CN111153991A - Human SARS-CoV-2 monoclonal antibody and its preparation method and use - Google Patents

Human SARS-CoV-2 monoclonal antibody and its preparation method and use Download PDF

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CN111153991A
CN111153991A CN202010120349.4A CN202010120349A CN111153991A CN 111153991 A CN111153991 A CN 111153991A CN 202010120349 A CN202010120349 A CN 202010120349A CN 111153991 A CN111153991 A CN 111153991A
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赵风强
秦李娜
吴炬
王咚咚
董兵
张凤英
邬晓乐
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Beijing Biosynthesis Biotechnology Co ltd
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Abstract

The invention discloses a human SARS-CoV-2 monoclonal antibody, its preparation method is: using SARS-CoV Nucleocapsid recombinant protein as immunogen, immunizing BALB/c mouse, making fusion and subcloning of mouse spleen cell and mouse bone marrow tumour cell, then making lots of repeated screening and domestication of cell strain by using commercial products SARS-CoV-2Nucleocapsid and MERS Nucleocapsid, finally successfully obtaining high affinity and high specificity anti SARS-CoV-2N monoclonal antibody hybridoma cell strain, finally preparing and purifying ascites so as to obtain monoclonal antibody; the amino acid sequence of the SARS-CoV Nucleocapsid recombinant protein is shown in SEQ ID NO. 1. The invention also discloses the application of the monoclonal antibody in preparing SARS-CoV-2 virus detection products and in preparing SARS-CoV-2 virus inhibiting medicine. The monoclonal antibody of the invention can detect SARS-CoV-2 in human throat swab/lung secretion and other samples by a double antibody sandwich method, can be applied to a SARS-CoV-2 detection kit, and is suitable for diagnosis, prevention and control of SARS-CoV-2 virus infection, scientific research of virus and the like.

Description

Human SARS-CoV-2 monoclonal antibody and its preparation method and use
Technical Field
The invention relates to a human SARS-CoV-2 monoclonal antibody and its preparation method and application, belonging to the technical field of monoclonal antibody preparation.
Background
On day 9/1 of 2020, scientists observed under an electron microscope that the pathogen responsible for this pneumonia presented an enveloped typical coronavirus morphology with a coronal appearance. Meanwhile, the sequencing result of pathogenic genome shows that the nucleic acid sequence of the coronavirus is not completely consistent with the 6 coronavirus (such as SARS, MERS and the like) discovered before. Thus, the World Health Organization (WHO) named the new virus at 12.1/2020: 2019Novel Coronavirus (2019Novel Coronavir, 2019-nCoV), day 11 month 2, the International Committee for Classification of viruses (ICTV) named SARS-CoV-2. The virus has long incubation period and strong infectivity, and as long as 24 days 2, 18 and 18 in 2020, 74185 cases (severe cases 11977) have been diagnosed, 14376 cases are cured cumulatively, 2004 cases are died cumulatively, and 5248 cases are suspected. Close contacts 574418 were cumulatively tracked, close contacts 135881 who were still under medical observation (information from the national health Care official website). There have been 25 diagnosed cases in countries and regions worldwide (world health organization 2/10/2020), which have constituted the emergent public health event of international concern. At present, specific targeted drug treatment is not available, so that the prevention of healthy people, the diagnosis and isolation of suspected and confirmed cases and treatment work are very important.
Coronaviruses belong to the order of the nested viruses (Nidovirales), the family of Coronaviridae (Coronaviridae), and the genus coronaviruses (Coronavirus), and are the largest viruses of the RNA viruses known to humans at present, and have a length of 27 to 32 kb. Coronaviruses have at least 4 major structural proteins, including spike (S), membrane (M), envelope (E) and nucleocapsid (N) proteins, which are essential for binding of the virus to cellular receptors and are essential for the complete structure of the virus. Wherein the nucleocapsid protein (N) is a basic phosphoprotein, the central region of which is combined with the viral genome RNA to form a coiled nucleocapsid helix, which is the core structure of the viral genetic material coated therein and is one of the viral proteins with the highest expression in the infected cells.
Current laboratory diagnosis of viral infections generally involves 3 methods: molecular diagnostics based on PCR technology; immunological-based serological tests; pathogenic biological diagnosis based on in vitro virus culture. In vitro virus culture is generally not a routine diagnostic method because it is highly demanding both on assay conditions and on the skill of the operator. With the continuous innovation of gene sequencing technology in recent years, scientists complete the genome sequencing of viruses at the initial stage of the new coronary pneumonia epidemic situation, and then a plurality of nucleic acid detection kits are developed and put into use. At present, laboratory detection of suspected cases of the new coronavirus is multipurpose nucleic acid detection (PCR), but most of nucleic acid detection samples are upper respiratory tract samples (throat swab and sputum are main samples), and the collection process has great exposure risk to medical workers. And the experimental process is long, about 4-5 h, and pollution (namely false positive or false negative results) is easy to occur. And due to frequent virus variation, primer design and the like, the nucleic acid detection method also has the problems of high false negative, low coincidence rate of detection results of different manufacturers and the like in the practical application process. The diagnosis method for detecting the virus antigen can greatly reduce the risk, and because serum is generally adopted as a detection sample, the sample is convenient to collect and low in toxic content, meanwhile, a complex processing procedure of the sample in a laboratory is omitted, a detection result can be obtained in 15-20 min, the operation is simple, and the current huge clinical diagnosis and treatment pressure can be greatly relieved while medical care personnel are protected.
The key to the development of SARS-CoV-2 virus antigen detection kit is to find out the antibody with high specificity and high affinity. Compared with polyclonal antibodies, the monoclonal antibody has the advantage of high specificity, so that the detection specificity and the anti-interference capability of the constructed kit can be greatly improved. In addition, because the protein on the virus envelope is easy to be mutated, the N protein is relatively stable, is the main antigen part of the virus, and has stronger antigenicity than other proteins. And at present, no commercial diagnostic kit taking SARS-CoV-2N protein monoclonal antibody as a core raw material is available.
Disclosure of Invention
Aiming at the prior art, the invention provides a human SARS-CoV-2 monoclonal antibody, a preparation method and application thereof. The invention utilizes DNA recombination technology to synthesize SARS-CoV Nucleocapsid full-length gene, constructs into prokaryotic expression vector pET28a, and obtains a large amount of SARS-CoVNucleocapsid protein in vitro through optimized fermentation, purification and renaturation processes; the monoclonal antibody of anti-human SARS-CoVNucleucoapsid is successfully prepared by immunizing BALB/c female mice and adopting a hybridoma technology; then, a large amount of repeated screening and cell strain domestication are carried out on the commercialized products SARS-CoV-2Nucleocapsid and MERSNuclocapsid, and finally, the anti-SARS-CoV-2N monoclonal antibody with high affinity and high specificity is successfully obtained; on the basis of the above-mentioned diagnosis method, a quick and quick diagnosis method for SARS-CoV-2 and its diagnosis reagent are developed, so that it provides a quick and accurate method for clinically diagnosing SARS-CoV-2 infection, at the same time it also provides strong support for scientific research of new virus.
The invention is realized by the following technical scheme:
a human SARS-CoV-2 monoclonal antibody, using SARS-CoV nucleoapsid recombinant protein as immunogen to immunize mouse to obtain monoclonal antibody; the amino acid sequence of the SARS-CoV Nucleocapsid recombinant protein is shown as SEQID NO. 1.
The SARS-CoV Nucleocapsid recombinant protein is prepared by the following method: human SARS-CoV nucleoapsid gene (nucleotide sequence shown in SEQ ID NO.2) is inserted into expression vector pET28a to construct pET28a-SARS-CoV-N fusion expression plasmid, and then converted into colibacillus, and then induced by IPTG, purified and renatured to obtain SARS-CoV nucleoapsid recombinant protein.
Amino acid sequence of SARS-CoV nucleoapsid recombinant protein (SEQ ID NO. 1):
MSDNGPQSNQRSAPRITFGGPTDSTDNNQNGGRNGARPKQRRPQGLPNNTASWFTALTQHGKEELRFPRGQGVPINTNSGPDDQIGYYRRATRRVRGGDGKMKELSPRWYFYYLGTGPEASLPYGANKEGIVWVATEGALNTPKDHIGTRNPNNNAATVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRGNSRNSTPGSSRGNSPARMASGGGETALALLLLDRLNQLESKVSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKQYNVTQAFGRRGPEQTQGNFGDQDLIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYHGAIKLDDKDPQFKDNVILLNKHIDAYKTFPPTEPKKDKKKKTDEAQPLPQRQKKQPTVTLLPAADMDDFSRQLQNSMSGASADSTQA
nucleotide sequence of human SARS-CoV nucleoapsid gene (SEQ ID NO. 2):
atgtctgataatggaccccaatcaaaccaacgtagtgccccccgcattacatttggtggacccacagattcaactgacaataaccagaatggaggacgcaatggggcaaggccaaaacagcgccgaccccaaggtttacccaataatactgcgtcttggttcacagctctcactcagcatggcaaggaggaacttagattccctcgaggccagggcgttccaatcaacaccaatagtggtccagatgaccaaattggctactaccgaagagctacccgacgagttcgtggtggtgacggcaaaatgaaagagctcagccccagatggtacttctattacctaggaactggcccagaagcttcacttccctacggcgctaacaaagaaggcatcgtatgggttgcaactgagggagccttgaatacacccaaagaccacattggcacccgcaatcctaataacaatgctgccaccgtgctacaacttcctcaaggaacaacattgccaaaaggcttctacgcagagggaagcagaggcggcagtcaagcctcttctcgctcctcatcacgtagtcgcggtaattcaagaaattcaactcctggcagcagtaggggaaattctcctgctcgaatggctagcggaggtggtgaaactgccctcgcgctattgctgctagacagattgaaccagcttgagagcaaagtttctggtaaaggccaacaacaacaaggccaaactgtcactaagaaatctgctgctgaggcatctaaaaagcctcgccaaaaacgtactgccacaaaacagtacaacgtcactcaagcatttgggagacgtggtccagaacaaacccaaggaaatttcggggaccaagacctaatcagacaaggaactgattacaaacattggccgcaaattgcacaatttgctccaagtgcctctgcattctttggaatgtcacgcattggcatggaagtcacaccttcgggaacatggctgacttatcatggagccattaaattggatgacaaagatccacaattcaaagacaacgtcatactgctgaacaagcacattgacgcatacaaaacattcccaccaacagagcctaaaaaggacaaaaagaaaaagactgatgaagctcagcctttgccgcagagacaaaagaagcagcccactgtgactcttcttcctgcggctgacatggatgatttctccagacaacttcaaaattccatgagtggagcttctgctgattcaactcaggcataa
the preparation method of the human SARS-CoV-2 monoclonal antibody comprises the following steps: using SARS-CoV Nucleocapsid recombinant protein as immunogen to immunize BALB/c mouse, then making fusion and subcloning of mouse spleen cell and mouse myeloma cell, and making specific screening and domestication by using SARS-CoV-2Nucleocapsid and MERS Nucleocapsid to finally obtain monoclonal cell strain with good specificity and high potency, then making ascites preparation and purification so as to obtain a large quantity of monoclonal antibody resisting human SARS-CoV Nucleocapsid.
Preferably, female mice 8-12 weeks old are selected for said BALB/c mice.
Preferably, the screening method is an ELISA method (enzyme-linked immunosorbent assay).
Preferably, the purification method is Protein a affinity purification.
The human SARS-CoV-2 monoclonal antibody is applied in preparing SARS-CoV-2 virus detecting product.
The human SARS-CoV-2 monoclonal antibody is applied in preparing medicine for inhibiting SARS-CoV-2 virus.
The human SARS-CoV-2 monoclonal antibody is applied in preparing medicine preparation for preventing and treating SARS-CoV-2 virus infection caused diseases.
A method for detecting the level of SARS-CoV-2 virus for non-diagnostic purposes: contacting the sample to be detected with the human SARS-CoV-2 monoclonal antibody, and detecting the immunoreaction of the sample and the human SARS-CoV-2 monoclonal antibody. The sample to be detected is selected from sputum, oral/nasopharyngeal secretions, pulmonary secretions and the like.
A reagent kit for detecting SARS-CoV-2 virus, which comprises the above-mentioned human SARS-CoV-2 monoclonal antibody.
A medicine for inhibiting SARS-CoV-2 virus contains the above-mentioned human SARS-CoV-2 monoclonal antibody.
The invention provides an expression and purification method of human SARS-CoV Nucleocapsid in vitro recombinant protein, which solves the problems of difficult acquisition of natural human SARS-CoV Nucleocapsid protein, and in vitro expression yield, purity and stability. Moreover, the SARS-CoV Nucleocapsid recombinant protein is used to immunize animal, so that it can prepare wider anti-SARS-CoVNucleocapsid antibody, and through the specific screening and domestication of SARS-CoV-2Nucleocapsid and MERS Nucleocapsid, it can prepare mouse anti-human SARS-CoV-2N monoclonal antibody with high affinity and good specificity.
The monoclonal antibody of the invention can detect SARS-CoV-2 in human throat swab/lung secretion and other samples by a double antibody sandwich method, can be applied to a SARS-CoV-2 detection kit, and is suitable for diagnosis, prevention and control of SARS-CoV-2 virus infection, scientific research of virus and the like.
The invention obtains the hybridoma cell strain N6A8 and hybridoma cell strain N7A7 which can generate the anti-human SARS-CoV-2N monoclonal antibody, and the anti-human SARS-CoV-2N monoclonal antibody N6A8 and N7A7 by screening, the antibody can detect the SARS-CoV-2N concentration in the human body fluid by a double antibody sandwich method, and has important application prospect. The invention establishes the ELISA method for detecting the human SARS-CoV-2N protein, realizes high sensitivity (pg level) and specificity of detection, can quickly and accurately determine the content of the human SARS-CoV-2N protein in samples such as throat swab/lung secretion and the like, and can provide reliable clinical reference value for early diagnosis and early treatment of SARS-CoV-2 infected patients.
The various terms and phrases used herein have the ordinary meaning as is well known to those skilled in the art.
Drawings
FIG. 1: the recombinant plasmid pET28a-SARS-CoV-N small sample induces expression of SDS-PAGE identification result, wherein, M: the pre-stained protein Marker, lanes 1-3 are respectively pET28a-SARS-CoV-N bacterial liquid post-ultrasonic precipitation after induction, pET28a-SARS-CoV-N bacterial liquid ultrasonic supernatant after induction, and pET28a-SARS-CoV-N bacterial liquid before induction.
FIG. 2: the result of SDS-PAGE identification after the purification and renaturation of human SARS-CoV-N recombinant protein, wherein, M: pre-staining a protein Marker; lane 1: after purification and renaturation, human SARS-CoV-N recombinant protein is obtained.
FIG. 3: the WB identification result of the monoclonal antibody (N7A7) against human SARS-CoV-2N protein.
FIG. 4: the WB identification result of the anti-human SARS-CoV-2N protein monoclonal antibody (N6A8), wherein 1, recombinant human SARS-CoV-2N protein (E.coli); 2. recombinant human SARS-CoV N protein (e.coli); 3. recombinant human MERS-CoV N protein (Baculovirus-instect Cells).
FIG. 5: anti-human SARS-CoV-2N protein monoclonal antibody and recombinant prokaryotic expression SARS-CoV-2-N, SARS-CoV-N and MERS-CoV-N protein antigen ELISA detection result.
Detailed Description
The present invention will be further described with reference to the following examples. However, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention.
The instruments, reagents, materials and the like used in the following examples are conventional instruments, reagents, materials and the like in the prior art and are commercially available in a normal manner unless otherwise specified. Unless otherwise specified, the experimental methods, detection methods, and the like described in the following examples are conventional experimental methods, detection methods, and the like in the prior art.
EXAMPLE 1 preparation of human SARS-CoV Nucleocapsid recombinant protein
(1) Construction of human SARS-CoV nucleoapsid prokaryotic expression vector
Human SARS-CoV nucleoapsid gene sequence SEQ ID NO.2 was synthesized artificially, inserted into NdeI and XhoI double-digested prokaryotic expression plasmid pET2a, and transformed into competent Escherichia coli BL21(DE 3). Screening positive clone on 0.1g/L kanamycin resistant plate, inducing with 1mM IPTG at 16 deg.C, 25 deg.C, 30 deg.C, 37 deg.C, preferably 25 deg.C, and shaking culturing at 200rpm for 14h to induce expression of human SARS-CoV nucleoapsid recombinant protein. The results of the final SDS-PAGE identification are shown in FIG. 1.
(2) Expression of pET28a-SARS-CoV-N fusion protein
The correctly identified pET28a-SARS-CoV-N strain was transferred to LB medium containing 100ug/ml kanamycin for overnight culture, transferred to fresh LB medium containing 100ug/ml kanamycin at a ratio of 1:100 the next day, and cultured with shaking at 37 ℃ and 200rpm until OD600 became 0.4-0.6, IPTG (final concentration of 1mmol/L) was added, and the temperature was adjusted to 25 ℃ to induce expression for 14 hours. Centrifuging at 4 deg.C and 10000r/min for 10min, and collecting precipitate.
(3) purification of pET28a-SARS-CoV-N fusion protein
A. The cell pellet was resuspended in lysis Buffer A (20mM Tris-HCl (pH8.0), 1mM EDTA, 0.1% Triton X-100), sonicated at 600W, centrifuged at 10000rpm for 30min, and the supernatant was collected.
B. And (3) purifying the thallus cracking supernatant through Ni column affinity chromatography: the invention adopts an AKTA Pure25 purifier to purify a protein sample.
Column assembling: 5ml of Ni Sepharose excel (GE) affinity packing were transferred to a XK 16/20Colum (GE) column, and the nickel column was equilibrated with 5 column volumes of binding buffer I (20mM Tris-HCl (pH 8.0)).
Loading: filtering a protein sample by a 0.45 mu m filter membrane to prepare a sample, wherein the flow rate is 2 ml/min; finally, 5-10 column volumes of buffer III were used for equilibration.
And (3) elution: elution was carried out with eluent I (20mM Tris-HCl (pH8.0), 20mM imidazole), eluent II (20mM Tris-HCl (pH8.0), 100mM imidazole), eluent III (20mM Tris-HCl (pH8.0), 250mM imidazole) and eluent IV (20mM Tris-HCl (pH8.0), 500mM imidazole), respectively, and different elution peaks were collected.
Balancing and storing: the nickel column was equilibrated with 5 column volumes of binding buffer I, purified water, 20% ethanol, respectively, and stored at 4 ℃.
(4) Protein dialysis renaturation: dialyzing the collected target peak into a proper buffer solution for storage, wherein the renaturation buffer solution is as follows: 10mm PBS (pH7.4), finally obtaining the soluble human SARS-CoV-2N recombinant protein.
(5) SDS-PAGE identification
SDS-PAGE was used to identify the samples after the purification and renaturation of the recombinant human SARS-CoV-N protein, the results are shown in FIG. 2.
EXAMPLE 2 preparation of monoclonal antibody against human SARS-CoV-2N
(1) Animal immunization
Using purified human SARS-CoV-N recombinant protein as immunogen, immunizing BALB/c female mouse of 8-12 weeks age by conventional method, emulsifying with Freund's complete adjuvant and antigen for the first time, and performing subcutaneous and abdominal multipoint immunization at dosage of 100 μ g/mouse; boosting the immunity once every two weeks for three times, wherein the adjuvant is Freund incomplete adjuvant, and the dosage is 100 mu g/time; detection of mouse tail blood titer by indirect ELISA method>1:104Three days before fusion, 100 mug of human SARS-CoV-2N antigen is boosted by intraperitoneal injection.
(2) Cell fusion
Mixing immune splenocytes with myeloma cells (SP2/0) at a ratio of 5:1, centrifuging at 1000rpm for 10min, and discarding supernatant; resuspending the cells in RPMI-1640 medium, centrifuging at 1000rpm for 10min, discarding the supernatant, sucking out the residual liquid with a dropper, tapping the bottom of the dropper to loosen and homogenize the precipitated cells, preheating in a 37 ℃ water bath, adding 1ml of preheated PEG1500(Roche) within 60s, then slowly dripping the RPMI-1640 medium to 20ml, centrifuging at 1000rpm for 10min, and discarding the supernatant. Cells were resuspended in HAT-containing medium and plated in 96-well cell culture plates and placed at 37 ℃ in 5% CO2Culturing in an incubator.
(3) Cell selection and monoclonalization
The indirect ELISA method is adopted to screen the positive culture hole specifically combined with the human SARS-CoV-N recombinant protein, and the specific process is as follows:
① preparation of antigen ELISA plate
Diluting human SARS-CoV-2N recombinant protein to 2 microgram/ml with 50mM pH9.6 carbonate buffer solution, adding to 96-well enzyme label plate, 100 microliter/well, coating overnight at 4 deg.C; after washing 3-5 times with PBST (10mM PBS (pH7.4), 0.05% Tween-80), spin-drying, blocking with blocking solution (10mM PBS (pH7.4), 5% BSA) at 37 ℃ for 2h for use.
② cell supernatant ELISA detection
Respectively taking 100 mu l of cell supernatant of 96-well cells to be screened, adding the cell supernatant into a prepared antigen ELISA plate of 96-well, incubating for 1h at 37 ℃, and washing the plate for 3-5 times by PBST. Further, 100. mu.l of goat anti-mouse-HRP diluted 1:5000 was added to each well, incubated at 37 ℃ for 45min, and the plate was washed 3-5 times with PBST. Finally adding substrate H2O2100. mu.l/well of TMB ((3,3 ', 5, 5' -tetramethylbenzidine)), developed in the dark for 10min, stopped by adding 50 ul/well of 2M sulfuric acid, and measured for absorbance of 450nM in a microplate reader.
③ cell monoclonality
And (3) selecting the wells with positive ELISA detection, and performing cell monoclonality by adopting a limiting dilution method to ensure that each culture well contains at most one hybridoma cell. When the cells grow to the culture holes 1/3-1/2, the cells are screened by an indirect ELISA method again, and finally, the single cell strain can be ensured to stably secrete the antibody of the anti-human SARS-CoV N recombinant protein.
(3) Hybridoma cell rescreening and monoclonalization
The method is the same as the operation method of the cell screening and monoclonality of the (3), the recombinant human SARS-CoV-2Nucleocapsid (bs-41408P, purchased from Beijing Boaosen biotechnology Co., Ltd.) and the recombinant human MERS-CoVNucleocsid (40068-V08B, purchased from Beijing Yiwangshan) are respectively coated on an enzyme label plate, the supernatant of the hybridoma is detected by adopting an indirect ELISA method, the hybridoma cell strain which has no cross with the MERS-CoV Nucleocapsid and has stronger reaction with the SARS-CoV-2Nucleocapsid is selected, and the hybridoma cell strain which has specific reaction with the human SARS-CoV-2Nucleocapsid and high affinity and secretes the anti-SARS-CoV-2 Nucleocapsid monoclonal antibody is finally obtained by multi-round screening.
(4) Cell cryopreservation
Cell cryopreservation solution: 90% fetal bovine serum + 10% DMSO, the hybridoma cells were centrifuged and resuspended in a pre-chilled cell cryopreservation solution at a cell density of 107cells/ml, 1 ml/count, subpackaging into freezing tubes, placing the freezing tubes into a programmed cooling box, placing the freezing tubes into a refrigerator at minus 80 ℃, and placing the freezing tubes into liquid nitrogen for long-term storage the next day.
(5) Preparation of monoclonal antibodies
In the invention, the monoclonal antibody of the anti-human SARS-CoV-2N recombinant protein is obtained by preparing a mouse ascites method, and the specific steps are as follows:
firstly, BALB/c male sensitization (intraperitoneal injection of liquid paraffin, 0.5 ml/mouse) of 8-12 weeks old is performed, hybridoma cells obtained in the mode of 2 weeks later are inoculated in an abdominal cavity (at least one time of serum-free culture and washing), and the inoculation amount is 106And (4) repeatedly extracting abdominal ascites and 4000rmp after the abdominal distension of the mice is obvious, centrifuging for 30min, collecting supernatant, and subpackaging at-20 ℃ for later use.
EXAMPLE 3 monoclonal antibody purification of recombinant protein against human SARS-CoV-2N
(1) Purification of monoclonal antibodies
The monoclonal antibody ascites obtained in example 2 was purified by Protein a affinity chromatography, comprising the following steps:
the method comprises the following basic steps: equilibration-loading-equilibration-elution-equilibration-preservation.
Balance liquid: 20mM PB (pH7.2) buffer;
eluent: 0.1M Glycine-HCl buffer pH 3.0;
neutralization buffer: 1M, pH9.0, Tris-HCl buffer;
preservation solution: 20% absolute ethyl alcohol.
Collecting 3-5ml ascites, precipitating with 50% saturated ammonium sulfate for more than 2 hr, centrifuging for 20min at 10000rmp, and removing supernatant; dissolving the precipitate with balance solution, filtering with 0.45 μm filter membrane, and preparing for sample loading; the Protein A affinity column is balanced by 5 times of column volume of balance liquid in advance, then the Protein precipitated by ammonium sulfate is loaded at the flow rate of 2ml/min, and finally the balance liquid with 5 times of column volume is used for balancing; and (3) replacing the eluent for elution, collecting the target protein, and then performing antibody: neutralizing the buffer at a volume ratio of 20:1 to obtain a purified monoclonal antibody against human SARS-CoV-2N recombinant protein.
Example 4: antibody pairing detection
(1) Antibody Horse Radish Peroxidase (HRP) labeling
HRP labeling was performed by sodium periodate oxidation. The labeled antibody is subjected to titer detection by an indirect ELISA method, and is stored at-20 ℃ for later use.
(2) Antibody pair screening
Antibody matching is carried out by adopting a chessboard titration method, and human SARS-CoV-2N recombinant proteins with the concentrations of 0.3ng/ml, 0.6ng/ml, 1.25ng/ml, 2.5ng/ml, 5ng/ml, 10ng/ml and 20ng/ml are respectively selected to screen the optimal matching antibody. Coating 24 enzyme-labeled antibodies with an empirical concentration of 200 ng/well, standing overnight at 4 ℃, washing for 3-5 times by using PBST, adding 300 mu l/well of a sealing solution, sealing for 2h at 37 ℃, and washing for 3-5 times by using PBST; adding a fixed value of human SARS-CoV-2N recombinant protein, incubating for 1h at 37 ℃, and washing for 3-5 times by PBST; respectively adding 13 HRP labeled antibodies diluted by 1:500 times, incubating for 45min at 37 ℃, and washing for 3-5 times by PBST; adding substrate TMB, developing in dark at 37 deg.C for 10min, adding stop solution 2M sulfuric acid 50 μ l, and measuring light absorption value of 450nM in microplate reader. The results are shown in Table 1, and the best match results were determined.
TABLE 1 optimal paired antibody detection results screened by checkerboard titration
Figure BDA0002392773740000081
Example 5 detection of specific reactivity of anti-N protein antibodies with viral antigens
To screen for antibodies specifically reactive with SARS virus. Therefore, the N protein of the recombinant expressed SARS-CoV-2 and MERS are selected to carry out SDS-PAGE electrophoresis, then the transformation and the sealing are carried out, Western Blot is respectively carried out with the N protein monoclonal antibody of the SARS-CoV-2, and finally the specificity of the N protein monoclonal antibody of the SARS-CoV-2 is determined; at the same time, the recombinant expressed SARS-CoV N protein and the recombinant expressed SARS-CoV-2N protein are respectively coated, and the two monoclonal antibodies of N6A8 and N7A7 described in example 4 are used to react with them, and the result shows that the recombinant expressed SARS-CoVN protein and the recombinant expressed SARS-CoV-2N protein have the same structure and activity, and do not cross with MERS-CoV N protein, and the details are shown in FIGS. 3-5.
The above examples are provided to those of ordinary skill in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications apparent to those skilled in the art are intended to be within the scope of the appended claims.
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Claims (10)

1. A human SARS-CoV-2 monoclonal antibody, which is characterized in that: using SARS-CoV nucleoapsid recombinant protein as immunogen to immunize mouse to obtain monoclonal antibody; the amino acid sequence of the SARS-CoV Nucleocapsid recombinant protein is shown in SEQ ID NO. 1.
2. The human SARS-CoV-2 monoclonal antibody of claim 1, wherein: the SARS-CoVNucleucoapsid recombinant protein is prepared by the following method: human SARS-CoV nucleoapsid gene whose nucleotide sequence is shown in SEQ ID No.2 is inserted into expression vector pET28a to construct pET28a-SARS-CoV-N fusion expression plasmid, and then the Escherichia coli is transformed, and then the induced, purified and renatured by IPTG to obtain SARS-CoV nucleoapsid recombinant protein.
3. The method for preparing human SARS-CoV-2 monoclonal antibody according to claim 1 or 2, wherein: using SARS-CoV Nucleocapsid recombinant protein as immunogen to immunize BALB/c mouse, then making fusion and subcloning of mouse spleen cell and mouse myeloma cell, and making specific screening and domestication by using SARS-CoV-2Nucleocapsid and MERS Nucleocapsid to finally obtain monoclonal cell strain with good specificity and high potency, then making ascites preparation and purification so as to obtain a large quantity of monoclonal antibody resisting human SARS-CoV Nucleocapsid.
4. The method for preparing human SARS-CoV-2 monoclonal antibody according to claim 3, wherein: selecting female mice 8-12 weeks old from the BALB/c mice;
or/and: the screening method is an ELISA method;
or/and: the purification method is Protein A affinity purification.
5. Use of the human SARS-CoV-2 monoclonal antibody of claim 1 or 2 in the preparation of SARS-CoV-2 virus detection products.
6. Use of the human SARS-CoV-2 monoclonal antibody of claim 1 or 2 for the preparation of a medicament for inhibiting SARS-CoV-2 virus.
7. Use of the human SARS-CoV-2 monoclonal antibody of claim 1 or 2 for the preparation of a pharmaceutical preparation for the prevention or treatment of a disease caused by SARS-CoV-2 virus infection.
8. A method for detecting the level of SARS-CoV-2 virus for non-diagnostic purposes, comprising: contacting the sample to be detected with the human SARS-CoV-2 monoclonal antibody as described in claim 1 or 2, and detecting the immunoreaction of the sample with the human SARS-CoV-2 monoclonal antibody.
9. A kit for detecting SARS-CoV-2 virus comprising the human SARS-CoV-2V monoclonal antibody of claim 1 or 2.
10. A medicament for inhibiting SARS-CoV-2 virus comprising the human SARS-CoV-2 monoclonal antibody of claim 1 or 2.
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