirred egg yolk, wherein the precipitation time is controlled to be 0.5 hour; b 6: centrifuging the precipitated egg yolk by a centrifuge, and adding a 0.45um membrane for filtration during centrifugation; b 7: adding a precipitator into the supernatant, stirring, performing secondary centrifugation through a centrifuge, and dissolving and precipitating the supernatant after the secondary centrifugation for at least 12 hours; b 8: adding additives to regulate the content of IgY (chicken yolk immunoglobulin); b 9: adding 0.22um membrane for filtration and sterilization; b10 sterilizing by Pasteur for 0.5 hr, filling and packaging.
And a step a8, adding additives, regulating the content of IgY (chicken egg yolk immunoglobulin), adding a sodium oxide reagent into the diluted egg yolk filtered in the step a7, adding the sodium oxide reagent, precipitating, and fixing the volume after precipitation to ensure that the content of the IgY (chicken egg yolk immunoglobulin) is about 90 percent.
IgY detection methods, including electrophoresis, SEC, binding activity, pseudovirus neutralization activity, ACE2 competition activity and HPLC.
In the description of the present invention, it is to be understood that the indicated orientations or positional relationships are based on the orientations or positional relationships shown in the drawings and are only for convenience in describing the present invention and simplifying the description, but are not intended to indicate or imply that the indicated devices or elements must have a particular orientation, be constructed and operated in a particular orientation, and are not to be construed as limiting the present invention.
In the present invention, unless otherwise explicitly specified or limited, for example, it may be fixedly attached, detachably attached, or integrated; can be mechanically or electrically connected; the terms may be directly connected or indirectly connected through an intermediate, and may be communication between two elements or interaction relationship between two elements, unless otherwise specifically limited, and the specific meaning of the terms in the present invention will be understood by those skilled in the art according to specific situations.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (6)
1. Based on the industrial application of gene recombination anti-Covid-19 and other coronavirus IgY antibodies and other small molecule antibodies, the method is characterized by comprising the following steps:
s1: cloning and expressing a new coronavirus S protein gene;
s2: preparing an antigen, namely performing Baculoviral-Insect Cell expression and His-tag (polyhistidine tag) purification of Insect cells through a bioreactor to ensure that the purity of the antigen is kept at 93%;
s3: selecting animals, namely selecting high-yield hens, wherein the age of the hens is the best age of the hens which just lay eggs;
s4: immunizing selected animals by using an oil adjuvant and an immunopotentiator, wherein the immunization period is about 2 months, the immunization frequency is 3 times, and after the third immunization is finished, eggs produced by immunized hens are collected at the time interval of 2 weeks;
s5: detecting the antibody of the hen, and detecting whether the antibody is produced or not and the titer of each hen by using an ELISA (enzyme-linked immunosorbent assay) method, wherein the ELISA detects the OD (optical density) value, and the formula is as follows: judging the sample OD value/negative control OD value is greater than 2.1 to be positive, taking the reciprocal of the dilution multiple of the hole corresponding to the minimum positive value as the highest titer, and making an antibody titer change table in the immune cycle;
s6: preparing an antibody;
s7: measuring the content of the antibody and the purity of the antibody;
s8: adding various additives including antiseptic and stabilizer, adjusting IgY content to be more than 3mg/ml, filtering with 0.2um filter membrane for sterilization, inactivating virus, and packaging.
2. The industrial application of anti-Covid-19 and other coronavirus IgY antibodies and other small molecule antibodies based on gene recombination according to claim 1, which is characterized in that: cloning and expressing the new coronavirus S protein gene in the step S1, cloning the new coronavirus S protein gene according to a known probe, labeling the probe with radioactivity or nonradioactive label, hybridizing the probe with genomic DNA treated by different endonucleases, and finally cutting the identified fragment from the gel and cloning the fragment to a specific vector.
3. The industrial application of anti-Covid-19 and other coronavirus IgY antibodies and other small molecule antibodies based on gene recombination according to claim 1, which is characterized in that: the step S6 comprises primary centrifugation and secondary centrifugation, wherein the primary centrifugation comprises the following steps:
a 1: weighing the collected eggs; a 2: PBS (phosphate buffered saline); a 3: beating the eggs into a glass ware, and separating egg yolk from egg white; a 4: stirring the separated yolk, wherein the stirring time is controlled to be 0.5 hour; a 5: precipitating the stirred egg yolk, wherein the precipitation time is controlled to be 0.5 hour; a 6: centrifuging the precipitated egg yolk by a centrifuge; a, 7: filtering the centrifuged egg yolk; a 8: adding an additive, adjusting the content of IgY (egg yolk immunoglobulin), carrying out constant volume for 12 hours, a 9: adding 0.45um and 0.22um membranes into the constant-volume egg yolk, and filtering and sterilizing; a 10: performing pasteurization for 0.5 hr, and packaging.
4. The industrial application of anti-Covid-19 and other coronavirus IgY antibodies and other small molecule antibodies based on gene recombination according to claim 3, characterized in that: the secondary centrifugation comprises the following steps:
b1 weighing the collected eggs; b 2: PBS (phosphate buffered saline); b 3: beating the eggs into a glass ware, and separating egg yolk from egg white; b 4: stirring the separated yolk, wherein the stirring time is controlled to be 0.5 hour; b 5: precipitating the stirred egg yolk, wherein the precipitation time is controlled to be 0.5 hour; b 6: centrifuging the precipitated egg yolk by a centrifuge, and adding a 0.45um membrane for filtration during centrifugation; b 7: adding a precipitator into the supernatant, stirring, performing secondary centrifugation through a centrifuge, and dissolving and precipitating the supernatant after the secondary centrifugation for at least 12 hours; b 8: adding additives to regulate the content of IgY (chicken yolk immunoglobulin); b 9: adding 0.22um membrane for filtration and sterilization; b10 sterilizing by Pasteur for 0.5 hr, filling and packaging.
5. The industrial application of anti-Covid-19 and other coronavirus IgY antibodies and other small molecule antibodies based on gene recombination according to claim 3, characterized in that: and a step a8, adding an additive, regulating the content of IgY (chicken egg yolk immunoglobulin), adding a sodium oxide reagent into the diluted egg yolk filtered in the step a7, adding the sodium oxide reagent, precipitating, and fixing the volume after precipitation to ensure that the content of the IgY (chicken egg yolk immunoglobulin) is about 90 percent.
6. The industrial application of anti-Covid-19 and other coronavirus IgY antibodies and other small molecule antibodies based on gene recombination according to claim 1, which is characterized in that: the IgY detection method comprises electrophoresis, SEC, binding activity, pseudovirus neutralization activity, ACE2 competition activity and HPLC.
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