CN113234146A - Preparation method of coronavirus spike protein monoclonal antibody - Google Patents
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Abstract
The invention discloses a preparation method of a coronavirus spike protein monoclonal antibody, which is characterized in that the obtained spike protein is concentrated by adopting a tangential flow ultrafiltration method, and the concentrated spike protein is purified by adopting a chromatography purification method; immunizing a mouse by using the purified spike protein receptor binding domain, and fusing a mouse spleen cell suspension and myeloma cells after immunization to obtain hybridoma cells; coating the spike protein receptor binding domain into an enzyme label plate, and then carrying out indirect ELISA screening on the hybridoma cells to obtain positive clones; and subcloning the positive clone, adopting a stable hybridoma cell strain screened by serum-free fermentation culture, and purifying to obtain the spike protein monoclonal antibody, so that the cure rate of the coronavirus can be improved.
Description
Technical Field
The invention relates to the technical field of monoclonal antibody preparation, in particular to a preparation method of a coronavirus spike protein monoclonal antibody.
Background
Based on current epidemiological investigations, the latency of the current epidemiological virus is generally 3-7 days, and the maximum is not more than 14 days. The clinical manifestations of this epidemic disease mainly include fever, hypodynamia and dry cough. A few patients have nasal obstruction, watery nasal discharge, diarrhea, etc. Some patients only show low fever, slight weakness, etc., and no pulmonary inflammation, and most of them recover after 1 week. The world health organization names 2019 novel coronavirus which is SARS-CoV-2 virus, the pathogen causing this viral pneumonia case is SARS-CoV-2 virus, the spike protein is surface membrane protein of SARS-CoV-2, and comprises two subunits, S1 and S2, S1 mainly comprises a receptor binding domain which is responsible for recognizing cell surface receptor, and S2 comprises essential elements required for membrane fusion.
The SARS-CoV-2 virus at present has extremely strong infection ability, no effective medicine appears, and the SARSCoV-2 virus can be eliminated, so that the patient can be cured only by the self resistance, and the cure rate of the SARS-CoV-2 virus is greatly reduced.
Disclosure of Invention
The invention aims to provide a preparation method of a coronavirus spike protein monoclonal antibody, which improves the cure rate of SARS-CoV-2 virus.
In order to achieve the above object, the present invention provides a method for preparing a coronavirus spike protein monoclonal antibody, comprising the steps of:
concentrating the obtained spike protein by adopting a tangential flow ultrafiltration method, and purifying the concentrated spike protein by adopting a chromatography purification method;
immunizing a mouse by using the purified spike protein receptor binding domain, and fusing a mouse spleen cell suspension and myeloma cells after immunization to obtain hybridoma cells;
coating the spike protein receptor binding domain into an enzyme label plate, and then carrying out indirect ELISA screening on the hybridoma cells to obtain positive clones;
and subcloning the positive clone, adopting serum-free fermentation culture to screen out a hybridoma stable cell strain, and purifying to obtain the spike protein monoclonal antibody.
The spike protein is the surface membrane protein of SARS-CoV-2, and comprises two subunits S1 and S2, S1 mainly comprises a receptor binding domain which is responsible for recognizing cell surface receptor, S2 comprises essential elements required for membrane fusion, and the S protein plays a key role in inducing, reacting with antibody and T cell and protective immunity, so that the spike protein monoclonal antibody can be well combined with the spike protein to form a compound which can be digested or degraded, thereby inactivating SARS-CoV-2 virus, achieving the effect of killing SARS-CoV-2 virus and improving the cure rate of SARS-CoV-2 virus.
Wherein, the method of tangential flow ultrafiltration is adopted to concentrate the obtained spike protein, and the method of chromatography purification is adopted to purify the concentrated spike protein, comprising the following steps:
acquiring spike protein required by preparation, repeatedly intercepting the spike protein by a 50kDa ultrafiltration membrane under the environment that the vacuum pump pressure is 0.1MPa and the reflux pressure is 0.05MPa, and collecting the spike protein in a sample collecting cup until the concentration volume is 20 mL;
and purifying the concentrated spike protein for multiple times by using an agarose gel filtration chromatographic column, and collecting eluent.
The purity of the obtained spike protein is improved, and the quality and the efficiency of the obtained antibody are ensured.
Wherein, the purified spike protein receptor binding domain is used for immunizing a mouse, and the spleen cell suspension of the immunized mouse is fused with myeloma cells to obtain hybridoma cells, which comprises the following steps:
immunizing a mouse for multiple times by using the purified spike protein receptor binding domain, wherein the immunization time interval is two weeks each time;
mixing the spleen cells of the mice after the immunization is finished for a set time length with the obtained myeloma cells of the mice according to the volume ratio of 1: 1;
and (3) carrying out cell fusion by using a cell fusion instrument, paving the fused cells into a 96-hole cell culture plate, adding HAT screening reagent, and carrying out primary complete liquid change after culturing for 5 days to obtain the hybridoma.
Through multiple immunizations of the mice, more immunized splenocytes can be ensured to be obtained as much as possible, and the quantity of the obtained antibodies is increased.
Wherein before the mouse spleen cells after the immunization is completed for a set time length and the obtained mouse myeloma cells are mixed in a volume ratio of 1:1, the method further comprises the following steps:
taking the spleens of the mice 1 week after multiple immunizations, putting the spleens of the mice into a culture dish containing 5ml of MEM solution, crushing and filtering to obtain a spleen cell suspension.
The purpose of crushing is to facilitate fusion with myeloma cells, and extraction of antibodies.
Wherein, the spike protein receptor binding domain is coated into an enzyme label plate, and then the hybridoma cells are subjected to indirect ELISA screening to obtain positive clones, which comprise:
coating the spike protein receptor binding domain into an enzyme label plate, performing overnight culture at the temperature of 4 ℃, washing with PBST (Poly-p-phenylene-succinate) with the pH of 7.4 for three times, adding a sealing solution at the temperature of 37 ℃, sealing for 2 hours, and then removing the sealing solution at the temperature of 37 ℃ and drying;
adding 100 mu L of supernatant of the hybridoma cells into the ELISA plate per well, incubating for 60min at the temperature of 36-37 ℃, and adding 100 mu L of goat anti-mouse IgG enzyme-labeled secondary antibody with the mass fraction of 0.5 per mill into the ELISA plate per well;
and (3) incubating for 60min at the temperature of 36-37 ℃, adding 100 mu L of TMB single-component developing solution into each hole of the ELISA plate, reacting for 15-20min at room temperature, adding 50 mu L of stop solution into each hole, performing indirect ELISA screening, and keeping positive clones.
The positive rate and specificity of the general selection are improved.
Wherein the confining liquid is one or more of 5% skimmed milk, 1% BSA, 1% OVA and 2% gelatin.
The invention relates to a preparation method of a coronavirus spike protein monoclonal antibody, which is characterized in that the obtained spike protein is concentrated by adopting a tangential flow ultrafiltration method, and the concentrated spike protein is purified by adopting a chromatography purification method; immunizing a mouse by using the purified spike protein receptor binding domain, and fusing a mouse spleen cell suspension and myeloma cells after immunization to obtain hybridoma cells; coating the spike protein receptor binding domain into an enzyme label plate, and then carrying out indirect ELISA screening on the hybridoma cells to obtain positive clones; and subcloning the positive clone, adopting a stable hybridoma cell strain screened by serum-free fermentation culture, and purifying to obtain the spike protein monoclonal antibody, wherein the cure rate of the monoclonal antibody to SARS-CoV-2 virus can be improved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic diagram of the steps of a method for preparing a coronavirus spike protein monoclonal antibody provided by the invention.
FIG. 2 is a schematic flow chart of a method for preparing a coronavirus spike protein monoclonal antibody provided by the invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.
Referring to fig. 1 and 2, the present invention provides a method for preparing a coronavirus spike protein monoclonal antibody, comprising the following steps:
s101, concentrating the obtained spike protein by adopting a tangential flow ultrafiltration method, and purifying the concentrated spike protein by adopting a chromatography purification method.
Specifically, the spike protein required by antibody preparation is obtained, and the spike protein is repeatedly intercepted by a 50kDa ultrafiltration membrane and collected in a sample collection cup under the environment that the vacuum pump pressure is 0.1MPa and the reflux pressure is 0.05MPa until the concentration volume is 20 mL; and (4) sampling filtered waste liquid in the test process, and freezing at-20 ℃ to be detected.
Purifying spike protein with Sepharose 4Fast Flow (4FF) filtration chromatography column of GE, wherein the Sepharose filtration column has a length of about 150cm and a diameter of about 2cm, and the sample loading volume is no more than 10% of the column area, balancing the whole column with PBS buffer solution before use, setting the Flow rate at 0.3mL/min, simultaneously monitoring OD280 absorbance in real time by using Berlol biological liquid chromatography system, respectively collecting samples with two peaks for detection, and performing the whole operation at 4 deg.C.
In order to determine the effectiveness of the purification method combining ultrafiltration and sepharose gel filtration chromatography (TFF-SEC), a fluorescent quantitative PCR method is adopted to respectively quantify samples collected by two corresponding peaks of virus culture supernatant before ultrafiltration concentration, concentrated waste liquid and filtered waste liquid after concentration and sepharose gel chromatography molecular sieve, and the purification effect of the ultrafiltration and sepharose gel molecular sieve technology is evaluated from the gene level; TCID50 is used to analyze whether the treated virus can ensure the activity of the virus, meanwhile, phosphotungstic acid is negatively infected, the treated virus particles are observed by an electron microscope, and SDS-PAGE and Westernblot are used to analyze the purified virus.
The gel filtration chromatography technology has the advantages of simple operation, mild operation conditions, simple operation method and good repeatability. The operator can conveniently collect the sample peak; and the cost is low, the operation can be carried out by only one peristaltic pump and one ultraviolet detector, and an expensive liquid chromatography system is not needed.
By adopting ultrafiltration combined with agarose gel column, most of biogenic impurities such as hybrid protein, glycolipid, nucleic acid and the like in the culture medium can be efficiently removed, and the concentration, enrichment and purification of the immunocompetent components of the virus are completed.
S102, immunizing a mouse by using the purified spike protein receptor binding domain, and fusing the immunized mouse spleen cell suspension and myeloma cells to obtain hybridoma cells.
Specifically, the RBD region sequence of the Skipe protein is subjected to whole gene synthesis, subcloned to a eukaryotic expression vector, and the C end of the expression vector is added with 8 × His tag to express the purified spike protein receptor binding domain SP-RBD-His. Immunizing Balb/c mice with a spike protein receptor binding domain SP-RBD-His, wherein each Balb/c mouse is immunized with 50 mug for 3 times at an interval of two weeks, and the immunization specifically comprises the following steps:
mixing a spike protein receptor binding domain SP-RBD-His and a Freund complete adjuvant according to a mass ratio of 1:1 to prepare a first mixed solution, carrying out subcutaneous injection on a 6-8-week-old Balb/c mouse by using the mixed solution, wherein the number of injection sites is 6-8, and the injection amount is 50 mu g each time to obtain a primary immune mouse, mixing the spike protein receptor binding domain SP-RBD-His and the Freund incomplete adjuvant according to a mass ratio of 1:1 to prepare a second mixed solution, and repeatedly immunizing the primarily immunized mouse for two weeks by using the second mixed solution for 2 times, wherein the interval is two weeks each time.
The specific steps of cell fusion are as follows: taking mouse spleen 1 week after three times of immunization, putting the mouse spleen into a culture dish containing 5ml of MEM liquid, crushing the spleen or putting the spleen into corresponding grinding equipment, grinding at a set rotating speed for a set grinding time, then filtering by using a nylon net or a 50-mesh screen to obtain a single cell suspension, mixing the single cell suspension and a logarithmic phase mouse myeloma cell SP2/0 cell liquid in a volume ratio of 1:1, carrying out cell fusion by using a cell fusion instrument, paving the fused cells into a 96-hole cell culture plate, adding an HAT screening reagent, and carrying out one-time total liquid change after 5 days of culture.
Wherein, the sequence of the RBD region of the Skipe protein is as follows:
RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNHHHHHHHH。
s103, coating the spike protein receptor binding domain into an enzyme label plate, and then performing indirect ELISA screening on the hybridoma cells to obtain positive clones.
Specifically, a spike protein receptor binding domain SP-RBD-His is coated into an ELISA plate, after overnight culture is carried out at the temperature of 4 ℃, PBST with the pH value of 7.4 is used for three times, a confining liquid is added at the temperature of 37 ℃ for sealing for 2 hours, then the confining liquid is discarded and dried at the temperature of 37 ℃, 100 microliter of supernatant of fused cells is added into the ELISA plate, after incubation is carried out for 60 minutes at the temperature of 36-37 ℃, 100 microliter of goat anti-mouse IgG enzyme-labeled secondary antibody with the mass fraction of 0.5 thousandths is added into the ELISA plate per well, after incubation is carried out for 60 minutes at the temperature of 36-37 ℃, 100 microliter of TMB single-component developing solution per well is added into the ELISA plate, after the incubation is carried out at the room temperature for 15-20 minutes, 50 microliter of stop solution is added into each well, indirect ELISA screening is carried out, and positive clones are retained. Wherein the confining liquid is one or more of 5% skimmed milk, 1% BSA, 1% OVA and 2% gelatin.
In order to observe the culture effect conveniently, a goat anti-mouse antibody or a goat anti-pig antibody can be marked by using 100 mu L of HRP or FITC diluted by 1:100 times, and then the positive clone obtained by screening is detected by a specific detection method: IFA judges the negative and positive result directly under the fluorescence microscope through detecting whether there is green fluorescence point, and IPMA's result needs to add 100 mu LAEC color development liquid per hole to develop color, use DDW to stop developing color, observe the test result under the microscope, select the positive clone qualified to detect, carry on the subsequent preparation process, select the qualified positive clone, can improve the quality and preparation efficiency of the antibody obtained, meanwhile, reduce the cost of the reagent that the subsequent preparation process needs to use.
S104, subcloning the positive clone, adopting serum-free fermentation culture to screen out a hybridoma stable cell strain, and purifying to obtain the spike protein monoclonal antibody.
Specifically, the positive clones are subjected to cloning culture and screening of positive subclones in time by adopting a limiting dilution method for detecting positive hybridoma cells until the positive detection rate is 100%, so that a hybridoma stable cell strain is obtained.
Culturing a hybridoma stable cell strain by adopting serum-free fermentation, collecting cell culture supernatant, mixing the supernatant, ProntenA and a phosphate buffer solution with the pH of 7.4, adding the mixture into a chromatographic column, eluting an antibody by using a citric acid solution with the pH of 4.0 and using a phosphate buffer solution with the pH of 7.4 as a balancing solution, adjusting the pH value to 7.0 by using a Tris-HCl buffer solution with the pH of 9.0, and collecting the antibody to obtain the spike protein monoclonal antibody.
Performing whole gene synthesis through a Skipe protein RBD region sequence, performing subcloning on a gene onto a carrier by taking a eukaryotic cell as the carrier, adding 8 His labels to the C end of the carrier to prepare a spike protein receptor binding domain SP-RBD-His, immunizing a 6-8-week-old Balb/C mouse by taking the spike protein receptor binding domain SP-RBD-His as an antigen, performing 3 times of immunization, taking the spleen of the mouse after one week of immunization to prepare a spleen single-cell suspension, performing cell fusion with a mouse logarithmic myeloma cell SP2/0, further culturing the fused cell, performing indirect ELISA screening on the fused cell, performing subcloning on a positive clone after retaining the positive clone to ensure that the positive detection rate is 100 percent to obtain a stable hybridoma cell strain, culturing the hybridoma cell strain, performing affinity purification through Pronteina, the obtained spike protein monoclonal antibody is the surface membrane protein of SARS-CoV-2, and contains two subunits S1 and S2, S1 mainly contains a receptor binding domain, and is responsible for recognizing cell surface receptor, S2 contains essential elements required for membrane fusion, and S protein plays a key role in inducing, reacting with antibody and T cell and protective immunity, so that the spike protein monoclonal antibody can be well combined with spike protein to form a compound, and the compound can be digested or degraded, thereby inactivating SARS-CoV-2 virus, and achieving the effect of killing SARS-CoV-2 virus.
The parameters such as temperature and the like involved in the invention can be up to plus or minus about 5 ℃ on the numerical value disclosed by the invention, and are mainly used for fitting the actual preparation environment temperature and improving the operation flexibility. In addition, the related additive reagents can be replaced by other reagents with the same pH value and concentration, so that the selectivity of the reagents is improved.
The invention relates to a preparation method of a coronavirus spike protein monoclonal antibody, which is characterized in that the obtained spike protein is concentrated by adopting a tangential flow ultrafiltration method, and the concentrated spike protein is purified by adopting a chromatography purification method; immunizing a mouse by using the purified spike protein receptor binding domain, and fusing a mouse spleen cell suspension and myeloma cells after immunization to obtain hybridoma cells; coating the spike protein receptor binding domain into an enzyme label plate, and then carrying out indirect ELISA screening on the hybridoma cells to obtain positive clones; and subcloning the positive clone, adopting a stable hybridoma cell strain screened by serum-free fermentation culture, and purifying to obtain the spike protein monoclonal antibody, wherein the cure rate of the monoclonal antibody to SARS-CoV-2 virus can be improved.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (6)
1. A preparation method of a coronavirus spike protein monoclonal antibody is characterized by comprising the following steps:
concentrating the obtained spike protein by adopting a tangential flow ultrafiltration method, and purifying the concentrated spike protein by adopting a chromatography purification method;
immunizing a mouse by using the purified spike protein receptor binding domain, and fusing a mouse spleen cell suspension and myeloma cells after immunization to obtain hybridoma cells;
coating the spike protein receptor binding domain into an enzyme label plate, and then carrying out indirect ELISA screening on the hybridoma cells to obtain positive clones;
and subcloning the positive clone, adopting serum-free fermentation culture to screen out a hybridoma stable cell strain, and purifying to obtain the spike protein monoclonal antibody.
2. The method of claim 1, wherein the step of concentrating the obtained spike protein by tangential flow ultrafiltration and the step of purifying the concentrated spike protein by chromatography comprises:
acquiring spike protein required by preparation, repeatedly intercepting the spike protein by a 50kDa ultrafiltration membrane under the environment that the vacuum pump pressure is 0.1MPa and the reflux pressure is 0.05MPa, and collecting the spike protein in a sample collecting cup until the concentration volume is 20 mL;
and purifying the concentrated spike protein for multiple times by using an agarose gel filtration chromatographic column, and collecting eluent.
3. The method of claim 1, wherein immunizing a mouse with said purified spike protein receptor binding domain and fusing a suspension of mouse spleen cells after immunization with myeloma cells to obtain hybridoma cells comprises:
immunizing a mouse for multiple times by using the purified spike protein receptor binding domain, wherein the immunization time interval is two weeks each time;
mixing the spleen cells of the mice after the immunization is finished for a set time length with the obtained myeloma cells of the mice according to the volume ratio of 1: 1;
and (3) carrying out cell fusion by using a cell fusion instrument, paving the fused cells into a 96-hole cell culture plate, adding HAT screening reagent, and carrying out primary complete liquid change after culturing for 5 days to obtain the hybridoma.
4. The method of claim 3, wherein the method further comprises, before mixing the spleen cells of the mouse after completion of the immunization for a predetermined period of time and the myeloma cells of the mouse obtained at a volume ratio of 1:
taking the spleens of the mice 1 week after multiple immunizations, putting the spleens of the mice into a culture dish containing 5ml of MEM solution, crushing and filtering to obtain a spleen cell suspension.
5. The method of claim 1, wherein the monoclonal antibody against a coronavirus spike-protein receptor is coated on a microplate, and the hybridoma cells are subjected to indirect ELISA screening to obtain positive clones, comprising:
coating the spike protein receptor binding domain into an enzyme label plate, performing overnight culture at the temperature of 4 ℃, washing with PBST (Poly-p-phenylene-succinate) with the pH of 7.4 for three times, adding a sealing solution at the temperature of 37 ℃, sealing for 2 hours, and then removing the sealing solution at the temperature of 37 ℃ and drying;
adding 100 mu L of supernatant of the hybridoma cells into the ELISA plate per well, incubating for 60min at the temperature of 36-37 ℃, and adding 100 mu L of goat anti-mouse IgG enzyme-labeled secondary antibody with the mass fraction of 0.5 per mill into the ELISA plate per well;
and (3) incubating for 60min at the temperature of 36-37 ℃, adding 100 mu L of TMB single-component developing solution into each hole of the ELISA plate, reacting for 15-20min at room temperature, adding 50 mu L of stop solution into each hole, performing indirect ELISA screening, and keeping positive clones.
6. The method of claim 5, wherein the coronavirus spike protein monoclonal antibody is produced by a single-chain reaction,
the confining liquid is one or more of 5% skimmed milk, 1% BSA, 1% OVA and 2% gelatin.
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