CN111909262B - New coronavirus RBD specific monoclonal antibody and application - Google Patents
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Abstract
The invention belongs to the technical field of monoclonal antibodies, and particularly discloses a new coronavirus RBD specific monoclonal antibody and application of the new coronavirus RBD specific monoclonal antibody. The invention has important scientific significance and application prospect for the prevention and clinical treatment of diseases caused by the novel coronavirus SARS-CoV-2 and the research and development of diagnostic reagents.
Description
Technical Field
The invention belongs to the technical field of monoclonal antibodies, and particularly relates to a novel coronavirus RBD specific monoclonal antibody and application thereof.
Background
Antibodies are immunoglobulin molecules composed of four polypeptide chains, including two heavy chains (H chains) and two light chains (L chains). The H chain consists of a heavy chain variable region (VH) consisting of three regions, CH1, CH2 and CH3, and a heavy chain constant region. The L chain consists of an L variable region (VL) and a light chain constant region consisting of a CL region. VH and VL can be further divided into hypervariable regions called Complementarity Determining Regions (CDRs) and conserved regions called Framework Regions (FR) that alternate.
The research shows that: the novel coronavirus (SARS-CoV-2) has four major structural proteins, spike protein (S protein), nucleocapsid protein (N protein), membrane protein (M protein), and envelope protein (E protein), respectively, wherein the S protein has two subunits: s1 and S2, receptor Binding Sites (RBDs) are located on the S1 subunit, and their primary function is to recognize host cell surface receptors, mediating fusion with host cells.
At present, specific drug-specific treatment is not available for new pathogen COVID-19, and the development of vaccines requires a current day. The plasma of a patient who is cured and discharged recently contains high-concentration specific antigen neutralizing antibodies, and after the plasma is input into the body of the patient, the plasma can neutralize new corona pathogens and mediate effective immune response, so that the plasma in the recovery period is expected to provide an effective treatment means for treating the patient infected with new corona viruses, the death rate is reduced, and the life safety of the patient is guaranteed.
Chinese invention patent application with application publication number CN111303280A discloses a highly neutralizing anti-SARS-CoV-2 fully human monoclonal antibody, which provides a fully human antibody with a recognition region of S1 non-RBD region, but the obtained fully human monoclonal antibody has limited virus blocking effect because the new coronavirus invades into the host cell and is combined with ACE2 of the host cell through the RBD, and the obtained antibody cDNA is obtained by marking plasma cells, but compared with the plasma cells, the antibody cDNA reacts rapidly after memory B cells are activated, so the memory B cells can initiate a faster and stronger humoral immune response than the primary reaction, and the humoral immune response initiated by the plasma cells is limited.
Disclosure of Invention
In order to achieve the aim, the invention provides a new coronavirus RBD specific monoclonal antibody, and particularly, the heavy chain amino acid sequence of the antibody is shown as SEQ ID NO. 1; the light chain amino acid sequence is shown as SEQ ID NO:2 (monoclonal antibody 1-CQTS 066). The heavy chain amino acid sequence can also be shown as SEQ ID NO. 3; the light chain amino acid sequence can also be shown as SEQ ID NO:4 (monoclonal antibody 2-CQTS 067). The heavy chain amino acid sequence can also be shown as SEQ ID NO. 5; the light chain amino acid sequence can also be shown as SEQ ID NO:6 (monoclonal antibody 3-CQTS 068). The heavy chain amino acid sequence can also be shown as SEQ ID NO. 7; the light chain amino acid sequence can also be shown as SEQ ID NO:8 (monoclonal antibody 4-CQTS 069). The heavy chain amino acid sequence can also be shown as SEQ ID NO. 9; the light chain amino acid sequence can also be shown as SEQ ID NO:10 (monoclonal antibody 5-CQTS 070). The heavy chain amino acid sequence can also be shown as SEQ ID NO. 11; the light chain amino acid sequence can also be shown as SEQ ID NO:12 (monoclonal antibody 6-CQTS 071). The heavy chain amino acid sequence can also be shown as SEQ ID NO 13; the light chain amino acid sequence can also be shown as SEQ ID NO:14 (monoclonal antibody 7-CQTS 072). The heavy chain amino acid sequence can also be shown as SEQ ID NO. 15; the light chain amino acid sequence can also be shown as SEQ ID NO:16 (monoclonal antibody 8-CQTS 073). The heavy chain amino acid sequence can also be shown as SEQ ID NO. 17; the light chain amino acid sequence may also be shown as SEQ ID NO:18 (mAb 9-CQTS 074). The heavy chain amino acid sequence can also be shown as SEQ ID NO. 19; the light chain amino acid sequence can also be shown as SEQ ID NO:20 (monoclonal antibody 10-CQTS 075). The heavy chain amino acid sequence can also be shown as SEQ ID NO 21; the light chain amino acid sequence can also be shown as SEQ ID NO:22 (monoclonal antibody 11-CQTS 076). The heavy chain amino acid sequence can also be shown as SEQ ID NO. 23; the light chain amino acid sequence can also be shown as SEQ ID NO:24 (monoclonal antibody 12-CQTS 077). The heavy chain amino acid sequence can also be shown as SEQ ID NO. 25; the light chain amino acid sequence may also be shown as SEQ ID NO:26 (mAb 13-CQTS 078). The heavy chain amino acid sequence can also be shown as SEQ ID NO. 27; the light chain amino acid sequence can also be shown as SEQ ID NO:28 (monoclonal antibody 14-CQTS 079). The heavy chain amino acid sequence can also be shown as SEQ ID NO. 29; the light chain amino acid sequence can also be shown as SEQ ID NO:30 (monoclonal antibody 15-CQTS 080). The heavy chain amino acid sequence can also be shown as SEQ ID NO. 31; the light chain amino acid sequence can also be shown as SEQ ID NO:32 (monoclonal antibody 16-CQTS 081). The heavy chain amino acid sequence can also be shown as SEQ ID NO. 33; the light chain amino acid sequence may also be shown as SEQ ID NO:34 (monoclonal antibody 17-CQTS 082). The heavy chain amino acid sequence can also be shown as SEQ ID NO. 35; the light chain amino acid sequence can also be shown as SEQ ID NO:36 (monoclonal antibody 18-CQTS 083). The heavy chain amino acid sequence can also be shown as SEQ ID NO 37; the light chain amino acid sequence can also be shown as SEQ ID NO:38 (mAb 19-CQTS 086). The heavy chain amino acid sequence can also be shown as SEQ ID NO: 39; the light chain amino acid sequence may also be shown as SEQ ID NO:40 (mAb 20-CQTS 090).
The invention also provides the application of the monoclonal antibody with the RBD specificity of the new coronavirus in the preparation of reagents or vaccines or medicaments for detecting or diagnosing SARS-CoV-2, wherein the medicaments comprise the monoclonal antibody with the RBD specificity of the new coronavirus as described in any one of claims 1-20 and pharmaceutically acceptable excipients, diluents or carriers; also provides a nucleic acid molecule for encoding the new coronavirus RBD specific monoclonal antibody; also provides an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule; also provides the application of the expression cassette, the recombinant vector, the recombinant bacterium or the transgenic cell line in the preparation of products.
The invention also provides a product, which comprises the new coronavirus RBD specific monoclonal antibody; the product application is any one of the following (b 1) to (b 4): (b 1) binding to novel coronavirus SARS-CoV-2; (b 2) detecting binding of the novel coronavirus SARS-CoV-2; (b 3) binding to the S protein of the novel coronavirus SARS-CoV-2; (b 4) detecting the S protein of the novel coronavirus SARS-CoV-2.
Preferably, the RBD-specific monoclonal antibody of the novel coronavirus is obtained by sorting RBD-specific memory B cells and obtaining antibody variable region cDNA from mRNA of the RBD-specific memory B cells.
The principle and the beneficial effects of the invention are as follows:
(1) The monoclonal antibody provided by the invention has RBD specificity, and compared with a monoclonal antibody aiming at an S1 non-RBD region, the monoclonal antibody provided by the invention is combined with RBD, thereby providing wider application values for screening antibody drugs, diagnosing, preventing and treating new coronary pneumonia.
(2) The monoclonal antibody provided by the invention is obtained by sorting RBD specific memory B cells, and compared with the prior art of sorting plasma cells, the monoclonal antibody prepared by the invention can trigger stronger humoral immune response. In addition, the invention only aims at RBD specific memory B cells to carry out subsequent RT-PCR, nested PCR and antibody function analysis, thereby greatly improving the specific binding capacity of the monoclonal antibody and the RBD.
Drawings
FIG. 1 is a diagram of cell sorting by flow cytometry analysis of memory B cells;
FIG. 2 is a diagram of cell sorting by flow cytometry analysis of RBD-specific memory B cells;
FIG. 3 is a gel electrophoresis of the PCR product of the antibody gene of a single cell;
FIG. 4 is a photograph of agarose gel electrophoresis following PCR amplification of an antibody gene expression cassette containing the CMV promoter, WPRE-gamma or WPRE-kappa element;
FIG. 5 is a graph showing the results of an experiment on the specificity of RBD.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of 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.
Example 1
This example provides a monoclonal antibody specific to a novel coronavirus RBD, the heavy chain amino acid sequence of which is shown in SEQ ID NO 1; the light chain amino acid sequence is shown as SEQ ID NO. 2.
The embodiment also provides the application of the new coronavirus RBD specific monoclonal antibody in the preparation of a reagent or a medicament for detecting or diagnosing SARS-CoV-2.
In practical production, the new coronavirus RBD-specific monoclonal antibody obtained in this embodiment may be used to prepare a nucleic acid molecule, or prepare an expression cassette, a recombinant vector, a recombinant bacterium, or a transgenic cell line containing the nucleic acid molecule, or prepare a pharmaceutical composition comprising the new coronavirus RBD-specific monoclonal antibody and a pharmaceutically acceptable excipient, diluent, or carrier.
In application, the related product prepared by the monoclonal antibody specific to the new coronavirus RBD obtained in the embodiment can have the application of any one of the following (b 1) to (b 4): (b 1) binds to novel coronavirus SARS-CoV-2; (b 2) detecting binding of the novel coronavirus SARS-CoV-2; (b 3) binding to the S protein of the novel coronavirus SARS-CoV-2; (b 4) detecting the S protein of the novel coronavirus SARS-CoV-2.
Examples 2 to 20
Examples 2-20 differ from example 1 in that: the amino acid sequences of the monoclonal antibodies specific to the RBD of the novel coronavirus are different, and the amino acid sequences of examples 2 to 20 are shown in the following table:
experimental group | Heavy chain amino acid sequence | Light chain amino acid sequence |
Example 1 CQTS066 | SEQ ID NO:1 | SEQ ID NO:2 |
Example 2 CQTS067 | SEQ ID NO:3 | SEQ ID NO:4 |
Example 3 CQTS068 | SEQ ID NO:5 | SEQ ID NO:6 |
Example 4 CQTS069 | SEQ ID NO:7 | SEQ ID NO:8 |
Example 5 CQTS070 | SEQ ID NO:9 | SEQ ID NO:10 |
Example 6 CQTS071 | SEQ ID NO:11 | SEQ ID NO:12 |
Example 7 CQTS072 | SEQ ID NO:13 | SEQ ID NO:14 |
Example 8 CQTS073 | SEQ ID NO:15 | SEQ ID NO:16 |
Example 9 CQTS074 | SEQ ID NO:17 | SEQ ID NO:18 |
Example 10 CQTS075 | SEQ ID NO:19 | SEQ ID NO:20 |
Example 11 CQTS076 | SEQ ID NO:21 | SEQ ID NO:22 |
Example 12 CQTS077 | SEQ ID NO:23 | SEQ ID NO:24 |
Example 13 CQTS078 | SEQ ID NO:25 | SEQ ID NO:26 |
Example 14 CQTS079 | SEQ ID NO:27 | SEQ ID NO:28 |
Example 15 CQTS080 | SEQ ID NO:29 | SEQ ID NO:30 |
Example 16 CQTS081 | SEQ ID NO:31 | SEQ ID NO:32 |
Example 17 CQTS082 | SEQ ID NO:33 | SEQ ID NO:34 |
Example 18 CQTS083 | SEQ ID NO:35 | SEQ ID NO:36 |
Example 19 CQTS086 | SEQ ID NO:37 | SEQ ID NO:38 |
Example 20 CQTS090 | SEQ ID NO:39 | SEQ ID NO:40 |
The novel coronavirus RBD specific monoclonal antibodies provided in examples 1-20 above were obtained by the following methods: firstly, single RBD specific memory B cells are obtained by separating peripheral blood of a new coronary pneumonia rehabilitation patient, then mRNA of the RBD specific memory B cells is obtained, then an antibody variable region gene expression cassette is constructed through RT-PCR and nested PCR, the antibody variable region gene expression cassette is transduced into 293T cells to express an antibody, supernatant is collected, RBD specificity of the supernatant is detected through an ELISA method, and a new coronary virus RBD specific monoclonal antibody is obtained through screening.
The method specifically comprises the following steps:
s1, collecting peripheral blood of a plurality of patients with the new coronary pneumonia, separating to obtain PBMC, and freezing and storing in a refrigerator at the temperature of-80 ℃ for later use.
S2, firstly removing Dead cells of PBMC obtained in the S1 by adopting Dead cell removing Dye (Dead Dye), then adopting CD19, mIg-G, mIg-D and S-RBD to stain and mark the memory B cells with high RBD specificity and binding capacity in the PBMC, and screening the memory B cells specific to the RBD; specific memory B cells were sorted using a flow cytometric sorter onto 96-well plates, one specific memory B cell per well, and frozen at-80 ℃ in a freezer for use.
Specifically, the preferred concentration range of the Dead Dye staining in this embodiment is 1-2 μ g/mL, and the preferred concentration range of the Dead Dye staining in this embodiment is 1.5 μ g/mL; CD19 is a B cell marker produced by Biolegend and is stained at a concentration ranging from 1 to 2. Mu.g/mL, and in this example, CD19 is preferably stained at a concentration of 1.5. Mu.g/mL. mIg-G is a B cell surface receptor produced by Biolegend, and the concentration range of the mIg-G during staining is 1-2 mu G/mL, and the concentration of the mIg-G during staining is 1.5 mu G/mL in the embodiment; mIg-D is B cell surface receptor produced by Biolegend, and the concentration range when staining is 1-2 μ g/mL, and the concentration when staining mIg-D is 1.5 μ g/mL is preferred in the embodiment; S-RBD is a novel coronavirus produced by sinobiological, is a protein receptor domain, and is stained at a concentration ranging from 1 to 2. Mu.g/mL, and the concentration of S-RBD staining is preferably 1.5. Mu.g/mL in this example.
Cell sorting of PBMC by CD19, mIg-G, mIg-D and S-RBD for RBD specific memory B cells sorting by flow cytometry the cell sorting profiles for B cells with specific memory for S-RBD are shown in FIGS. 1 and 2, where Batch ID 0428, 0505, 0522, 0528 in FIG. 2 are screening batches. The principle of screening RBD-specific memory B cells by CD19, mIg-G, mIg-D and S-RBD in this example is as follows: removing PBMC from dead tissueStaining with cell Dye (Dead Dye), CD19 as a B cell marker, mIg-G positive and mIg-D negative as a memory B cell marker, and RBD-specific IgG expressing memory B cells, then dividing the cell population into CD19 cell populations using a flow cytometer, and then dividing the mIg-G populations from the CD19 positive cell populations + mIg-D - Cell population from mIg-G + mIg-D - Dividing the cell group into RBD positive memory B cells, and sorting the RBD positive memory B cells by a flow cytometry sorter.
S3, sorting to obtain mRNA of a single RBD specific memory B cell, and performing RT-PCR amplification to obtain antibody variable region cDNA. Specifically, when RT-PCR is used to amplify cDNA in the variable region of the antibody, the primer front segment of the primer designed in this example is designed with a universal Leader (see primer sequence listing one and primer sequence listing two), which effectively improves the amplification rate of the antibody gene, and the experimental result is shown in fig. 3.
S4, amplifying the cDNA of the antibody variable region obtained from the S1-S3 by adopting nested PCR to construct an antibody variable region gene expression cassette.
S3 and S4 are performed by the following six sections in total: (1) extracting mRNA of RBD specific memory B cells; (2) single cell mRNA Reverse Transcription (RT); (3) adding G tail (TDT); (4) first round PCR (1 st PCR); (5) second round PCR (2 nd PCR); (6) PCR amplification of BCR-ORF to construct a gene expression cassette; (7) CMV, WPRE-gamma/kappa/l fragment amplification and CMV, BCR-Vgamma/kappa/l ((6) product), WPRE-gamma/kappa/l Overlap PCR (Overlap PCR) pre-connection; (8) BCR-gamma ORF, BCR-kappa ORF and BCR-lPCR amplification.
The preparation and reaction conditions of each part of reaction liquid are as follows:
(1) Using Dynabeads TM mRNA DIRECT TM The single cell mRNA extraction is carried out by a Purification Kit (Thermo Fisherscientific), and the method specifically comprises the following steps:
(1) centrifuging: taking out the 96-well plate sorted with single RBD specific memory B cells from a refrigerator at-80 ℃, and centrifuging the plate at 600 Xg for 30s to enable the cells to be centrifuged at the bottom of the well;
(2) cleaning: taking out a Dynabeads oligo (dT) 25 microsphere bottle, uniformly mixing by vortex, sucking enough microspheres according to 2 mu l/hole, placing on a magnet block, standing for 30s, discarding supernatant, and resuspending by using 500 mu l lysine Buffer;
(3) preparation: adding the mixture into a 50mL centrifuge tube according to 9. Mu.l/hole lysine Buffer, adding the 500. Mu.l microsphere suspension into the centrifuge tube, and uniformly blowing by using a gun;
(4) subpackaging: subpackaging the microspheres by using an eight-connecting tube, and then adding the microspheres into a cell plate according to 9 mu l/hole by using a row gun;
(5) rinsing: pasting a film on a 96-hole plate, then rinsing the periphery of the tube wall for 2 cycles;
(6) and (3) incubation: standing at room temperature for 5min to fully release and combine mRNA of the RBD specific memory B cells to the microspheres, and after the incubation is finished, performing 600 Xg instantaneous centrifugation to enable the microspheres to be centrifuged at the bottom of the hole. Place 96-well plates in DynaMag TM -96side Magnet magnetic plate, pipette off supernatant;
(7) wash A: adding Washing Buffer A according to 8 mul/hole, walking the plate back and forth for 7-8 times to fully wash the microspheres, and discarding the supernatant;
(8) wash B: wash Buffer B was added at 8. Mu.l/well, the plate was walked back and forth 7-8 times to wash the microspheres thoroughly, the supernatant was discarded, and then the pre-prepared Reverse Transcription (RT) reaction was added at 10. Mu.l/well. The reagent preparation and reaction conditions are described in the following (2).
(2) Reverse Transcription (RT) (10. Mu.l system)
The reagents required for formulation are shown in table 1 below:
name of reagent | Volume of |
DEPC-H 2 O | 4.5μl |
5×primerscript Buffer | 2.0μl |
2.5mM dNTP | 2.0μl |
RNase Inhibitor | 1μl |
Sample | beads |
PrimerScriptⅡRTase | 0.5μl |
Total volume | 10μl |
The reaction conditions are as follows: 42 ℃ for 60min (mixing every 20 min);
after the reaction was completed, the 96-well plate was instantaneously centrifuged at 600 Xg, and then the 96-well plate was placed in DynaMag TM On a 96-side Magnet magnetic plate, the supernatant was aspirated off by a discharge gun, and then a previously prepared TDT reaction solution was added at 10. Mu.l/well, and the reagent preparation and reaction conditions were as described in (3) below.
(3) Add G tail (TDT) (10. Mu.l system)
The reagents required for formulation are shown in table 2 below:
name of reagent | Volume of |
H 2 O | 6.4μl |
5×TdT buffer | 2.0μl |
10mM dGTP | 0.5μl |
0.1%BSA | 1.0μl |
Sample | beads |
TdT | 0.1μl |
Total volume | 10μl |
The reaction conditions are as follows: 37 ℃ for 40min (mix every 20 min).
After the reaction, the reaction mixture was centrifuged at 600 Xg in a 96-well plate and then placed in DynaMag TM On a 96-side Magnet magnetic plate, the supernatant was aspirated off by a pipette, and then a first PCR (1 st PCR) reaction solution prepared in advance was added at 10. Mu.l/well, and the reagent preparation and reaction conditions were as described in (4) below.
(4) 1st PCR (10. Mu.l System) (see primer sequence Listing)
The reagents required for formulation are shown in table 3 below:
based on the PCR principle, the experimental reaction conditions of 1st PCR are as follows: (1) pre-denaturation at 95 ℃ for 3min; (2) denaturation at 95 ℃ for 15sec, annealing at 60 ℃ for 5sec, elongation at 72 ℃ for 1min,30-35cycles, preferably 30cycles in this example; (3) circulating at 72 deg.C for external extension for 5min, and storing at 4 deg.C.
(5) Second round PCR (2 nd PCR) (10. Mu.l system) (see primer sequence Listing I and primer sequence Listing II)
The reagents required for formulation are shown in table 4 below:
name of reagent | Volume of |
H 2 O | 1.5μl |
2×GC Buffer | 5μl |
2.5mM dNTP | 1μl |
FP:MAC-AP3/AP3(10μM) | 0.5μl |
RP:Cg-nest/K20/CI-nest(10μM) | 0.5μl |
PrimesTAR | 0.5μl |
sample | 1μl |
Total volume | 10μl |
Based on the PCR principle, the experimental reaction conditions of 2nd PCR are as follows: (1) pre-denaturation at 95 ℃ for 3min; (2) denaturation at 95 ℃ for 15sec, annealing at 60 ℃ for 5s, elongation at 72 ℃ for 1min,30-35cycles, preferably 35cycles in this example; extension is carried out for 5min at 72 ℃ in a circulating way, and the product is stored at 4 ℃.
After the PCR is finished: mu.l of each well was subjected to 1.5% agarose gel electrophoresis. The wells of the paired Gamma and Kappa or Lamada chains were sequenced.
(6) Amplification and construction of antibody expression cassettes (BCR-ORF)
PCR amplification promoter region (CMV promoter), WPRE-gamma (antibody gamma chain) and WPRE-kappa (antibody kappa chain) with the following PCR amplification system shown in Table 5 below:
the PCR amplification conditions were: (1) pre-denaturation at 95 ℃ for 3min; (2) denaturation at 95 ℃ for 15sec, annealing at 56 ℃ for 15sec, elongation at 72 ℃ for 1min,30cycles; (3) extension is carried out for 5min at 72 ℃ in a circulating way, and the product is stored at 12 ℃.
(7) Amplification of CMV, WPRE-gamma/kappa/l fragment and pre-ligation of CMV, BCR-Vgamma/kappa/l, WPRE-gamma/kappa/l Overlap PCR (Overlap PCR)
The experimental system is shown in table 6 below:
the PCR amplification conditions were: pre-denaturation at 95 ℃ for 3min; denaturation at 95 ℃ for 15sec, annealing at 50 ℃ for 15sec, extension at 72 ℃ for 1.5min,10cycles; extension is carried out for 5min at 72 ℃ in a circulating way, and the product is stored at 12 ℃.
(8) BCR-gamma ORF, BCR-kappa ORF, BCR-l PCR amplification
The experimental system is shown in table 7 below:
PCR amplification procedure: pre-denaturation at 95 ℃ for 3min; denaturation at 95 ℃ for 15sec, annealing at 58 ℃ for 15sec, extension at 72 ℃ for 1.5min,30cycles; extension is carried out for 5min at 72 ℃ in a circulating way, and the product is stored at 12 ℃.
After amplification, agarose gel electrophoresis is adopted, whether the size of the obtained antibody variable region gene is correct or not is analyzed by gel imaging, the experimental result is shown in figure 4, the Marker is in the middle position, and the band is in the position of 5000 bp.
BCR-gamma ORF and BCR-kappa/ORF ethanol precipitation: placing 30 μ l of PCR products of BCR-gamma ORF and BCR-kappa ORF in 8 connecting tubes respectively, adding 120 μ l of anhydrous ethanol and 6 μ l of sodium acetate solution, mixing well, and standing at-80 deg.C for 30min;10000rpm, centrifuging for 20min, discarding the supernatant, sequentially rinsing with 200 μ l of 70% ethanol and anhydrous ethanol once respectively, fully volatilizing the ethanol at 56 deg.C, adding 40 μ l of sterile water, oscillating to fully dissolve the precipitate, and detecting the concentration of antibody variable region gene.
The Leader primers used in S3 and S4 are described in the following primer sequence I:
the J-region primers used for S3 and S4 are described in the second primer sequence:
primer ID | sequence |
IGHJ_01 | GATGGGCCCTTGGTGGAGGGTGAGGAGACGGTGACCAGGGTGCCCTGGCCCCAGT |
IGHJ_02 | GATGGGCCCTTGGTGGAGGGTGAGGAGACAGTGACCAGGGTGCCACGGCCCCAGA |
IGHJ_03 | GATGGGCCCTTGGTGGAGGGTGAAGAGACGGTGACCATTGTCCCTTGGCCCCAGA |
IGHJ_04 | GATGGGCCCTTGGTGGAGGGTGAGGAGACGGTGACCGTGGTCCCTTGCCCCCAGA |
IGKJ_01 | GATGGTGCAGCCACAGTTCGTTTGATTTCCACCTTGGTCCCTTGGCCGAACGTCC |
IGKJ_02 | GATGGTGCAGCCACAGTTCGTTTGATTTCCACCTTGGTCCCTTGGCCGAACGTCC |
IGKJ_03 | GATGGTGCAGCCACAGTTCGTTTGATATCCACTTTGGTCCCAGGGCCGAAAGTGA |
IGKJ_04 | GATGGTGCAGCCACAGTTCGTTTGATCTCCACCTTGGTCCCTCCGCCGAAAGTGA |
IGKJ_05 | GATGGTGCAGCCACAGTTCGTTTAATCTCCAGTCGTGTCCCTTGGCCGAAGGTGA |
IGLJ_01 | GGGGCAGCCTTGGGCTGACCTAGGACGGTGACCTTGGTCCCAGTTCCGAAGACAT |
IGLJ_02 | GGGGCAGCCTTGGGCTGACCTAGGACGGTCAGCTTGGTCCCTCCGCCGAATACCA |
IGLJ_03 | GGGGCAGCCTTGGGCTGACCTAAAATGATCAGCTGGGTTCCTCCACCAAATACAA |
IGLJ_04 | GGGGCAGCCTTGGGCTGACCTAGGACGGTCAGCTCGGTCCCCTCACCAAACACCC |
IGLJ_05 | GGGGCAGCCTTGGGCTGACCTAGGACGGTCAGCTCCGTCCCCTCACCAAACACCC |
IGLJ_06 | GGGGCAGCCTTGGGCTGACCGAGGACGGTCACCTTGGTGCCACTGCCGAACACAT |
IGLJ_07 | GGGGCAGCCTTGGGCTGACCGAGGACGGTCAGCTGGGTGCCTCCTCCGAACACAG |
IGLJ_08 | GGGGCAGCCTTGGGCTGACCGAGGGCGGTCAGCTGGGTGCCTCCTCCGAACACAG |
and S5, transducing the antibody variable region gene expression cassette obtained in the S4 into 293T cells for 48 hours to express the antibody, collecting the supernatant, detecting the RBD specificity of the supernatant by using an ELISA method, and screening the RBD specific fully human monoclonal antibody.
(A) Antigen was diluted with PBS (final concentration 2. Mu.g/mL), 10. Mu.l/well, and coated onto 384-well ELISA plates overnight at 4 ℃ or 2h at 37 ℃ (4 ℃ overnight is preferred in this example). NOTE: after the addition, the liquid is instantly centrifuged to ensure that the liquid is at the bottom.
The experimental system is shown in table 8 below:
name of reagent | Goods number | Original concentration | Final concentration | Dilution ratio |
SARS-COV-2RBD | Cat:40592-V08H | 200μg/mL | 2μg/mL | 1:100 |
Goat pab to Hu IgG-ALP | Cat:ab97221 | 1mg/mL | 2μg/mL | 1:500 |
(B) PBST (0.05% Tween 20, cat #, TB220) was prepared: 1L of PBS was added with 0.5mL of Tween 20;
PBST machine washed plates (Thermoscientific wellwash versa) or hand washed (plates that were machine washed were still manually clapped/centrifuged using a microplate centrifuge (MPC-P25) for 1min to make the plates invisible to water and air bubbles).
And (3) sealing: 80 μ l of BSA (BioFroxx, cat. NO:4240GR 100) at 5% (prepared in PBST) was added to the washed plate and incubated at 37 ℃ for 1 hour in an incubator. PBST machine washing board or hand washing.
(C) Sample adding and standard substance. Wherein, the standard substance: 10 μ l/well stock concentration 1 μ g/mL, gradient dilutions 250ng/mL, 125ng/mL, 62.5ng/mL, 31.25ng/mL, 15.63ng/mL, 7.81ng/mL, 3.9ng/mL, and 1.95ng/mL. (blocking solution dilution); sample preparation: cell supernatants transfected with antibody genes. Negative control/blank wells: blocking solution 10. Mu.l/well.
Incubate at 37 ℃ for 30min. PBST machine washing board or hand washing.
(D) Secondary antibody was added at a concentration of 10. Mu.l/well, followed by incubation at 37 ℃ for 30min.
The experimental system is shown in table 9 below:
name of secondary antibody | Goods number | Original concentration | Final concentration | Dilution ratio |
goat-anti-human IgG-ALP | A18808 | 1.5mg/ml | 0.3μg/ml | 1:5000 |
Goat pab to Hu IgG-ALP | Ab98532 | 0.5mg/ml | 0.25μg/ml | 1:2000 |
PBST machine washing board or hand washing. Mu.l/well of PNPP (disodium p-nitrophenylphosphate) and OD (450 mm) values of 5min,10 min, 15min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min and 60min were measured using (Thermoscientific Muttiskan GO). 50mg PNPP powder (Thermo, prod # 34045) +40mL ddH 2 O +10mL of Diethylhanol amine substrate Buffer (5X), and PNPP was stored at 4 ℃ protected from light.
As shown in FIG. 5, the OD value greater than 0.1 was positive.
The foregoing is merely a preferred embodiment of this invention, which is intended to be illustrative, not limiting; those skilled in the art will appreciate that many variations, modifications, and even equivalent variations are possible within the spirit and scope of the invention as defined in the appended claims.
Claims (6)
1. The monoclonal antibody with the specificity of the new coronavirus RBD is characterized in that the amino acid sequence of a heavy chain is shown as SEQ ID NO 1; the light chain amino acid sequence is shown as SEQ ID NO. 2.
2. The monoclonal antibody specific to RBD of the novel coronavirus of claim 1, wherein the antibody variable region cDNA is obtained by sorting RBD-specific memory B cells and then passing the mRNA of the RBD-specific memory B cells.
3. Use of the monoclonal antibody specific to the novel coronavirus RBD of claim 1 in the preparation of a reagent or a medicament for detecting or diagnosing SARS-CoV-2, wherein the medicament comprises the monoclonal antibody specific to the novel coronavirus RBD of claim 1, and a pharmaceutically acceptable excipient, diluent or carrier.
4. A nucleic acid molecule encoding the novel coronavirus RBD-specific monoclonal antibody of claim 1.
5. An expression cassette, recombinant vector, recombinant bacterium or transgenic cell line comprising the nucleic acid molecule of claim 4.
6. The use of the expression cassette, recombinant vector, recombinant strain or transgenic cell line of claim 5 in the preparation of a product for any of the following uses (b 1) to (b 4): (b 1) binds to novel coronavirus SARS-CoV-2; (b 2) detecting a novel coronavirus SARS-CoV-2; (b 3) binding to the S protein of the novel coronavirus SARS-CoV-2; (b 4) detecting the S protein of the novel coronavirus SARS-CoV-2.
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