CN115141272A - Novel coronavirus monoclonal antibody XY1 and application thereof - Google Patents
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Abstract
The invention discloses a novel coronavirus monoclonal antibody XY1 and application thereof. The full-humanized antibody XY1 resisting SARS-Cov-2S1 or RBD protein is screened, and the antibody of the invention has important scientific significance and clinical application prospect for the prevention, treatment and diagnosis of SARS-Cov-2.
Description
The application has the application number of 2021101325814, the application date of 1 month 31 of 2021, the name of the invention is: a novel coronavirus monoclonal antibody and application thereof.
The technical field is as follows:
the invention belongs to the technical field of monoclonal antibody screening and preparation, and particularly relates to a novel coronavirus monoclonal antibody XY1 and application thereof.
Background art:
SARS-Cov-2 is a linear single-strand positive-strand RNA virus whose structural proteins include spike protein (S), small envelope protein (E), envelope protein (M) and nucleoprotein (N). The S protein is composed of S1 and S2 subunits.
SARS-Cov-2 binds to the membrane protein of Angiotensin-converting enzyme 2 (ACE 2) on the surface of lung epithelial cells via spike protein (S protein) on the surface of virus particles, and ACE2 then undergoes a change in shape structure, leading to the virus entering the cell. Based on a new coronavirus pathogenic mechanism, the neutralizing antibody of the S protein blocks the combination of the S protein and ACE2 on the cell surface, thereby blocking the virus from entering cells.
When SARS-Cov-2 invades the body, it will induce the body to produce corresponding effect B cell and memory B cell, the effect B cell will produce antibody, the antibody can be used for diagnosing and treating SARS-Cov-2. The blood plasma obtained from SARS-Cov-2 convalescent patient is transfused into SARS-Cov-2 severe patient, and the symptoms can be obviously improved. However, in the face of a huge number of patients, the plasma number of SARS-Cov-2 convalescent patients is limited, and the monoclonal antibody can be produced in vitro in a large scale by screening the SARS-Cov-2 convalescent patients for effective neutralizing antibody against SARS-Cov-2 and by genetic engineering and protein expression technology.
The invention content is as follows:
the main purpose of the invention is to provide a specific monoclonal antibody aiming at SARS-Cov-2 and the application thereof. The mononuclear cell is obtained from the SARS-Cov-2 rehabilitative person to construct phage library, and the specific antibody against SARS-Cov-2 is quickly screened, and the antibody can be used for clinical detection, diagnosis, prevention and treatment of novel coronavirus SARS-Cov-2.
In order to achieve the purpose, the invention is realized by the following technical scheme:
extracting peripheral blood from SARS-Cov-2 patient, obtaining mononuclear cell and extracting total RNA, synthesizing cDNA, using heavy chain variable region and light chain variable region doubling primer to amplify heavy chain variable region and light chain variable region, randomly combining obtained heavy chain variable region and light chain variable region, recombining with phage vector, converting recombined product into XL1-Blue, adding auxiliary phage VCSM13 to obtain titer about 10 13 Size SARS-Cov-2 bacteriophagyA somatic antibody library. And (3) screening an anti-Fab antibody by using the antigen S1 or RBD, and sequencing to obtain a heavy chain variable region sequence and a light chain variable region sequence which can be combined with the S1 or RBD, wherein the heavy chain variable region is assembled with an Fc fragment of IgG1, and the heavy chain and the light chain of the antibody are simultaneously expressed in mammalian cells to express the fully humanized anti-S1 or RBD antibody. Through in vitro affinity determination and pseudovirus neutralization experiment, monoclonal antibody for SARS-Cov-2 detection, diagnosis, prevention or treatment is screened.
The novel coronavirus antibody screened by the invention comprises 11 antibodies in total, namely XY1, XY2, XY3, XY4, XY5, XY6, XY7, XY8, XY9, XY10 and XY 11.
The invention screens the following:
the XY1 antibody heavy chain variable region comprises:
CDRH1 amino acid sequence: EDTFTSHY
CDRH2 amino acid sequence: INPTGGSI
CDRH3 amino acid sequence: ARGGFTPDTSAPDV
Preferably the amino acid sequence of the variable region of the XY1 antibody heavy chain:
GSTGDQVQLVQSGAEVKKPGASVKVSCRASEDTFTSHYIHWVRQAPGQGLEWMGIINPTGGSISYAQKFQGRVAMTKDTSTSTVYMELSSLRSEDTAVYYCARGGFTPDTSAPMDVWGQGTMVTVSS；
the XY1 antibody light chain variable region comprises:
CDRL1 amino acid sequence: QGIRNS
CDRL2 amino acid sequence: DAS
CDRL3 amino acid sequence: QHYFGTPLT
Preferably the amino acid sequence of the variable region of the XY1 antibody light chain:
GSTGDAEIVMTQSPSSLSASEGDRVIITCRASQGIRNSLAWYQQKPGKAPKLLLYDASKLESGVPSRFSGSGSGTHFTLTIDSLQPEDFATYYCQHYFGTPLTFGGGTKVEIK；
the XY2 antibody heavy chain variable region comprises:
CDRH1 amino acid sequence: EDTFTSHY
CDRH2 amino acid sequence: INPTGGSI
CDRH3 amino acid sequence: ARGGFTPDTSAPDV
Preferably the amino acid sequence of the variable region of the XY2 antibody heavy chain:
GSTGDEVQLVESGAEVKKPGASVKVSCRASEDTFTSHYIHWVRQAPGQGLEWMGIINPTGGSISYAQKFQGRVAMTKDTSTSTVYMELSSLRSEDTAVYYCARGGFTPDTSAPMDVWGQGTMVTVSS；
the XY2 antibody light chain variable region comprises:
CDRL1 amino acid sequence: QTISKY
CDRL2 amino acid sequence: EAS
CDRL3 amino acid sequence: QQSYSSRFT
Preferably the amino acid sequence of the variable region of the XY2 antibody light chain:
GSTGDAAIRLTQSPSSLSASVGDTVTITCRASQTISKYLHWYQQKPGEAPKLLISEASTFQGGVSSRFSGSRSGTDFTLTIYSLQPEDSATYYCQQSYSSRFTFGPGTKVEIK；
the variable region amino acid sequence of the XY3 antibody heavy chain comprises:
CDRH1 amino acid sequence: EDTFTSHY
CDRH2 amino acid sequence: INPTGGSI
CDRH3 amino acid sequence: ARGGFTPDTSAPDV
Preferably the amino acid sequence of the heavy chain variable region of the XY3 antibody:
GSTGDEVQLVESGAEVKKPGASVKVSCRASEDTFTSHYIHWVRQAPGQGLEWMGIINPTGGSISYAQKFQGRVAMTKDTSTSTVYMELSSLRSEDTAVYYCARGGFTPDTSAPMDVWGQGTMVTVSS；
the variable region amino acid sequence of the XY3 antibody light chain comprises:
CDRL1 amino acid sequence: QGISSW
CDRL2 amino acid sequence: AAS
CDRL3 amino acid sequence: QANSFPLT
Preferably the amino acid sequence of the variable region of the XY3 antibody light chain:
GSTGDAAIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGQGTKLEIK；
the variable region amino acid of the XY4 antibody heavy chain comprises:
CDRH1 amino acid sequence: GGTFSSIA
CDRH2 amino acid sequence: IIPIFGTA
CDRH3 amino acid sequence: ardvietiygmdv
Preferably the amino acid sequence of the variable region of the XY4 antibody heavy chain:
GSTGDQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSIAINWVRQAPGQGLAWMGKIIPIFGTANYAQKFQGRVTMTADESTNTAYMELSSLRSEDTAVYYCARDVIEATIYGMDVWGQGTTVTVSS；
the variable region amino acid of the XY4 antibody light chain comprises:
CDRL1 amino acid sequence: QSISTY
CDRL2 amino acid sequence: GAS
CDRL3 amino acid sequence: QQSYSAPYT
Preferably the amino acid sequence of the variable region of the XY4 antibody light chain:
GSTGDADIVMTQSPSSLPASVGDRVTITCRTSQSISTYVNWYQQKSGNAPELLMYGASILQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPYTFAQGTKLEIR；
the variable region amino acid of the XY5 antibody heavy chain comprises:
CDRH1 amino acid sequence: EDTFTSHY
CDRH2 amino acid sequence: INPTGGSI
CDRH3 amino acid sequence: ARGGFTPDTSAPDV
Preferably the amino acid sequence of the variable region of the XY5 antibody heavy chain:
GSTGDQVQLVQSGAEVKKPGASVKVSCRASEDTFTSHYIHWVRQAPGQGLEWMGIINPTGGSISYAQKFQGRVAMTKDTSTSTVYMELSSLRSEDTAVYYCARGGFTPDTSAPMDVWGQGTMVTVSS；
the variable region amino acid sequence of the XY5 antibody light chain comprises:
CDRL1 amino acid sequence: QGVSSNY
CDRL2 amino acid sequence: AAS
CDRL3 amino acid sequence: QHYDSPPYT
Preferably the XY5 antibody light chain variable region amino acid sequence:
GSTGDAVIWMTQSPSSLSASMGDRVTITCRASQGVSNYLAWYQHKPGKAPELLIYAASTLQSGVPSRFSASRSGTDFTLTISSLQPEDIATYYCQHYDSPPYTFGQGTKLEVK；
the variable heavy chain amino acid sequence of the XY6 antibody comprises:
CDRH1 amino acid sequence: EDTFTSHY
CDRH2 amino acid sequence: INPTGGSI
CDRH3 amino acid sequence: ARGGFTPDTSAPDV
Preferably the amino acid sequence of the heavy chain variable region of the XY6 antibody:
GSTGDEVQLVQSGAEVKKPGASVKVSCRASEDTFTSHYIHWVRQAPGQGLEWMGIINPTGGSISYAQKFQGRVAMTKDTSTSTVYMELSSLRSEDTAVYYCARGGFTPDTSAPMDVWGQGTMVTVSS；
the variable region amino acid sequence of the XY6 antibody light chain comprises:
CDRL1 amino acid sequence: ALAKHF
CDRL2 amino acid sequence: KDT
CDRL3 amino acid sequence: QSPDTTGRI
Preferably the amino acid sequence of the variable region of the XY6 antibody light chain:
GSTGDASYELTQPPSVSVSPGQTARITCSGDALAKHFGHWYQQRPGQAPVLVIYKDTERPLGIPERFSGSSSGATVTLTISAVEAEDEADYYCQSPDTTGRIFGGGTKVTVLGQPKA；
the variable region amino acid sequence of the XY7 antibody heavy chain comprises:
CDRH1 amino acid sequence: EDTFTSHY
CDRH2 amino acid sequence: INPTGGSI
CDRH3 amino acid sequence: ARGGFTPDTSAPDV
Preferably the amino acid sequence of the variable region of the XY7 antibody heavy chain:
GSTGDQVQLVQSGAEVKKPGASVKVSCRASEDTFTSHYIHWVRQAPGQGLEWMGIINPTGGSISYAQKFQGRVAMTKDTSTSTVYMELSSLRSEDTAVYYCARGGFTPDTSAPMDVWGQGTMVTVSS；
the variable region amino acid sequence of the XY7 antibody light chain comprises:
CDRL1 amino acid sequence: QGISSW
CDRL2 amino acid sequence: AAS
CDRL3 amino acid sequence: QQSYSIPRT
Preferably the amino acid sequence of the variable region of the XY7 antibody light chain:
GSTGDAAIRLTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSIPRTFGQGTKLEIK；
the variable region amino acid sequence of the XY8 antibody heavy chain comprises:
CDRH1 amino acid sequence: EDTFTSHY
CDRH2 amino acid sequence: INPTGGSI
CDRH3 amino acid sequence: ARGGFTPDTSAPDV
Preferably the amino acid sequence of the heavy chain variable region of the XY8 antibody:
QVQLVQSGAEVKKPGASVKVSCRASEDTFTSHYIHWVRQAPGQGLEWMGIINPTGGSISYAQKFQGRVAMTKDTSTSTVYMELSSLRSEDTAVYYCARGGFTPDTSAPMDVWGQGTMVTVSS；
the variable region amino acid sequence of the XY8 antibody light chain comprises:
CDRL1 amino acid sequence: QDISDW
CDRL2 amino acid sequence: RAV
CDRL3 amino acid sequence: QQTNTFPIT
Preferably the amino acid sequence of the variable region of the XY8 antibody light chain:
GSTGDAVIWMTQSPIQMTQSPSSVSAYVGDRVTITCRASQDISDWLAWYQQAPGKAPKLLIYRAVTLQDDVPSRFSGSGSGTDFSLTITGLQREDFATYYCQQTNTFPITFGHGTRLEIK；
the variable region amino acid sequence of the XY9 antibody heavy chain comprises:
CDRH1 amino acid sequence: EDTFTSHY
CDRH2 amino acid sequence: INPTGGSI
CDRH3 amino acid sequence: ARGGFTPDTSAPDV
Preferably the amino acid sequence of the variable region of the XY9 antibody heavy chain:
GSTGDEVQLVQSGAEVKKPGASVKVSCRASEDTFTSHYIHWVRQAPGQGLEWMGIINPTGGSISYAQKFQGRVAMTKDTSTSTVYMELSSLRSEDTAVYYCARGGFTPDTSAPMDVWGQGTMVTVSS；
the variable region amino acid sequence of the XY9 antibody light chain comprises:
CDRL1 amino acid sequence: SSDVGSYNL
CDRL2 amino acid sequence: EVT (EVT)
CDRL3 amino acid sequence: ISYAGNNNLV
Preferably the amino acid sequence of the variable region of the XY9 antibody light chain:
GSTGDAQSALTQPPSVSGAPGQTVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLIIIEVTKRPPGVPDRFSGSKSGNTASLTVTGLQAEDEADYHCISYAGNNNLVFGGGTQLTVLGQPKA；
the variable region amino acid sequence of the XY10 antibody heavy chain comprises
CDRH1 amino acid sequence: EDTFTSHY
CDRH2 amino acid sequence: INPTGGSI
CDRH3 amino acid sequence: ARGGFTPDTSAPDV
Preferably the amino acid sequence of the heavy chain variable region of the XY10 antibody:
GSTGDQVQLVQSGAEVKKPGASVKVSCRASEDTFTSHYIHWVRQAPGQGLEWMGIINPTGGSISYAQKFQGRVAMTKDTSTSTVYMELSSLRSEDTAVYYCARGGFTPDTSAPMDVWGQGTMVTVSS；
the variable region amino acid sequence of the XY10 antibody light chain comprises:
CDRL1 amino acid sequence: SSDIGRSS
CDRL2 amino acid sequence: RNN
CDRL3 amino acid sequence: AAWDNTLRGYV
Preferably the amino acid sequence of the variable region of the XY10 antibody light chain:
GSTGDASYELTQLPSASGTPGQRVTISCSGSSSDIGRSSVNWYQQLPGTAPKLLIYRNNQRPSGVPDRLSGSKSGTSGSLAISGLQSEDEADYYCAAWDNTLRGYVFGTGTKVTVLGQPKA；
the amino acid sequence of the heavy chain variable region of the XY11 antibody comprises:
CDRH1 amino acid sequence: EDTFTSHY
CDRH2 amino acid sequence: INPTGGSI
CDRH3 amino acid sequence: ARGGFTPDTSAPDV
Preferably the amino acid sequence of the heavy chain variable region of the XY11 antibody:
GSTGDQVQLVQSGAEVKKPGASVKVSCRASEDTFTSHYIHWVRQAPGQGLEWMGIINPTGGSISYAQKFQGRVAMTKDTSTSTVYMELSSLRSEDTAVYYCARGGFTPDTSAPMDVWGQGTMVTVSS；
the variable region amino acid sequence of the XY11 antibody light chain comprises:
CDRL1 amino acid sequence: QSVLFSPNNKNY
CDRL2 amino acid sequence: WAS
CDRL3 amino acid sequence: QQYDSSPWT
Preferably the amino acid sequence of the variable region of the XY11 antibody light chain:
GSTGDADIQLTQSPDSLAVSLGERATINCKSSQSVLFSPNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYDSSPWTFGQGTKVEIK。
the antibodies of the invention comprise conventional constant regions in addition to the heavy and light chain variable regions described above.
The novel coronavirus antibody disclosed by the invention comprises XY1, XY2, XY3, XY4, XY5, XY6, XY7, XY8, XY9, XY10 and XY11 antibodies, and through detection affinity and in-vitro neutralization experiments, the 11 antibodies are found to have more excellent affinity, reach nM level, and have a neutralizing effect on pseudoviruses; further preferably, the antibody composition comprises a combination of XY4 and XY10 antibodies, and the additive neutralization effect of the two antibodies is very significant.
The novel coronavirus antibody of the invention, preferably at least one of XY1, XY4, XY7 and XY10 antibodies is an antibody for novel coronavirus treatment; the IC50 values of these four antibodies have significant advantages; it is further preferred that the XY4 and XY10 antibody combination is an antibody for use in novel coronavirus therapy. According to the ELISA detection results of different antibody combinations, XY4 is suitable for being used as a detection phase for detecting the antigen; at least one of the XY1, XY2, XY3, XY5, XY6, XY7, XY8, XY9 antibodies is suitable for use as a coating phase antibody in the detection of an antigen.
The invention further preferably provides the application of the antibody combination of the coating phase and the detection phase in preparing a reagent for detecting the novel coronavirus:
the coating phase antibody XY1 combination detects at least one of phase antibodies XY4, XY7, XY8, XY9, XY10, XY11, preferably detects at least one of phase antibodies XY4, XY10, and more preferably detects phase antibody XY4.
Detecting at least one of phase antibodies XY4, XY9 and XY10 by combining the coating phase antibodies XY2, preferably at least one of phase antibodies XY4 and XY10; further preferably, the detection phase antibody XY4.
The coating phase antibody XY3 is combined with at least one of detection phase antibodies XY4 and XY10, preferably detection phase antibody XY4.
The coating phase antibody XY4 is combined with at least one of detection phase antibodies XY2, XY8 and XY10, preferably detection phase antibody XY10;
coating phase antibody XY5 combined detection phase antibody XY4, XY10 at least one, preferably detection phase antibody XY4;
the coating phase antibody XY6 is combined with at least one of detection phase antibodies XY4 and XY10, preferably detection phase antibody XY4;
coating phase antibody XY7 combined detection phase antibody XY4, XY10 at least one, preferably detection phase antibody XY4;
coating phase antibody XY8 combined detection phase antibody XY4, XY10 at least one, preferably detection phase antibody XY4;
coating phase antibody XY9 combined detection phase antibody XY4, XY10 at least one, preferably detection phase antibody XY4;
coating phase antibody XY10 combined detection phase antibody XY4;
the coating phase antibody XY11 is combined with the detection phase antibody XY4.
The novel coronavirus neutralizing antibody of the invention further comprises sequences with the amino acid sequences of the heavy chain variable region and the light chain variable region being more than 80% identical to the amino acid sequences of the heavy chain variable region and the light chain variable region of the 11 antibodies.
Antibodies that include conservative sequence variants of the amino acid sequence of the antibody are also included within the scope of the invention. Conservative amino acid sequence variants include modifications to the amino acid sequence that do not significantly alter the properties of the neutralizing antibodies of the invention, such as variants resulting from similar amino acid substitutions, deletions of amino acids, or additions as are well known in the art.
The neutralizing antibodies of the present invention also include both human and non-human antibodies, as well as all antibodies that have the same function or are engineered and optimized as the neutralizing antibodies described above. Further comprising: glycosylation or polyethylene glycol modification, etc.
The novel coronavirus monoclonal antibody further comprises any one of Fab, fab '-SH, fv, scFv and (Fab') 2 fragments, and comprises any one of the combined fragments of the heavy chain variable region and the light chain variable region of the antibody.
Fab refers to the portion of an antibody molecule that contains one light chain variable and constant region and one heavy chain variable and constant region that are disulfide bonded.
Fab' -SH refers to a Fab fragment that contains part of the hinge region.
Fv refers to the smallest antibody fragment containing the variable regions of the antibody heavy and light chains and having all antigen binding sites.
scFv refers to an engineered antibody in which the variable region of the light chain is linked directly to the variable region of the heavy chain or via a peptide chain.
(Fab ') 2 refers to the dimer of Fab'.
The invention also provides the nucleic acid sequences of the variable regions of the heavy chain and the light chain of the novel coronavirus neutralizing antibody.
Also included are nucleic acid sequences that are 80% or more identical thereto or nucleic acid sequences that contain degenerate codons that express the same amino acids as the sequences.
The invention also provides a preparation for detecting, preventing or treating the novel coronavirus, which comprises the novel coronavirus neutralizing antibody.
The invention also provides the application of the novel coronavirus neutralizing antibody, which comprises any one or more of the following components;
(1) Used for preparing a novel coronavirus detection preparation;
(2) For the preparation of a formulation for the prevention of a novel coronavirus infection;
(3) Can be used for preparing preparation for treating new type coronavirus infection.
The invention obtains mononuclear cells from a Xinguan rehabilitator, extracts RNA, then reversely transcribes the RNA into cDNA, amplifies heavy chain and light chain of an antibody by combining a heavy chain variable region primer and a light chain variable region primer, and then assembles the heavy chain variable region and the light chain variable region primer on a phage vector to construct a SARS-Cov-2 Fab phage antibody library; using antigen S1 or RBD (RBD is a section of protein in S1 protein and plays a key role in binding with ACE 2), screening anti-Fab antibody, and sequencing to obtain heavy chain variable region and light chain variable region sequences capable of binding with S1 or RBD, wherein the heavy chain variable region is assembled with Fc fragment of IgG1, and the heavy chain and the light chain of the antibody are simultaneously expressed in mammalian cells to express fully humanized anti-RBD or S1 antibody. Through in vitro affinity determination and pseudovirus neutralization experiment, monoclonal antibody with strong affinity and virus neutralization capacity and capable of being used for SARS-Cov-2 detection, diagnosis, prevention or treatment is screened. Plays an active promoting role in preventing and treating the novel coronavirus.
Drawings
FIG. 1 shows the electrophoretic band for the heavy chain variable region amplification of antibody;
FIG. 2 shows the amplified electrophoretic band of kappa chain in the light chain variable region of the antibody;
FIG. 3 is an electrophoretic band of the lambda chain amplification of the variable region of the antibody light chain;
FIG. 4 is an electrophoretic band after fusion of the heavy and light chains of an antibody;
FIG. 5 is an electrophoretic band after SfiI enzyme digestion of pC3C plasmid;
FIG. 6 shows the result of colony PCR identification of the fused pC3C plasmid-linked antibody heavy and light chains by product transformation XL 1-Blue;
FIG. 7 is the ELISA assay of enriched phage antibody library of round 5;
helper phage (VCMS 13) was added to the control wells, and enriched phage was added to the experimental group in round 5;
FIG. 8 shows the result of PCR identification of monoclonal bacteria liquid by adding RBD phage to XL 1-Blue;
FIG. 9 shows the expression of monoclonal antibodies and Coomassie blue staining;
FIG. 10 shows the result of ELISA assay for monoclonal antibodies;
no antibody was added to the control wells, but a secondary anti-human Fab-HRP antibody was added;
FIG. 11 is a monoclonal antibody IC50 assay;
FIG. 12 shows the detection of the additive neutralizing effect of antibodies using XY1, XY4, XY7, and XY10 combinations.
The specific implementation mode is as follows:
the invention is further described with reference to specific examples, which are not intended to be limiting.
The RBD protein of SARS-CoV-2 used in the examples of the present invention was purchased from Beijing Yi Qiao Shen Biotechnology Co., ltd, and S1 of SARS-Cov-2 was purchased from Beijing Baipu Sess Biotechnology Co., ltd.
The construction method of the phage antibody library of the invention is referred to as follows: dimitrov (ed.), generation and Selection of Rabbit Antibody Libraries by phase Display,2009, therapeutic μ tic Antibodies: methods and Protocols, vol.525, 101-128.
Example 1
1. Construction of phage antibody libraries
Obtaining 18 parts of peripheral blood of SARS-Cov-2 convalescent patients (blood samples are from convalescent patients within 1 month after the recovery discharge) and separating monocytes by using the human peripheral blood lymph separation medium. Extracting total RNA, mixing 18 parts of RNA in equal amount, carrying out reverse transcription to obtain cDNA, amplifying to obtain heavy chain variable region fragments and light chain variable region fragments by using a heavy chain variable region and light chain variable region doubling primer, randomly splicing the antibody heavy chain variable region fragments and light chain variable region fragments, connecting the antibody heavy chain variable region fragments and light chain variable region fragments with a phage vector, converting XL1-Blue bacteria (purchased from Toshiba Biotech Co., ltd.), and assembling into a phage antibody display library in the presence of a helper phage VSCM13 (purchased from NTCC national type culture Collection).
The method specifically comprises the following steps:
1. monocyte isolation
10ml of peripheral blood was collected from 18 SARS-Cov-2 patients and mononuclear cells were isolated using human peripheral blood-lymph separation medium (LTS 1077-1, a tertiary ocean biologicals science and technology, inc.; ltd.).
(1) Blood collection: 10mL of peripheral blood of a blood donor is aseptically extracted, anticoagulant (30 u/mL of heparin) is added, and the mixture is subpackaged in a 15mL centrifuge tube.
(2) A15 mL centrifuge tube containing anticoagulated whole blood was set at 3000rpm for 10min, at acceleration of 7 and deceleration of 6.
(3) The upper plasma layer was aspirated and the lower cells were diluted with an equal volume of PBS.
(4) A50 mL centrifuge tube was first filled with the same amount of separation medium as the blood sample.
(5) Carefully suck the blood sample with a pipette and apply it to the surface of the separation medium, 500-1100g (1800 rpm), centrifuge for 10min, accelerate 5, decelerate 4.
(6) After centrifugation, the centrifuge tube is divided into four layers from top to bottom; the second layer is a layer of annular milky white lymphocytes.
(7) The second layer of circular opalescent lymphocyte layer was carefully pipetted into a new 15mL centrifuge tube, 10mL of pbs was added to the resulting centrifuge tube and the cells were mixed.
(8) 1800rpm, 10min centrifugation, 5 acceleration, 4 deceleration.
(9) The supernatant was discarded, 5ml of erythrocyte lysate was added to each 15ml centrifuge tube, mixed slightly and left to stand at room temperature for 5min.
(10) 1800rpm, 10min centrifugation, 5 acceleration, 4 deceleration.
(11) The supernatant was discarded and 10mL of PBS was aspirated to resuspend the resulting cells.
(12) 1800rpm, 10min centrifugation, 5 acceleration, 4 deceleration.
(13) The supernatant was discarded and precipitated as PBMC.
2. Total RNA extraction
Subjecting the obtained mononuclear cells to a reaction at 5X10 6 Total RNA was extracted by adding 0.75ml of Trizol (Thermo fisher) to each cell, and 18 portions of RNA were mixed in equal amounts, each 1. Mu.g of RNA was taken.
(1)5X10 6 0.75ml Trizol was added to each cell.
(2) Mixing up and down for several times, and incubating for 5min on ice to ensure the cells to be fully cracked.
(3) Centrifuge at 12000rpm for 10 minutes at 4 ℃ and transfer the supernatant to a clean centrifuge tube.
(4) 0.2ml of chloroform was added to 1ml of Trizol, and the mixture was mixed well.
(5) Incubate on ice for 2-3min.
(6) Centrifuge at 12000rpm for 15 minutes at 4 ℃.
(7) Transfer the supernatant to a clean centrifuge tube.
(8) Adding isopropanol with the same volume into the supernatant and mixing evenly.
(9) Incubate at room temperature for 10min, centrifuge at 12000rpm at 4 ℃ for 10-15 min.
(10) The supernatant was discarded and the precipitate was retained.
(11) The precipitate was washed by adding 1ml of 75% ethanol.
(12) Centrifuge at 12000rpm for 5 minutes at 4 ℃.
(13) The supernatant was discarded as much as possible.
(15) Drying at room temperature for 5-10min, and no liquid can be seen on the tube wall.
(16) Add 20-50 μ l of RNAse free water to dissolve the RNA back.
(17) Taking 1. Mu.l of RNA for electrophoresis detection and determining the concentration of the RNA.
(18) RNA is subpackaged and stored in a refrigerator at the temperature of 80 ℃ below zero to avoid repeated freeze thawing.
3. Synthesis of cDNA
Mu.g of RNA was taken and used in a cDNA synthesis kit (K1612, thermo fisher).
(1) cDNA was synthesized in an amount of 5. Mu.g of RNA.
(2) The 10. Mu.l RNA/primer mix. Mu.re system is shown in Table 1 below, and includes:
TABLE 1
| System of ingredients | Volume of |
| RNA | 5μg |
| 50μM Oligo(dT) 20 | 1μl |
| Hex Random Primer | 1μl |
| 10mM dNTP | 1μl |
| DEPC-treated water | |
(3) Incubate at 65 ℃ for 5min, place on ice and stand for 1min.
(4) The cDNA Synthesis Mix was prepared for the next reaction by adding additional reagents as in Table 2 below.
TABLE 2
| Components of the |
1 reaction |
| 10xRT buffer | 2μl |
| 25mM MgCl 2 | 4μl |
| 0.1M DTT | 2μl |
| RNaseOΜT(40Μ/μl) | 1μl |
| Superscript III RT(200Μ/μl) | 1μl |
(5) Add 10. Mu.l of cDNA Synthesis Mix to the RNA/primer Mix gently mixed, centrifuge, and react according to the following conditions in Table 3:
TABLE 3
| |
50℃ | 50min |
| Step2 | 85℃ | 5min |
| Cooling on ice and centrifuging briefly | ||
| Step3 | RNaseH | 1μl |
| Step4 | 37℃ | 20min |
(6) The cDNA products were stored in aliquots at-20 ℃.
4. Antibody heavy chain variable region (VH) and light chain variable region (VL) amplification
The synthesized cDNA was diluted 10-fold and used to amplify the templates for the heavy and light chain variable regions.
(1) The system was formulated as in table 4 below, with VH as an example:
TABLE 4
(2) The reaction conditions are shown in the following table
TABLE 5
| Step1 | 98℃ | 15s |
| Step2 | 98℃ | 10s |
| Step3 | 55℃ | 5s |
| Step4 | 72℃ | 5s |
| Go to Step 2x34 | ||
| Step5 | 72℃ | 1m 30s |
| Step6 | 4℃ | Forever |
(3) Electrophoresis
Mu.l of the DNA was subjected to 1% agarose gel electrophoresis to determine whether the desired band was amplified and the size was about 400bp (see FIG. 1).
(4) All amplified VH's were mixed in equal amounts, added with 0.1 volume of 3M sodium acetate and 2.2 volumes of ethanol and mixed well, left overnight at-20 ℃.
(5) Centrifugation was carried out at 16000g for 15min at 4 ℃ and the supernatant removed, rinsed with 1ml of 70% ethanol (room temperature), dried at room temperature, redissolved in 200. Mu.l of water and gel-electrophoresed with 1% gelatin.
(6) The 400bp band was excised, recovered using QIAquick Gel Extraction Kit (QIAGEN, 28706), and the concentration was measured.
(7) The amplification and recovery of VL were performed according to the procedures (1) to (6), and the results are shown in FIG. 2 and FIG. 3.
(8) The nucleic acid concentration after VH and VL purification was adjusted to a final concentration of 100 ng/. Mu.l and stored at-20 ℃.
5. Amplification of C kappa-pelB
(1) Amplification of C.kappa. -pelB and CL-pelB with the vectors pC. Kappa.and pCL (vectors from add gene) (100 ng/. Mu.l) as templates
See Table 6 for an example of amplification of C.kappa. -pelB.
TABLE 6
| pCκ | 10μl |
| HCK (sequence see the aforementioned references) | 60μl |
| Pelb (sequence see the aforementioned references) | 60μl |
| ddH 2 O | 370μl |
| PrimerSTAR | 500μl |
| Total | 1000μl |
(2) The reaction conditions are shown in Table 7
TABLE 7
| Step1 | 98℃ | 15s |
| Step2 | 98℃ | 10s |
| Step3 | 55℃ | 5s |
| Step4 | 72℃ | 5s |
| Go to Step 2x34 | ||
| Step5 | 72℃ | 1m 30s |
| Step6 | 4℃ | Forever |
(3) 10 μ l of the product was electrophoretically detected to obtain a band of about 400bp in size.
(4) All amplified C.kappa. -pelB were mixed, added with 0.1 volume of 3M sodium acetate and 2.2 volumes of ethanol, and mixed well, left overnight at-20 ℃.
(5) Centrifugation is carried out at 16000g for 15min at 4 ℃, the supernatant is removed, the gel is rinsed with 1ml of 70% ethanol (room temperature), dried at room temperature, redissolved by adding 200. Mu.l of water and subjected to gel electrophoresis using 1% gel.
(6) The 400bp band was excised, recovered using QIAquick Gel Extraction Kit (QIAGEN, 28706), and the concentration was measured.
(7) The purified C.kappa. -pelB was diluted to a final concentration of 100 ng/. Mu.l and stored at-20 ℃.
(8) CL-pelB was amplified and the template pC κ was replaced with pCL by performing the procedures according to steps (1) to (7).
6. Human Vkappa/Human Ckappa/CL/Human VH or Human V lambda/Human CL/Human VH fusions exemplified by the Human Vkappa/Human Ckappa/CL/Human VH fusion
(1) Mixing different amounts of VL, VH, ck-pelB according to Table 8 below
TABLE 8
| VL | 10μl |
| VH | 10μl |
| Ck-pelB | 10μl |
| Primerstar Mix | 500μl |
| H 2 O | 470μl |
| Total | 1000μl |
(2) The reaction conditions are shown in Table 9
TABLE 9
| Step1 | 98℃ | 15s |
| Step2 | 98 | 10s |
| Step3 | ||
| 50℃ | 5s | |
| Step4 | 72℃ | 5s |
| Step5 | Go to Step 2x10 | |
| Step6 | 72℃ | 1m 30s |
| Step7 | 4℃ | Forever |
(3) Amplification of human VL/human Ck/human VH, see Table 10
| Spliced product | 50μl |
| C-5' SFIVL (sequence see the aforementioned references) | 4μl |
| c-3' sfivh (sequence see the aforementioned references) | 4μl |
| ddH 2 O | 17μl |
| PrimerSTAR | 25μl |
| Total | 100μl |
(4) The amplification conditions are shown in Table 11
TABLE 11
| Step1 | 98℃ | 15s |
| Step2 | 98℃ | 10s |
| Step3 | 55℃ | 5s |
| Step4 | 72℃ | 5s(Go to Step2 x 39) |
| Step5 | 72℃ | 1m 30s |
| Step6 | 4℃ | Forever |
(5) Electrophoretic detection
Taking 10 mu l of PCR product, carrying out electrophoresis detection, and fusing to obtain the target gene band with the size of 1.2Kb. (the results are shown in FIG. 4)
(6) All amplified human VL/human Ck/human VH were mixed with 0.1 volume of 3M sodium acetate and 2.2 volumes of ethanol and mixed together overnight at-20 ℃.
(7) Centrifugation was carried out at 16000g for 15min at 4 ℃ and the supernatant removed, rinsed with 1ml of 70% ethanol (room temperature), dried at room temperature, redissolved in 200. Mu.l of water and subjected to gel electrophoresis using 1% gel.
(8) The 1.2Kb sized band was excised, recovered using QIAquick Gel Extraction Kit (QIAGEN, 28706), and the concentration was measured.
(9) Human VL/h. Mu.man Ck/Human VH purified nucleic acid was diluted to a final concentration of 150 ng/. Mu.l and stored at-20 ℃.
(10) The fusion of human V.lamda/human CL/human VH was performed according to steps (1) - (9).
7. SfiI digestion of human Vkappa/human Ckappa/CL/human VH and human Vlambda/human CL/human VH
Taking the example of a human V kappa/human Ckappa/CL/human VH cleavage
(1) The enzyme digestion system is shown in Table 12
TABLE 12
| human Vκ/human Cκ/CL/human VH(150ng/μl) | 200μl |
| 10Xbuffer | 30μl |
| H 2 O | 60μl |
| SfiI 40u/μl | 10μl |
(2) Water bath at 50 ℃ and enzyme digestion reaction for 3h.
(3) The Gel was electrophoresed on a 1% Gel, and a 1.2kb band was excised and collected using QIAquick Gel Extraction Kit (QIAGEN, 28706).
(4) Detecting SfiI enzyme digestion, recovering the human VL/human Ck/human VH segment, adjusting the concentration to 50 ng/mu l, and storing at-20 ℃.
(5) The Human V.lamda/Human CL/Human VH was cleaved and the procedure was as in steps (1) to (4).
8. SfiI restriction enzyme pC3C (vector purchased from add gene)
(1) The enzyme digestion system is shown in Table 13
Watch 13
| pC3C 1μg/μl | 50μl |
| 10Xbuffer | 30μl |
| H 2 O | 208μl |
| SfiI 40u/μl | 12μl |
(2) Water bath at 50 ℃ and enzyme digestion reaction for 3h.
(3) Electrophoresis using a 1% gel will cut two bands, a 3.5Kb and a 1.2Kb sized fragment.
(see FIG. 5 for results)
(4) A3.5 Kb vector backbone was recovered using the QIAquick Gel Extraction Kit (QIAGEN, 28706).
(5) After Sfi enzyme digestion, the concentration of the recovered vector skeleton is adjusted to 100 ng/mul.
9. Connecting pC3C (SfiI) and Human VL/Human Ck/Human VH (SfiI) and Human V lambda/Human CL/Human VH
Exemplified are pC3C (SfiI) and human VL/human Ck/human VH (SfiI) linkages
(1) The connection system is shown in Table 14
TABLE 14
| 100 ng/. Mu.l SfiI digested pC3C | 1.5 |
| 50 ng/. Mu.l SfiI digested human VL/human Ck/human VH | 2μl |
| 10XT4 DNA ligase buffer | 2μl |
| H 2 O | 13.5μl |
| T4 DNA ligase 2000u/μl | 1μl |
The control group is shown in Table 15
Watch 15
| 100 ng/. Mu.l pC3C after SfiI enzyme digestion | 1.5μl |
| 10XT4 DNA ligase buffer | 2μl |
| H 2 O | 15.5μl |
| T4 DNA ligase 2000u/μl | 1μl |
(2) The reaction was carried out at 16 ℃ overnight.
(3) All ligation reactions were combined and 1/10 volume of 3M sodium acetate solution was added.
(4) 2.2 times volume of precooled absolute ethyl alcohol is added, mixed evenly up and down, and precipitated at-20 ℃ overnight.
(5) 16000g, centrifuge at 4 deg.C for 30min, carefully discard the supernatant.
(6) Adding 70% ethanol, gently cleaning the precipitate, centrifuging at 16000g and 4 deg.C for 5min, and removing the supernatant.
(7) Drying at room temperature.
(8) Dissolution with appropriate amount of water, typically 1 ligation reaction with 1. Mu.l volume of ddH 2 Dissolving O completely, and storing at-20 deg.C.
(9) The ligation of pC3C (SfiI) and Human V.lamda/Human CL/Human VH was performed according to the procedures (1) to (8).
10. Conversion of ligation product to XL1-Blue
(1) XL1-Blue shock-receptive ice is placed for 10min for dissolution.
(2) And adding 19 mu l of the precipitated and concentrated ligation product into a 1.5ml centrifuge tube, carrying out ice bath, adding 300 mu l of XL1-Blue electric shock competence, mixing uniformly, quickly transferring into a 2mm electric shock cup, and standing on ice for 1min.
(3) Electric shock conditions: 2.5KV,4ms
(4) 5ml (1ml +2ml) SOC culture medium is rapidly added after the electric conversion is finished, and the SOC culture medium is transferred to a 50ml conical centrifuge tube and cultured for 1h at 37 ℃ and 250 rpm. Mu.l of the bacterial liquid is added into 198 mu.l of LB, and meanwhile, the bacterial liquid is evenly coated on an LB plate containing 100 mu g/mu.l of carbenicillin, and the bacterial liquid is placed at 37 ℃ for overnight culture for calculating the transformation efficiency and the colony PCR identification positive rate. The PCR results are shown in FIG. 6.
(5) 10ml of SB medium, 3. Mu.l of 100. Mu.g/. Mu.l carbenicillin, and 30. Mu.l of 5. Mu.g/. Mu.l tetracycline were added. Incubated at 37 ℃ and 250rpm for 1h.
(6) Add 4.5. Mu.l of 100. Mu.g/. Mu.l carbenicillin, continue at 37 ℃,250rpm, incubate for 1-4h.
(7) The cultured bacteria were transferred to a 500ml culture flask, and 84ml of SB medium, 42.5. Mu.l of 100. Mu.g/. Mu.l carbenicillin, 170. Mu.l of 5. Mu.g/. Mu.l tetracycline, and 1ml of VCMS13 helper phage (10 ml) were added 11 -10 12 pfu/ml)，37℃，275rpm，90min。
(8) Mu.l of 50. Mu.g/. Mu.l kanamycin was added thereto, and cultured at 37 ℃ and 275rpm overnight.
(9) 3000g, and centrifugation at 4 ℃ for 15min.
(10) Precipitated phages
The supernatant (200 ml) was transferred to a 500ml clean centrifuge tube, and 8g of PEG-8000 and 6g of NaCl were added thereto, and the mixture was placed at 37 ℃ and 300rpm for 5min to promote dissolution. Standing on ice for 30min-1h.15000g, centrifuge at 4 ℃ for 15min. The supernatant was discarded, the flask was placed upside down on filter paper and allowed to dry for 10min, and excess liquid was carefully removed. The phage were resuspended in 2ml TBS containing 1% BSA (pipetted mix up and down), 16000g, centrifuged at 4 ℃ for 5min, and the supernatant was passed through a 0.22 μm filter (Millipore) and transferred to a 2ml centrifuge tube. Can be directly stored on ice in a short time, or added with 0.01 time of 2 percent sodium azide for 4 ℃ and added with 1 time of glycerol for long-time storage for-20 ℃.
2. Phage antibody library screening
Screening of RBD phage antibodies is exemplified
Using a 96-well plate coated with RBD protein, 5 rounds of screening were performed, and then single clones were selected to identify RBD-specific antibodies.
1. Antigen coating
(1) RBD was added to carbonate buffer at a concentration of 2. Mu.g/ml,
(2) Adding 50 mu l RBD into each well of a corning high protein adsorption enzyme-linked immunosorbent assay 96 pore plate, and standing overnight at 4 ℃;
(3) Discarding supernatant, adding 5% skimmed milk powder 200 μ l/well, standing at 37 deg.C for 1 hr;
(4) The supernatant was discarded and washed 5 times with 0.05% TBST.
2. RBD antibody screening
(1) Diluting the phage antibody library with 2% skimmed milk powder by 10 times, taking 2RBD antigen-coated 96-well micropores, adding 100 μ l diluted phage, and standing at 37 deg.C for 1h.
(2) Discard the supernatant, wash 5 times with 0.05% TBST, add 100. Mu.l 100mM glycine, stand at 37 ℃ for 15min, blow-beat continuously, add 9. Mu.l 1M Tris.
(3) And (3) adding the phage eluted in the step (2) into 2ml of XL1-Blue bacteria, and standing for 15min at room temperature.
(4) 6ml of SB medium was added, 1.6. Mu.l of 100. Mu.g/. Mu.l carbenicillin and 12. Mu.g of 5. Mu.g/. Mu.l tetracycline were added, and the mixture was incubated at 37 ℃ and 250rpm for 1 hour.
(5) Adding 2.4 μ l of carbenicillin 100 μ g/μ l, and culturing for 1 hr;
(6) Add 1ml of helper phage VCSM13 (10) 11 -10 12 pfu/ml), transferred to a 500ml flask, added with 91ml SB medium, added with 46. Mu.l of 100. Mu.g/. Mu.l carbenicillin, 184. Mu.l of 5. Mu.g/. Mu.l tetracycline, incubated at 37 ℃ for 1.5h at 250rpm, added with 140. Mu.l of 50. Mu.g/. Mu.l kanamycin, incubated at 37 ℃ overnight at 250 rpm.
(7) And (3) concentrating the phage antibody: centrifuging the bacterial liquid of the previous step to remove bacteria, and collecting supernatant (100 ml); 4g of PEG-8000 and 3g of NaCl were added thereto, and the mixture was heated at 37 ℃ and 300rpm for 5min to promote dissolution. Standing on ice for 30min-1h.15000g, centrifuge at 4 ℃ for 15min. The supernatant was discarded, the flask was placed upside down on filter paper and allowed to dry for 10min, and excess liquid was carefully removed. 2ml of TBS containing 1% BSA was used to resuspend the phage (pipetted up and down), 16000g, centrifuged at 4 ℃ for 5min, and the supernatant was passed through a 0.22 μm filter (Millipore) and transferred to a 2ml centrifuge tube. Can be directly stored on ice in a short time, or added with 0.01 time of 2 percent sodium azide for 4 ℃ and added with 1 time of glycerol for long-time storage for-20 ℃.
(8) And (5) taking the phage in the step (7), and performing 5 rounds of screening according to the steps (1) to (7).
(9) ELISA identification of five rounds of screened antibody libraries:
1) ELISA 96-well plate, each well coated with 100ng of RBD;
2) Adding 100 μ l phage (diluted 10 times with 2% skimmed milk powder) and incubating at 37 deg.C for 1h;
3) Washing 5 times with 0.05% TBST;
4) Adding 100 μ l of anti-M13-HRP antibody (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., ltd.) diluted 2000 times with 5% skimmed milk powder, and incubating at 37 deg.C for 1h;
5) Washing 5 times with 0.05% TBST;
6) Adding 100 μ l TMB, and developing in dark for 5min;
7) Add 50. Mu.l of 1M H 2 SO 4 The reaction was terminated.
8) Absorbance was measured at 450nm using a microplate reader.
The results are shown in FIG. 7.
3. Identification of RBD monoclonal antibody
(1) Taking RBD phage selected in the 5 th round, diluting 10 -7 Adding 1 μ l into 200 μ l XL1-Blue bacteria, standing at room temperature for 15min;
(2) Completely coating the bacteria in the step (1) on an LB individual culture medium containing 100 mu g/ml carbenicillin, and culturing at 37 ℃ overnight;
(3) 200 monoclonals are selected and added into 6ml of liquid LB culture medium containing 10 mu g/ml tetracycline and 100 mu g/ml carbenicillin, and cultured at 37 ℃ and 250rpm for XY4 hours, a small amount of bacterial liquid is taken for PCR identification of the bacterial liquid, and the clone with the band size of 1200bp can be selected and amplified (the result is shown in figure 8).
The colony PCR amplification system is shown in Table 16:
TABLE 16
| Bacterial liquid | 0.5μl |
| VHSEQ (sequences see the aforementioned references) | 0.2μl |
| vlseq (sequence see the aforementioned references) | 0.2μl |
| ddH 2 O | 4.1μl |
| PrimerSTAR | 5μl |
| Total | 10μl |
Colony PCR reaction conditions are shown in table 17:
TABLE 17
| Step1 | 98℃ | 3min |
| Step2 | 98 | 10s |
| Step3 | ||
| 50℃ | 5s | |
| Step4 | 72℃ | 15s |
| Step5 | Go to Step 2x34 | |
| Step6 | 72℃ | 1m 30s |
| Step7 | 4℃ | Forever |
(4) Uniformly dividing the positive clone bacterial liquid obtained in the step (3) into 2 parts, and adding 3 mu l of helper phage VCSM13 (10) 11 -10 12 pfu/ml), left at room temperature for 20min, 2.1. Mu.l of 50. Mu.g/. Mu.l kanamycin was added, and the other aliquot was incubated overnight at 37 ℃ and 250rpm without any substance;
(5) Centrifuging the bacteria added with the helper phage at 4000rpm for 10min, transferring the supernatant into a clean centrifuge tube, diluting the supernatant by 10 times, adding 100 mu l of the diluted supernatant into an enzyme-linked immunosorbent assay (ELISA) plate coated with RBD, incubating at 37 ℃ for 1h, washing 5 times with 0.05% TBST, adding 2000 times diluted antibody (purchased from Beijing Yinqiao Hibiscus biotechnology, inc.), incubating at 37 ℃ for 1h, washing 5 times with 0.05% TBST, adding 100 mu l TMB, developing for 2min, adding 50 mu l of 1M H, and adding 2 SO 4 The reaction is stopped, and the absorbance is measured at 450nm by using a microplate reader, and positive clones with the absorbance 2.1 times higher than that of the control are obtained.
(6) And (5) selecting the positive clones in the step (5), extracting plasmids of the bacteria without adding the helper phage, and sequencing to obtain sequences of the heavy chain variable region and the light chain variable region.
4. S1 phage antibody screening was performed according to steps 1-3.
3. Expression and activity identification of fully humanized antibody
1. Construction of fully humanized vector
(1) Designing corresponding primers according to the obtained sequences of the heavy chain variable region or the light chain variable region of the antibody, respectively amplifying, adding secretion signal peptides at the N ends of the heavy chain and the light chain, and adding an Fc fragment of IgG1 at the C end of the heavy chain;
(2) The heavy chain variable region and the light chain variable region were respectively homologously recombined into pcDNA3.4 vector. (pcDNA3.4 vector was purchased from Wuhan \28156
2. Expression of fully humanized antibodies
(1) Extracting corresponding heavy chain expression vector and light chain expression vector respectively by using an endotoxin removal medium extraction kit (purchased from OMEGA);
(2) When 293T cells (purchased from GmbH, gmbH) reached 80% confluence, PEI was used for transfection;
(3) 12h after transfection, replacement with serum-free protein expression medium, 37 ℃ C., 5% CO 2 Culturing for 7 days;
(4) The antibody was purified using protein A/G packing. The results are shown in FIG. 9.
3. Identification of fully humanized antibody Activity
Exemplified by RBD antibody Activity
(1) ELISA identification
The obtained antibody was adjusted to a concentration of 1mg/ml, diluted 2000-fold with 5% skim milk powder, added to an enzyme-linked immunosorbent assay well coated with RBD antigen, incubated at 37 ℃ for 1h, washed 5 times with 0.05% TBST, diluted 2000-fold anti-human Fab-HRP secondary antibody (purchased from Beijing Solibao Biotech Co., ltd.) with 5% skim milk powder, incubated at 37 ℃ for 1h, washed 5 times with 0.05% TBST, added with 100. Mu.l TMB, developed for 2min, added with 50. Mu.l 1M H 2 SO 4 The reaction was stopped and measured at 450nm using a microplate readerAnd (5) determining the absorbance. The results are shown in FIG. 10.
(2) Fully humanized antibody pseudovirus neutralization assay
1) Mixing 200 TCID50 pseudoviruses expressing SARS-Cov-2S protein with a series of antibodies with different dilution concentrations in equal amount, adding the mixture into a 96-well cell culture plate with the total volume of 100 mu l, incubating the mixture at 37 ℃ for 1h, and adding 100 mu l 293T cells containing 20000 over-expressed ACE 2; control without antibody, equal amount of pseudovirus was mixed with 293T overexpressing ACE 2; mixing pseudoviruses with 293T cells in the same amount as background; 5% CO at 37 ℃ 2 After standing for 48h, the fluorescence intensity was measured. Inhibition efficiency = (experimental group-background)/(control-background) × 100%. The results are shown in FIG. 11.
2) The combination of fully humanized antibodies XY1, XY4, XY7 and XY10 is used for detecting the antibody superposition neutralization effect as follows: 200 TCID50 pseudoviruses expressing SARS-Cov-2S protein are mixed with a series of antibodies with different dilution concentrations in equal volume (single antibody concentration is 0.009766 mu g/ml; when two antibodies are combined, the equal volume is mixed, the total concentration is 0.009766 mu g/ml), the total volume is 100 mu l, the pseudoviruses are added into a 96-well cell culture plate, the culture plate is incubated at 37 ℃ for 1h, and 100 mu l of 293T cells containing 20000 over-expressed ACE2 are added; control without antibody, equivalent pseudovirus was mixed with ACE2 overexpressing 293T; mixing pseudoviruses with 293T cells in the same amount as background; 5% CO at 37 ℃ 2 After standing for 48h, the fluorescence intensity was measured. Inhibition efficiency = (experimental group-background)/(control-background) × 100%.
The results are shown in FIG. 12.
(3) Fully humanized antibody affinity assay
Determination of antibody affinity was measured using BIAcore 8k (BIAcore, cytiva) and antibody diluted to a concentration of 20 μ g/ml injected at a flow rate of 10 μ l/min for a period of 20s to produce a response of 1000 μm. Binding affinity to SARS-CoV-2RBD protein was measured at a flow rate of 50. Mu.L/min and an injection time of 2 minutes. The affinity results are shown in Table 18.
TABLE 18 antibody affinity assay
(4) Antibody combination detection of RBD of SARS-Cov-2
The detection is carried out by adopting an ELISA sandwich method, monoclonal fully-humanized antibodies XY1, XY2, XY3, XY4, XY5, XY6, XY7, XY8, XY9, XY10 and XY11 coat an ELISA plate, SARS-Cov-2 antigen RBD is added, XY1, XY2, XY3, XY4, XY5, XY6, XY7, XY8, XY9, XY10 and XY11 phage antibodies are added for different combinations, an antibody with HRP-marked anti-M13 is added for detection color development, the darker the color indicates the stronger the capturing capability on SARS-Cov-2 antigen, and the pair of antibodies is suitable for detecting SARS-Cov antigen. The results are shown in Table 19.
The specific operation is as follows:
1) ELISA 96-well plates coated with 100ng of fully humanized antibody per well;
2) Add 100. Mu.l antigen (10 pg/ml), incubate 1h at 37 ℃;
3) Washing 5 times with 0.05% TBST;
4) Adding phage antibody diluted by 10 times with 2% skimmed milk powder, and incubating at 37 deg.C for 1h;
5) Washing 5 times with 0.05% TBST;
6) Adding anti-M13-HRP antibody (purchased from Beijing Yinqiao Shenzhou Biotechnology Co., ltd.) diluted 2000 times with 5% skimmed milk powder, and incubating at 37 deg.C for 1h;
7) Adding 100 μ l TMB, and developing in dark for 5min;
8) Add 50. Mu.l of 1M H 2 SO 4 The reaction was terminated.
9) Absorbance was measured at 450nm using a microplate reader.
10 Detection of S1 antigen was performed according to steps (1) to (9).
In the following table 19, the column is a monoclonal fully humanized antibody, the column is a phage antibody, and the heavy chain variable region and the light chain variable region of the phage antibody and the full antibody are the same, except that the same antibody has different forms, and the combination of the underlined data in the following table shows more excellent effects.
TABLE 19 detection of RBD proteins by cross-combination of different monoclonal antibodies
| XY1 | XY2 | XY3 | XY4 | XY5 | XY6 | XY7 | XY8 | XY9 | XY10 | XY11 | Control of | |
| XY1 | 0.8848 | 0.7326 | 3.497333 | 0.7735 | 0.475067 | 1.022033 | 1.0165 | 1.800633 | 3.002033 | 1.066633 | 0.07912 | |
| XY2 | 0.6635 | 0.7934 | 3.561467 | 0.551133 | 0.2924 | 0.631633 | 0.847033 | 1.0656 | 2.019467 | 0.958733 | 0.05423 | |
| XY3 | 0.3361 | 0.59015 | 3.0677 | 0.33515 | 0.17445 | 0.369 | 0.4125 | 0.5762 | 1.27265 | 0.64975 | 0.06881 | |
| XY4 | 0.8688 | 1.1878 | 0.90005 | 0.60735 | 0.3471 | 0.9918 | 1.03855 | 0.83135 | 2.4142 | 0.90515 | 0.05987 | |
| XY5 | 0.56505 | 0.79005 | 0.7313 | 2.83085 | 0.24635 | 0.69735 | 0.52915 | 0.7721 | 1.7483 | 0.6084 | 0.07762 | |
| XY6 | 0.42205 | 0.55845 | 0.54215 | 2.7278 | 0.4206 | 0.5086 | 0.48525 | 0.7785 | 1.3035 | 0.702 | 0.06931 | |
| XY7 | 0.398 | 0.73475 | 0.9844 | 3.1126 | 0.4679 | 0.1885 | 0.41515 | 0.92155 | 1.0476 | 0.6443 | 0.05542 | |
| XY8 | 0.37835 | 0.46405 | 0.5455 | 3.0873 | 0.50125 | 0.24975 | 0.4681 | 0.7979 | 1.71195 | 0.68565 | 0.08339 | |
| XY9 | 0.5695 | 0.5438 | 0.4639 | 2.3447 | 0.3912 | 0.206 | 0.6099 | 0.45535 | 1.32155 | 0.52985 | 0.06871 | |
| XY10 | 0.7053 | 0.75605 | 0.75415 | 3.2243 | 0.47525 | 0.17805 | 0.54015 | 0.5249 | 0.45935 | 0.5754 | 0.05324 | |
| XY11 | 0.27535 | 0.4699 | 0.4894 | 2.7212 | 0.27635 | 0.13865 | 0.3255 | 0.4718 | 0.69185 | 0.92985 | 0.04996 |
The column is the coating antibody, the row is the detection antibody, and the blank is not added with the phage antibody, but is added with the helper phage VCSM13 and the anti-M13-HRP antibody.
Through the series of activity identification, 11 novel coronavirus antibodies of monoclonal fully-humanized antibodies XY1, XY2, XY3, XY4, XY5, XY6, XY7, XY8, XY9, XY10 and XY11 are obtained.
Claims (8)
1. A novel coronavirus antibody characterized by being XY1,
the XY1 antibody heavy chain variable region comprises:
CDRH1 amino acid sequence: EDTFTSHY
CDRH2 amino acid sequence: INPTGGSI
CDRH3 amino acid sequence: arggfftpdtsapmdv;
the XY1 antibody light chain variable region comprises:
CDRL1 amino acid sequence: QGIRNS
CDRL2 amino acid sequence: DAS
CDRL3 amino acid sequence: QHYFGTPLT.
2. The novel coronavirus antibody according to claim 1, wherein the XY1 antibody heavy chain variable region amino acid sequence:
GSTGDQVQLVQSGAEVKKPGASVKVSCRASEDTFTSHYIHWVRQAPGQGLEWMGIINPTGGSISYAQKFQGRVAMTKDTSTSTVYMELSSLRSEDTAVYYCARGGFTPDTSAPMDVWGQGTMVTVSS；
XY1 antibody light chain variable region amino acid sequence:
GSTGDAEIVMTQSPSSLSASEGDRVIITCRASQGIRNSLAWYQQKPGKAPKLLLYDASKLESGVPSRFSGSGSGTHFTLTIDSLQPEDFATYYCQHYFGTPLTFGGGTKVEIK。
3. the novel coronavirus antibody according to claim 1,
XY1 is an antibody for use in the treatment of novel coronaviruses.
4. The type of antibody fragment of any one of claims 1 to 3, comprising Fab, fab '-SH, fv, scFv, (Fab') 2 fragments.
5. Any nucleic acid capable of expressing the novel coronavirus antibody of any one of claims 1-3.
6. The application of the novel coronavirus antibody combination of the coating phase and the detection phase in the preparation of a reagent for detecting novel coronavirus is characterized in that: the coating phase antibody XY1 is combined to detect any one of phase antibodies XY4, XY7, XY8, XY9, XY10, and XY 11.
7. An agent for detecting, preventing or treating a novel coronavirus, comprising the novel coronavirus antibody according to any one of claims 1 to 3.
8. Use of the novel coronavirus antibody of any one of claims 1-3, comprising any one or more of the following;
(1) Used for preparing a novel coronavirus detection preparation;
(2) For the preparation of a formulation for the prevention of a novel coronavirus infection;
(3) Can be used for preparing preparation for treating new type coronavirus infection.
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| CN115109150A (en) | 2022-09-27 |
| CN113150130B (en) | 2022-10-18 |
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| CN113150130A (en) | 2021-07-23 |
| CN115141272B (en) | 2024-06-25 |
| CN115141271A (en) | 2022-10-04 |
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| CN115109151A (en) | 2022-09-27 |
| CN115109151B (en) | 2024-06-11 |
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