CN115109150A - Novel coronavirus monoclonal antibody XY10 and application thereof - Google Patents

Novel coronavirus monoclonal antibody XY10 and application thereof Download PDF

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CN115109150A
CN115109150A CN202210703223.9A CN202210703223A CN115109150A CN 115109150 A CN115109150 A CN 115109150A CN 202210703223 A CN202210703223 A CN 202210703223A CN 115109150 A CN115109150 A CN 115109150A
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antibody
amino acid
acid sequence
variable region
novel coronavirus
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徐义
罗均利
蔡开妹
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Xiangya Hospital of Central South University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention discloses a novel coronavirus monoclonal antibody XY10 and application thereof. The full-humanized antibody XY10 of anti 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

Novel coronavirus monoclonal antibody XY10 and application thereof
The application has the application number of 2021101325814, the application date of 2021, 1 month and 31 days, and the invention name is as follows: 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 XY10 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 (spike protein, S protein) on the surface of viral particles, and ACE2 then undergoes a change in shape structure, resulting in the virus entering the cell. Based on a novel coronavirus pathogenic mechanism, the neutralizing antibody of the S protein blocks the combination of the S protein and ACE2 on the cell surface, so that the virus is blocked from entering cells.
When SARS-Cov-2 invades the body, it will induce the body to produce corresponding effector B cell and memory B cell, and the effector B cell will produce antibody, which 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 present invention is to provide a specific monoclonal antibody against SARS-Cov-2 and its application. 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 combined primer to amplify heavy chain variable region and light chain variable region, randomly combining obtained heavy chain variable region and light chain variable region, and recombining with phage vectorThe recombinant product is converted into XL1-Blue, and a helper phage VCSM13 is added to obtain a titer of about 10 13 Size SARS-Cov-2 phage antibody library. The antigen S1 or RBD is used to screen anti-Fab antibody, and the heavy chain variable region and light chain variable region sequences capable of binding with S1 or RBD are obtained by sequencing, the heavy chain variable region is assembled with the Fc fragment of IgG1, the heavy chain and the light chain of the antibody are expressed in mammalian cells at the same time, and the antibody of fully humanized anti-S1 or RBD is expressed. 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 antibodies screened by the invention comprise 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: ARGGFTPDTSAPMDV
Preferably the amino acid sequence of the heavy chain variable region of the XY1 antibody:
GSTGDQVQLVQSGAEVKKPGASVKVSCRASEDTFTSHYIHWVRQAPGQGLEWMGIINPTGGSISYAQKFQGRVAMTKDTSTSTVYMELSSLRSEDTAVYYCARGGFTPDTSAPMDVWGQGTMVTVSS；
the variable region of the light chain of the XY1 antibody comprises:
CDRL1 amino acid sequence: QGIRNS
CDRL2 amino acid sequence: DAS
CDRL3 amino acid sequence: QHYFGTPLT
Preferably the amino acid sequence of the light chain variable region of the XY1 antibody:
GSTGDAEIVMTQSPSSLSASEGDRVIITCRASQGIRNSLAWYQQKPGKAPKLLLYDASKLESGVPSRFSGSGSGTHFTLTIDSLQPEDFATYYCQHYFGTPLTFGGGTKVEIK；
the XY2 antibody heavy chain variable region comprises:
CDRH1 amino acid sequence: EDTFTSHY
CDRH2 amino acid sequence: INPTGGSI
CDRH3 amino acid sequence: ARGGFTPDTSAPMDV
Preferably the amino acid sequence of the heavy chain variable region of the XY2 antibody:
GSTGDEVQLVESGAEVKKPGASVKVSCRASEDTFTSHYIHWVRQAPGQGLEWMGIINPTGGSISYAQKFQGRVAMTKDTSTSTVYMELSSLRSEDTAVYYCARGGFTPDTSAPMDVWGQGTMVTVSS；
the variable region of the light chain of the XY2 antibody comprises:
CDRL1 amino acid sequence: QTISKY
CDRL2 amino acid sequence: EAS
CDRL3 amino acid sequence: QQSYSSRFT
Preferably the amino acid sequence of the light chain variable region of the XY2 antibody:
GSTGDAAIRLTQSPSSLSASVGDTVTITCRASQTISKYLHWYQQKPGEAPKLLISEASTFQGGVSSRFSGSRSGTDFTLTIYSLQPEDSATYYCQQSYSSRFTFGPGTKVEIK；
the amino acid sequence of the heavy chain variable region of the XY3 antibody comprises:
CDRH1 amino acid sequence: EDTFTSHY
CDRH2 amino acid sequence: INPTGGSI
CDRH3 amino acid sequence: ARGGFTPDTSAPMDV
Preferably the amino acid sequence of the heavy chain variable region of the XY3 antibody:
GSTGDEVQLVESGAEVKKPGASVKVSCRASEDTFTSHYIHWVRQAPGQGLEWMGIINPTGGSISYAQKFQGRVAMTKDTSTSTVYMELSSLRSEDTAVYYCARGGFTPDTSAPMDVWGQGTMVTVSS；
the amino acid sequence of the variable region of the light chain of the XY3 antibody comprises:
CDRL1 amino acid sequence: QGISSW
CDRL2 amino acid sequence: AAS
CDRL3 amino acid sequence: QQANSFPLT
Preferably the amino acid sequence of the light chain variable region of the XY3 antibody:
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: ARDVIEATIYGMDV
Preferably the amino acid sequence of the heavy chain variable region of the XY4 antibody:
GSTGDQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSIAINWVRQAPGQGLAWMGKIIPIFGTANYAQKFQGRVTMTADESTNTAYMELSSLRSEDTAVYYCARDVIEATIYGMDVWGQGTTVTVSS；
the variable region amino acid of the light chain of the XY4 antibody comprises:
CDRL1 amino acid sequence: QSISTY
CDRL2 amino acid sequence: GAS
CDRL3 amino acid sequence: QQSYSAPYT
Preferably the amino acid sequence of the light chain variable region of the XY4 antibody:
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: ARGGFTPDTSAPMDV
Preferably the amino acid sequence of the heavy chain variable region of the XY5 antibody:
GSTGDQVQLVQSGAEVKKPGASVKVSCRASEDTFTSHYIHWVRQAPGQGLEWMGIINPTGGSISYAQKFQGRVAMTKDTSTSTVYMELSSLRSEDTAVYYCARGGFTPDTSAPMDVWGQGTMVTVSS；
the amino acid sequence of the variable region of the light chain of the XY5 antibody comprises:
CDRL1 amino acid sequence: QGVSSNY
CDRL2 amino acid sequence: AAS
CDRL3 amino acid sequence: QHYDSPPYT
Preferably the amino acid sequence of the light chain variable region of the XY5 antibody:
GSTGDAVIWMTQSPSSLSASMGDRVTITCRASQGVSNYLAWYQHKPGKAPELLIYAASTLQSGVPSRFSASRSGTDFTLTISSLQPEDIATYYCQHYDSPPYTFGQGTKLEVK；
the amino acid sequence of the heavy chain variable region of the XY6 antibody comprises:
CDRH1 amino acid sequence: EDTFTSHY
CDRH2 amino acid sequence: INPTGGSI
CDRH3 amino acid sequence: ARGGFTPDTSAPMDV
Preferably the amino acid sequence of the heavy chain variable region of the XY6 antibody:
GSTGDEVQLVQSGAEVKKPGASVKVSCRASEDTFTSHYIHWVRQAPGQGLEWMGIINPTGGSISYAQKFQGRVAMTKDTSTSTVYMELSSLRSEDTAVYYCARGGFTPDTSAPMDVWGQGTMVTVSS；
the amino acid sequence of the variable region of the light chain of the XY6 antibody comprises:
CDRL1 amino acid sequence: ALAKHF
CDRL2 amino acid sequence: KDT
CDRL3 amino acid sequence: QSPDTTGRI
Preferably the amino acid sequence of the light chain variable region of the XY6 antibody:
GSTGDASYELTQPPSVSVSPGQTARITCSGDALAKHFGHWYQQRPGQAPVLVIYKDTERPLGIPERFSGSSSGATVTLTISAVEAEDEADYYCQSPDTTGRIFGGGTKVTVLGQPKA；
the amino acid sequence of the heavy chain variable region of the XY7 antibody comprises:
CDRH1 amino acid sequence: EDTFTSHY
CDRH2 amino acid sequence: INPTGGSI
CDRH3 amino acid sequence: ARGGFTPDTSAPMDV
Preferably the amino acid sequence of the heavy chain variable region of the XY7 antibody:
GSTGDQVQLVQSGAEVKKPGASVKVSCRASEDTFTSHYIHWVRQAPGQGLEWMGIINPTGGSISYAQKFQGRVAMTKDTSTSTVYMELSSLRSEDTAVYYCARGGFTPDTSAPMDVWGQGTMVTVSS；
the amino acid sequence of the variable region of the light chain of the XY7 antibody comprises:
CDRL1 amino acid sequence: QGISSW
CDRL2 amino acid sequence: AAS
CDRL3 amino acid sequence: QQSYSIPRT
Preferably the amino acid sequence of the light chain variable region of the XY7 antibody:
GSTGDAAIRLTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSIPRTFGQGTKLEIK；
the amino acid sequence of the heavy chain variable region of the XY8 antibody comprises:
CDRH1 amino acid sequence: EDTFTSHY
CDRH2 amino acid sequence: INPTGGSI
CDRH3 amino acid sequence: ARGGFTPDTSAPMDV
Preferably the amino acid sequence of the heavy chain variable region of the XY8 antibody:
QVQLVQSGAEVKKPGASVKVSCRASEDTFTSHYIHWVRQAPGQGLEWMGIINPTGGSISYAQKFQGRVAMTKDTSTSTVYMELSSLRSEDTAVYYCARGGFTPDTSAPMDVWGQGTMVTVSS；
the amino acid sequence of the variable region of the light chain of the XY8 antibody comprises:
CDRL1 amino acid sequence: QDISDW
CDRL2 amino acid sequence: RAV
CDRL3 amino acid sequence: QQTNTFPIT
Preferably the amino acid sequence of the light chain variable region of the XY8 antibody:
GSTGDAVIWMTQSPIQMTQSPSSVSAYVGDRVTITCRASQDISDWLAWYQQAPGKAPKLLIYRAVTLQDDVPSRFSGSGSGTDFSLTITGLQREDFATYYCQQTNTFPITFGHGTRLEIK；
the amino acid sequence of the heavy chain variable region of XY9 antibody comprises:
CDRH1 amino acid sequence: EDTFTSHY
CDRH2 amino acid sequence: INPTGGSI
CDRH3 amino acid sequence: ARGGFTPDTSAPMDV
Preferably the amino acid sequence of the heavy chain variable region of the XY9 antibody:
GSTGDEVQLVQSGAEVKKPGASVKVSCRASEDTFTSHYIHWVRQAPGQGLEWMGIINPTGGSISYAQKFQGRVAMTKDTSTSTVYMELSSLRSEDTAVYYCARGGFTPDTSAPMDVWGQGTMVTVSS；
the amino acid sequence of the variable region of the light chain of the XY9 antibody comprises:
CDRL1 amino acid sequence: SSDVGSYNL
CDRL2 amino acid sequence: EVT (EVT)
CDRL3 amino acid sequence: ISYAGNNNLV
Preferably the amino acid sequence of the light chain variable region of the XY9 antibody:
GSTGDAQSALTQPPSVSGAPGQTVTISCTGTSSDVGSYNLVSWYQQHPGKAPKLIIIEVTKRPPGVPDRFSGSKSGNTASLTVTGLQAEDEADYHCISYAGNNNLVFGGGTQLTVLGQPKA；
the amino acid sequence of the variable region of the XY10 antibody heavy chain comprises
CDRH1 amino acid sequence: EDTFTSHY
CDRH2 amino acid sequence: INPTGGSI
CDRH3 amino acid sequence: ARGGFTPDTSAPMDV
Preferably the amino acid sequence of the heavy chain variable region of the XY10 antibody:
GSTGDQVQLVQSGAEVKKPGASVKVSCRASEDTFTSHYIHWVRQAPGQGLEWMGIINPTGGSISYAQKFQGRVAMTKDTSTSTVYMELSSLRSEDTAVYYCARGGFTPDTSAPMDVWGQGTMVTVSS；
the amino acid sequence of the variable region of the light chain of the XY10 antibody comprises:
CDRL1 amino acid sequence: SSDIGRSS
CDRL2 amino acid sequence: RNN
CDRL3 amino acid sequence: AAWDNTLRGYV
Preferably the amino acid sequence of the light chain variable region of the XY10 antibody:
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: ARGGFTPDTSAPMDV
Preferably the amino acid sequence of the heavy chain variable region of the XY11 antibody:
GSTGDQVQLVQSGAEVKKPGASVKVSCRASEDTFTSHYIHWVRQAPGQGLEWMGIINPTGGSISYAQKFQGRVAMTKDTSTSTVYMELSSLRSEDTAVYYCARGGFTPDTSAPMDVWGQGTMVTVSS；
the amino acid sequence of the variable region of the light chain of the XY11 antibody comprises:
CDRL1 amino acid sequence: QSVLFSPNNKNY
CDRL2 amino acid sequence: WAS
CDRL3 amino acid sequence: QQYDSSPWT
Preferably the amino acid sequence of the light chain variable region of the XY11 antibody:
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 comprises XY1, XY2, XY3, XY4, XY5, XY6, XY7, XY8, XY9, XY10 and XY11, and 11 antibodies are detected to have more excellent affinity and reach nM level through in vitro neutralization experiments, and have neutralization effect on pseudoviruses; further preferably, the combination of XY4 and XY10 antibodies is included, and the additive neutralization effect of the two antibodies is very significant.
The novel coronavirus antibody of the present invention, preferably at least one of XY1, XY4, XY7 and XY10 is an antibody for novel coronavirus treatment; the IC50 values for 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 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 the reagent for detecting the novel coronavirus:
the coating phase antibody XY1 is combined with at least one of detection phase antibodies XY4, XY7, XY8, XY9, XY10 and XY11, preferably at least one of detection phase antibodies XY4 and XY10, and more preferably detection phase antibody XY 4.
Coating phase antibody XY2 combined with at least one of detection phase antibodies XY4, XY9 and XY10, preferably at least one of detection phase antibodies XY4 and XY 10; further preferably, the detection phase antibody XY 4.
The coating phase antibody XY3 is combined with at least one of detection phase antibodies XY4 and XY10, preferably detection phase antibody XY 4.
Coating phase antibody XY4 combined with at least one of detection phase antibodies XY2, XY8, XY10, preferably detection phase antibody XY 10;
coating phase antibody XY5 combined with at least one of detection phase antibodies XY4, XY10, preferably detection phase antibody XY 4;
coating phase antibody XY6 combined with at least one of detection phase antibodies XY4, XY10, preferably detection phase antibody XY 4;
coating phase antibody XY7 combined with at least one of detection phase antibodies XY4 and XY10, preferably detection phase antibody XY 4;
coating phase antibody XY8 combined with at least one of detection phase antibodies XY4, XY10, preferably detection phase antibody XY 4;
coating phase antibody XY9 combined with at least one of detection phase antibodies XY4, XY10, preferably detection phase antibody XY 4;
coating phase antibody XY10 combined with detection phase antibody XY 4;
coating phase antibody XY11 was combined with detection phase antibody XY 4.
The novel coronavirus neutralizing antibody of the present invention further comprises sequences having 80% or more identity between the amino acid sequences of the heavy chain variable region and the light chain variable region and the amino acid sequences of the 11 antibody heavy chain variable regions and the amino acid sequences of the antibody light chain variable regions.
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 antibody of the present invention also includes human and non-human antibodies, and all antibodies having the same function as the above neutralizing antibody or modified and optimized. 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 region of the antibody heavy chain, the variable region of the light chain and having all antigen binding sites.
scFv refers to an engineered antibody in which the variable region of the light chain is linked to the variable region of the heavy chain either directly 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 the body of a Xinguan rehabilitative person, extracts RNA, then carries out reverse transcription to form cDNA, amplifies heavy chains and light chains of antibodies by combining heavy chain variable region primers and light chain variable region primers, and assembles the heavy chain variable regions and the light chain variable regions onto 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 and plays a key role in binding with ACE2), screening anti-Fab antibody, and sequencing to obtain the 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 is the antibody light chain variable region kappa chain amplification electrophoresis band;
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 the electrophoresis band after SfiI enzyme digestion of pC3C plasmid;
FIG. 6 shows the result of colony PCR identification of the product after fusion of pC3C plasmid-linked antibody heavy and light chains, transformed into XL 1-Blue;
FIG. 7 is the ELISA assay of enriched phage antibody library of round 5;
helper phage (VCMS13) was added to the control wells, and the experimental groups were added with enriched phage 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 combinations of antibodies XY1, XY4, XY7, XY 10.
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 by phase Display, 2009, therapeutic μ tic Antibodies: Methods and Protocols, vol.525, 101-.
Example 1
First, construction of phage antibody library
Obtaining 18 peripheral blood (blood sample from recovered patient within 1 month of recovery discharge) of SARS-Cov-2 patient using human peripheral blood lymph separation liquid to separate mononuclear cells. Extracting total RNA, mixing 18 parts of RNA in equal amount, carrying out reverse transcription to obtain cDNA, amplifying to obtain fragments of a heavy chain variable region and a light chain variable region by using a heavy chain variable region and light chain variable region merger primer, randomly splicing the antibody heavy chain variable region and light chain variable region, connecting the spliced fragments with a phage vector, converting XL1-Blue bacteria (purchased from Toshiba Biotech limited), 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 (LTS1077-1, 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 is set at 3000rpm for 10min, accelerated speed is 7, and decelerated speed is 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 aspirate the blood sample with a pipette onto the surface of the separation fluid, 500-1100g (1800rpm), centrifuge for 10min, acceleration 5, deceleration 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) Carefully pipette the second layer of circular opalescent lymphocytes with a pipette and transfer to a new 15mL centrifuge tube, add 10mL of pbs to the resulting centrifuge tube and mix the cells well.
(8)1800rpm, 10min centrifugation, 5 acceleration, 4 deceleration.
(9) The supernatant was discarded, 5ml of erythrocyte lysate was added to each 15ml centrifuge tube, slightly mixed and allowed to stand at room temperature for 5 min.
(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 the treatment according to 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 taking 1. mu.g of RNA.
(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 2-3 min.
(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 uniformly.
(9) Incubate at room temperature for 10min, and 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 re-dissolve RNA.
(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 collected 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 Total volume 10. mu.l
(3) Incubate at 65 ℃ for 5min, place on ice and stand for 1 min.
(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 System 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
Step1 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. Amplification of antibody heavy chain variable region (VH) and light chain variable region (VL)
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
Figure BDA0003705150320000141
(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
10. 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 is carried out at 16000g for 15min at 4 ℃, the supernatant is removed, rinsing is carried out with 1ml of 70% ethanol (room temperature), drying is carried out at room temperature, 200. mu.l of water is added for redissolution, and gel electrophoresis is carried out with 1% gel.
(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
For an example of amplification of C.kappa. -pelB, see Table 6.
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. mu.l of the product was electrophoretically detected to give 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 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.
(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
VL concentration 100 ng/. mu.l, VH concentration 100 ng/. mu.l, Ckappa-pelB concentration 100 ng/. mu.l
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
Watch 10
Spliced product 50μl
C-5' SFIVL (sequences see the aforementioned references) 4μl
c-3' sfivh (sequences 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 1.2 Kb. (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 cut out, 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 enzyme digestion of human V kappa/human Ckappa/CL/human VH and human V lambda/human CL/human VH
Exemplified by the human Vkappa/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 3 h.
(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 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 3 h.
(3) Electrophoresis using a 1% gel will cut two bands, a 3.5Kb and a 1.2Kb sized fragment.
(the results are shown in FIG. 5)
(4) A3.5 Kb vector backbone was recovered using the QIAquick Gel Extraction Kit (QIAGEN, 28706).
(5) After the enzyme digestion of Sfi, the concentration of the recovered carrier skeleton is adjusted to 100 ng/mul.
9. pC3C (SfiI) and Human VL/Human Ck/Human VH (SfiI) and Human V lambda/Human CL/Human VH
Exemplified by 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 1.5μl
50 ng/. mu.l of 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 after SfiI enzyme digestion pC3C 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 volumes of 3M sodium acetate solution were 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 ℃ for 30min, carefully discard the supernatant.
(6) Adding 70% ethanol, gently cleaning the precipitate, 16000g, centrifuging at 4 deg.C for 5min, and discarding 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 connection of pC3C (SfiI) and Human V.lamda/Human CL/Human VH was performed according to the procedures (1) to (8).
10. Ligation product was converted to XL1-Blue
(1) XL1-Blue shock-receptive ice was left for 10min to dissolve.
(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 1 min.
(3) Electric shock conditions: 2.5KV and 4ms
(4) After the end of the electrotransfer, 5ml (1ml +2ml +2ml) of SOC medium was rapidly added, and the mixture was transferred to a 50ml conical centrifuge tube and cultured at 37 ℃ and 250rpm for 1 hour. 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 1 h.
(6) Adding 4.5. mu.l of 100. mu.g/. mu.l carbenicillin, continuing to incubate at 37 ℃ and 250rpm for 1-4 h.
(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, centrifuged at 4 ℃ for 15 min.
(10) Precipitated phages
The supernatant (200ml) was transferred to a 500ml clean centrifuge tube, and 8g PEG-8000 and 6g NaCl were added, and the mixture was placed at 37 ℃ and 300rpm for 5min to facilitate dissolution. Standing on ice for 30min-1 h. 15000g, centrifuge at 4 ℃ for 15 min. 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, the supernatant was filtered 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 ℃.
Screening of phage antibody library RBD phage antibody screening is taken as an example
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 a 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 1 h.
(2) The supernatant was discarded, washed 5 times with 0.05% TBST, 100. mu.l of 100mM glycine was added, and the mixture was left at 37 ℃ for 15min, during which time pipetting was continued, and 9. mu.l of 1M Tris was added.
(3) Adding the phage eluted in the step (2) into 2ml 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) 1ml of the helper phage VCSM13 (10) was added 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 ℃, 250rpm for 1.5h, added with 140. mu.l of 50. mu.g/. mu.l kanamycin, incubated at 37 ℃, 250rpm overnight.
(7) And (3) concentrating the phage antibody: centrifuging the bacterial liquid of the previous step to remove bacteria, and collecting supernatant (100 ml); 4g PEG-8000 and 3g NaCl were added, and the mixture was allowed to stand at 37 ℃ and 300rpm for 5min to promote dissolution. Standing on ice for 30min-1 h. 15000g, centrifuge at 4 ℃ for 15 min. 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, the supernatant was filtered 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 plates coated with 100ng RBD per well;
2) adding 100 μ l phage (diluted 10 times with 2% skimmed milk powder) and incubating at 37 deg.C for 1 h;
3) washing 5 times with 0.05% TBST;
4) adding 100 μ l of 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 1 h;
5) washing 5 times with 0.05% TBST;
6) adding 100 μ l TMB, and developing in dark for 5 min;
7) 50 μ l of 1M H was added 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 15 min;
(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, 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 Terminating the reaction, and measuring the absorbance at 450nm by using a microplate reader, wherein the absorbance is higher thanPositive clones were obtained in a 2.1-fold comparison.
(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.
Expression and activity identification of fully humanized antibody
1. Construction of fully humanized vectors
(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 homologously recombined into pcDNA3.4 vector, respectively. (pcDNA3.4 vector from Wuhan vast Ling Biotech Co., Ltd.)
2. Expression of fully humanized antibodies
(1) Extracting corresponding heavy chain expression vector and light chain expression vector respectively by using endotoxin removing middle extraction kit (purchased from OMEGA);
(2) when the fusion level of 293T cells (purchased from Wuhan Punuoist Life technologies, Inc.) reached 80%, transfection was performed using PEI;
(3) 12h after transfection, the medium was changed to serum-free protein expression medium at 37 ℃ with 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 identification of RBD antibody Activity
(1) ELISA identification
Adjusting the obtained antibody to 1mg/ml, diluting with 5% skimmed milk powder 2000 times, adding into enzyme-linked immunosorbent assay well coated with RBD antigen, incubating at 37 deg.C for 1h, washing with 0.05% TBST for 5 times, diluting with 5% skimmed milk powder 2000 times antihuman Fab-HRP secondary antibody (purchased from Beijing Solebao Biotech, Ltd.), incubating at 37 deg.C for 1h, washing with 0.05% TBST for 5 times, adding 100 μ l TMB, developing for 2min, adding 50 μ l TMB, and mixingl1M H 2 SO 4 The reaction was terminated and absorbance was measured at 450nm using a microplate reader. The results are shown in FIG. 10.
(2) Fully humanized antibody pseudovirus neutralization assay
1) Mixing 200 TCID50 expressing SARS-Cov-2S protein pseudovirus with a series of antibodies with different dilution concentration in equal amount, adding into 96-well cell culture plate with total volume of 100 μ l, incubating at 37 deg.C for 1h, adding 100 μ l 293T cells containing 20000 over-expressed ACE 2; control no antibody added, equal amounts of pseudovirus were mixed with 293T overexpressing ACE 2; mixing background equivalent pseudoviruses with 293T cells; 5% CO at 37 ℃ 2 After standing for 48h, the fluorescence intensity was measured. Inhibition efficiency was 100% (experimental group-background)/(control-background). 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 0.009766. mu.g/ml; when two antibodies are combined, equal mixing is carried out, the total concentration is 0.009766. mu.g/ml), the total volume is 100. mu.l, the mixture is added into a 96-well cell culture plate, the incubation is carried out for 1h at 37 ℃, and 100. mu.l of 293T cells containing 20000 over-expressed ACE2 are added; control no antibody added, equal amounts of pseudovirus were mixed with 293T overexpressing ACE 2; mixing background equivalent pseudoviruses with 293T cells; 5% CO at 37 ℃ 2 After standing for 48h, the fluorescence intensity was measured. Inhibition efficiency ═ 100% of (experimental group-background)/(control-background).
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
Figure BDA0003705150320000241
Figure BDA0003705150320000251
(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 mark anti-M13 is added, the coloration is detected, the darker the color indicates that the capturing capability of the SARS-Cov-2 antigen is stronger, and the pair of antibodies is also indicated to be suitable for the detection of the 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 (10pg/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 1 h;
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 1 h;
7) adding 100 μ l TMB, and developing in dark for 5 min;
8) 50 μ l of 1M H was added 2 SO 4 The reaction was terminated.
9) Absorbance was measured at 450nm using a microplate reader.
10) Detection of the 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, but the same antibody has different forms, and the combination of 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
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 added with the helper phage VCSM13 and the anti-M13-HRP antibody.
Through the series of activity identification, 11 novel coronavirus antibodies including 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 XY 10;
the amino acid sequence of the variable region of the XY10 antibody heavy chain comprises
CDRH1 amino acid sequence: EDTFTSHY
CDRH2 amino acid sequence: INPTGGSI
CDRH3 amino acid sequence: ARGGFTPDTSAPMDV, respectively;
the amino acid sequence of the variable region of the light chain of the XY10 antibody comprises:
CDRL1 amino acid sequence: SSDIGRSS
CDRL2 amino acid sequence: RNN
CDRL3 amino acid sequence: AAWDNTLRGYV are provided.
2. The novel coronavirus antibody according to claim 1,
amino acid sequence of heavy chain variable region of XY10 antibody:
GSTGDQVQLVQSGAEVKKPGASVKVSCRASEDTFTSHYIHWVRQAPGQGLEWMGIINPTGGSISYAQKFQGRVAMTKDTSTSTVYMELSSLRSEDTAVYYCARGGFTPDTSAPMDVWGQGTMVTVSS；
amino acid sequence of light chain variable region of XY10 antibody:
GSTGDASYELTQLPSASGTPGQRVTISCSGSSSDIGRSSVNWYQQLPGTAPKLLIYRNNQRPSGVPDRLSGSKSGTSGSLAISGLQSEDEADYYCAAWDNTLRGYVFGTGTKVTVLGQPKA。
3. the novel coronavirus antibody according to claim 1,
XY10 is an antibody useful 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: coating phase antibody XY10 combined with detection phase antibody XY 4; or the detection phase antibody XY10 combined coating phase antibody XY1, XY2, XY3, XY4, XY5, XY6, XY7, XY8, XY 9.
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.
CN202210703223.9A 2021-01-31 2021-01-31 Novel coronavirus monoclonal antibody XY10 and application thereof Pending CN115109150A (en)

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