CN116514962A - Antibodies or functional fragments thereof that specifically bind to N protein and uses thereof - Google Patents

Antibodies or functional fragments thereof that specifically bind to N protein and uses thereof Download PDF

Info

Publication number
CN116514962A
CN116514962A CN202310133633.9A CN202310133633A CN116514962A CN 116514962 A CN116514962 A CN 116514962A CN 202310133633 A CN202310133633 A CN 202310133633A CN 116514962 A CN116514962 A CN 116514962A
Authority
CN
China
Prior art keywords
seq
amino acid
acid sequence
antibody
antigen
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202310133633.9A
Other languages
Chinese (zh)
Inventor
庾蕾
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Eighth Affiliated Hospital Of Guangzhou Medical University Guangzhou Eighth People's Hospital Guangzhou Liver Disease Hospital Guangzhou Institute Of Infectious Diseases
Original Assignee
Eighth Affiliated Hospital Of Guangzhou Medical University Guangzhou Eighth People's Hospital Guangzhou Liver Disease Hospital Guangzhou Institute Of Infectious Diseases
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Eighth Affiliated Hospital Of Guangzhou Medical University Guangzhou Eighth People's Hospital Guangzhou Liver Disease Hospital Guangzhou Institute Of Infectious Diseases filed Critical Eighth Affiliated Hospital Of Guangzhou Medical University Guangzhou Eighth People's Hospital Guangzhou Liver Disease Hospital Guangzhou Institute Of Infectious Diseases
Priority to CN202310133633.9A priority Critical patent/CN116514962A/en
Publication of CN116514962A publication Critical patent/CN116514962A/en
Pending legal-status Critical Current

Links

Classifications

    • 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
    • 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
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • 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]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host
    • 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 belongs to the field of biological medicine, and in particular relates to an antibody or a functional fragment thereof specifically binding to N protein and application thereof. The invention provides monoclonal antibodies and antigen binding fragments that target binding to the N protein of SARS-CoV-2 coronavirus, wherein the antibodies are one selected from the group consisting of N3, N5, N31, N83 and 3B7. The invention also relates to nucleic acids encoding the antibodies or antigen binding fragments and host cells comprising the same, as well as methods of making and uses of the antibodies or antigen binding fragments. The antibody provided by the invention has strong binding force with SARS-CoV-2N protein antigen, and does not generate cross reaction with N protein antigens of MERS virus, human coronavirus OC43 and HKU1, which has important significance for developing a rapid diagnosis test based on immune principle and designing an effective vaccine targeting SARS-CoV-2.

Description

Antibodies or functional fragments thereof that specifically bind to N protein and uses thereof
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to an antibody or a functional fragment thereof specifically binding to N protein and application thereof.
Background
The viral nucleocapsid (N) protein is one of four structural proteins of SARS-CoV-2, involved in viral replication, assembly and modulation of immune responses. It has two domains, an N-terminal domain (NTD) and a C-terminal domain (CTD), consisting of an N-arm, a central Ser/Arg-rich linker and a C-tail fragment, the N-protein can form dimers by interaction of two CTD monomers. Antibody responses targeting the SARS-CoV-2N protein were observed after natural infection and vaccination with inactivated virus vaccine, but the antibody levels after vaccination with inactivated virus vaccine were much lower compared to natural infection. Recently, research reports that nucleocapsid protein has protective effect on SARS-CoV-2 as a constituent of vaccine, so that research on N protein epitope has important significance for coping with novel coronavirus pneumonia (COVID-19) diseases.
Accurate epitope localization of SARS-CoV-2N protein relies on isolation of N-protein specific monoclonal antibodies (mAbs), which will aid in determining antigen structure in the case of antigen-antibody interactions. Although mabs of murine SARS-CoV-2N protein have been isolated previously, the targeting epitopes of these murine mabs may differ significantly from those derived from the infectious agent.
SARS-CoV-2, SARS-CoV and MERS-CoV belong to the genus Beta-coronavirus, common human coronaviruses (HuCoVs), such as HCoV-OC43 and HCoV-HKU1, also belong to the genus Beta. Among these HuCoVs, SARS-CoV-2 and the N protein of SARS-CoV are highly conserved, with similarities exceeding 90%, and since previously infected by other HuCoVs, the conserved N protein epitopes in subsequent SARS-CoV-2 infections may induce cross-reactions to protect or modulate the whole body immune response. It is currently unclear whether antigen cross-reactivity or past infection in patients with covd-19 would induce detection of antibodies specific for HuCoVs.
Therefore, it is of great importance to provide an antibody with excellent sensitivity, high research and diagnostic properties and industrial availability.
Disclosure of Invention
In view of the above, the present invention aims to provide an antibody or a functional fragment thereof that specifically binds to N protein and uses thereof. The invention successfully clones and identifies 5N protein specific monoclonal antibodies N3, N5, N31, N83 and 3B7 from patients in convalescence of COVID-19. Three antibodies (N3, N5 and N31) target the CTD domain of SARS-CoV-2N protein, while the other two antibodies (N83 and 3B 7) target the NTD domain; the ELISA method is used for determining the combination of the 5-strain antibody and other coronavirus N proteins, and the result shows that the 5-strain antibody is cross-combined with SARS virus N proteins, but does not cross-react with MERS virus, human coronavirus OC43 and HKU 1N proteins.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a first object of the present invention is to provide an isolated antibody or antigen-binding fragment thereof that specifically binds to SARS-CoV-2 coronavirus N protein, said antibody being one selected from the group consisting of N3, N5, N31, N83 and 3B 7; the antibody or antigen binding fragment thereof comprises a heavy chain hypervariable region and a light chain hypervariable region in an N3, N5, N31, N83, or 3B7 antibody;
wherein the N3, N5, N31, N83, or 3B7 antibody comprises heavy chain hypervariable regions CDR1, CDR2, and CDR3, and light chain hypervariable regions CDR1, CDR2, and CDR3;
the amino acid sequence of a heavy chain hypervariable region CDR1 of the N3 antibody is shown as SEQ ID NO.11, the amino acid sequence of a CDR2 is shown as SEQ ID NO.12, and the amino acid sequence of a CDR3 is shown as SEQ ID NO. 13; the amino acid sequence of a CDR1 of a light chain hypervariable region of the N3 antibody is shown as SEQ ID NO.14, the amino acid sequence of a CDR2 is shown as SEQ ID NO.15, and the amino acid sequence of a CDR3 is shown as SEQ ID NO. 16;
the amino acid sequence of a heavy chain hypervariable region CDR1 of the N5 antibody is shown as SEQ ID NO.17, the amino acid sequence of a CDR2 is shown as SEQ ID NO.18, and the amino acid sequence of a CDR3 is shown as SEQ ID NO. 19; the amino acid sequence of a CDR1 of a light chain hypervariable region of the N5 antibody is shown as SEQ ID NO.20, the amino acid sequence of a CDR2 is shown as SEQ ID NO.21, and the amino acid sequence of a CDR3 is shown as SEQ ID NO. 22;
the amino acid sequence of a heavy chain hypervariable region CDR1 of the N31 antibody is shown as SEQ ID NO.23, the amino acid sequence of a CDR2 is shown as SEQ ID NO.24, and the amino acid sequence of a CDR3 is shown as SEQ ID NO. 25; the amino acid sequence of a CDR1 of a light chain hypervariable region of the N31 antibody is shown as SEQ ID NO.26, the amino acid sequence of a CDR2 is shown as SEQ ID NO.27, and the amino acid sequence of a CDR3 is shown as SEQ ID NO. 28;
the amino acid sequence of a heavy chain hypervariable region CDR1 of the N83 antibody is shown as SEQ ID NO.29, the amino acid sequence of a CDR2 is shown as SEQ ID NO.30, and the amino acid sequence of a CDR3 is shown as SEQ ID NO. 31; the amino acid sequence of a CDR1 of a light chain hypervariable region of the N83 antibody is shown as SEQ ID NO.32, the amino acid sequence of a CDR2 is shown as SEQ ID NO.33, and the amino acid sequence of a CDR3 is shown as SEQ ID NO. 34;
the amino acid sequence of a CDR1 of a heavy chain hypervariable region of the 3B7 antibody is shown as SEQ ID NO.35, the amino acid sequence of a CDR2 is shown as SEQ ID NO.36, and the amino acid sequence of a CDR3 is shown as SEQ ID NO. 37; the amino acid sequence of CDR1 of the light chain hypervariable region of the 3B7 antibody is shown as SEQ ID NO.38, the amino acid sequence of CDR2 is shown as SEQ ID NO.39, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 40;
the amino acid sequence of the heavy chain variable region of the N3 antibody comprises a sequence shown as SEQ ID NO.1, and the amino acid sequence of the light chain variable region of the N3 antibody comprises a sequence shown as SEQ ID NO. 2;
the amino acid sequence of the heavy chain variable region of the N5 antibody comprises a sequence shown as SEQ ID NO.3, and the amino acid sequence of the light chain variable region of the N5 antibody comprises a sequence shown as SEQ ID NO. 4;
the amino acid sequence of the heavy chain variable region of the N31 antibody comprises a sequence shown as SEQ ID NO.5, and the amino acid sequence of the light chain variable region of the N31 antibody comprises a sequence shown as SEQ ID NO. 6;
the amino acid sequence of the heavy chain variable region of the N83 antibody comprises a sequence shown as SEQ ID NO.7, and the amino acid sequence of the light chain variable region of the N83 antibody comprises a sequence shown as SEQ ID NO. 8;
the amino acid sequence of the heavy chain variable region of the 3B7 antibody comprises a sequence shown as SEQ ID NO.9, and the amino acid sequence of the light chain variable region of the 3B7 antibody comprises a sequence shown as SEQ ID NO. 10.
QMQLVQSGAEVKRPGSSVKVSCKASGGTFSVYAISWVRQAPGQGLEWMGGIIPIFGTA NYAQKFQGRVTLTADESTSTAYMELSSLSSEDTAVYYCARGGYCSGANCPKWGEWSHSYN YMDVWGKGTTVTVSS(SEQ ID NO.1)；
DVVMTQSPDSLAVSLGERATINCKSSQSVFYSSNNKDYLAWYQQKPGQPPKLLIYWAS TRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYKSPGTFGQGTKLEIK(SEQ IDNO.2)；
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAIGWVRQAPGQGLEWMGAIIPIFGTT IFAQKFQGRVTITADESTTTAYMELSSLRSDDTAVYYCARGGGGVERGVILTRWFDPWGQG TLVTVSS(SEQ ID NO.3)；
QSVLTQPPSVSAAPGQKVTLSCSGSSSNIGNNHVSWYQQLPGTAPKLLIYDNNKRPSGI PDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDNSLSAGVFGGGTKLTVV(SEQ IDNO.4)；
QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYGISWVRQAPGQGLEWMGGIIPILGTA NYAQRFQGRIALTADESTSTAYMEVSSLRSEDTAVYYCARGMWSNPPGYCCSYMDVWGK GTTVTVSS(SEQ ID NO.5)；
EIVLTQSPATLSVSPGERVTLACRASQSLRSKLAWYQQKPGQAPRLLIYDASTRATGFPA RFSGSGSGTEFTLTISSLQSEDFAVYYCQQYSNWPRTFGQGTKLEIK(SEQ ID NO.6)；
QVQLQESGPRLVKSSETLSLTCTVSGGSISSGSYYWNWIRQPAGKGLEWIGRVYISGVT SYNPSLKSRVTISLDISKNQFSLQLTSVTAADSAVYYCARGGMAVGAPLYYYFYGMDVWG QGTTVTVSS(SEQ ID NO.7)；
DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYVDWYLQKPGQSPQLMIYLGSN RASGVPDRFSGSGSGTHFTLKISRVEAEDVGVYYCMQALQTLSITFGQGTRLEIK(SEQ IDNO.8)；
QMQLVQSGAEVKKPGASVRVSCKVSGYSLTELPMHWVRQAPGKGLEWVGSFDPQDA ETTYAQRFQGRVAMTEDTSTDTAYMELSSLRSEDTAVYYCATPAPTIVGVVIYALHIWGQGT MVTVSS(SEQ ID NO.9)；
EIVLTQSPGTLSLSPGERATLSCRASQRVSDNSLAWYQQKPGQAPSLLIFAASIRAPGIPDRISGTGSGTDFTLTINRLEPEDFVVYFCLQYTTSPPLTFGGGTKLEIK(SEQ ID NO.10)；
GGTFSVYA(SEQ ID NO.11)；
IIPIFGTA(SEQ ID NO.12)；
ARGGYCSGANCPKWGEWSHSYNYMDV(SEQ ID NO.13)；
QSVFYSSNNKDY(SEQ ID NO.14)；
WAS(SEQ ID NO.15)；
QQYYKSPGT(SEQ ID NO.16)；
GGTFSSYA(SEQ ID NO.17)；
IIPIFGTT(SEQ ID NO.18)；
ARGGGGVERGVILTRWFDP(SEQ ID NO.19)；
SSNIGNNH(SEQ ID NO.20)；
DNN(SEQ ID NO.21)；
GTWDNSLSAGV(SEQ ID NO.22)；
GGTFSSYG(SEQ ID NO.23)；
IIPILGTA(SEQ ID NO.24)；
ARGMWSNPPGYCCSYMDV(SEQ ID NO.25)；
QSLRSK(SEQ ID NO.26)；
DAS(SEQ ID NO.27)；
QQYSNWPRT(SEQ ID NO.28)；
GGSISSGSYY(SEQ ID NO.29)；
VYISGVT(SEQ ID NO.30)
ARGGMAVGAPLYYYFYGMDV(SEQ ID NO.31)；
QSLLHSNGYNY(SEQ ID NO.32)；
LGS(SEQ ID NO.33)
MQALQTLSIT(SEQ ID NO.34)；
GYSLTELP(SEQ ID NO.35)；
FDPQDAET(SEQ ID NO.36)；
ATPAPTIVGVVIYALHI(SEQ ID NO.37)；
QRVSDNS(SEQ ID NO.38)；
AAS(SEQ ID NO.39)；
LQYTTSPPLT(SEQ ID NO.40)。
Preferably, wherein the N3, N31, N83 or 3B7 antibody further comprises a heavy chain constant region comprising the amino acid sequence shown in SEQ ID No.41 and a light chain constant region comprising the amino acid sequence shown in SEQ ID No. 42.
ASTKGPSVFPLAPSSRSTSESTAALGCLVKDYFPEPVTVSWNSGSLTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYVCNVNHKPSNTKVDKRVEIKTCGGGSKPPTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPDVKFNWYVNGAEVHHAQTKPRETQYNSTYRVVSVLTVTHQDWLNGKEYTCKVSNKALPAPIQKTISKDKGQPREPQVYTLPPSREELTKNQVSLTCLVKGFYPSDIVVEWESSGQPENTYKTTPPVLDSDGSYFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSVSPGK(SEQ ID NO.41)；
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO.42)。
Preferably, wherein the N5 antibody further comprises a heavy chain constant region comprising the amino acid sequence shown in SEQ ID No.41 and a light chain constant region comprising the amino acid sequence shown in SEQ ID No. 43.
MRDLAAVASVGLPLLRRQAQVAAGRVLVVALFGGCGGLHSRLDGAAICLPGHCHGSR VEVTYETHQCGLVGLKLLTRGWEQSDRGGSLGLT(SEQ ID NO.43)。
Preferably, the antibody or antigen binding fragment thereof is a monoclonal antibody.
Preferably, the N3, N5, N31, N83 or 3B7 antibodies each specifically bind to the N protein antigen of SARS virus, but do not cross-react with the N protein antigens of MERS virus, human coronavirus OC43 and HKU1.
It is a second object of the present invention to provide an isolated polynucleotide encoding an antibody or antigen-binding fragment thereof according to any one of the above.
It is a third object of the present invention to provide an isolated vector comprising a polynucleotide according to the above.
It is a further object of the present invention to provide a host cell comprising a polynucleotide according to the above or a vector according to the above.
It is a further object of the present invention to provide a method of expressing an antibody or antigen-binding fragment thereof according to any one of the preceding claims, characterized in that the method comprises culturing a host cell according to the preceding claims under conditions suitable for expressing the antibody or antigen-binding fragment thereof, and optionally recovering the antibody or antigen-binding fragment thereof according to any one of the preceding claims from the host cell or from the culture medium.
It is another object of the present invention to provide the use of an antibody or antigen binding fragment thereof, said polynucleotide, said vector, said host cell or said method according to any one of the above in the preparation of a reagent for detecting SARS virus N protein.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides antibodies or functional fragments thereof which specifically bind to N protein and uses thereof, and specifically provides 5N protein specific monoclonal antibodies N3, N5, N31, N83 and 3B7, and determines immunodominant regions of SARS-CoV-2N protein, wherein the N3, N5 and N31 antibodies target CTD domains of SARS-CoV-2N protein, and the N83 and 3B7 antibodies target NTD domains of SARS-CoV-2N protein. The cross-reactions of the N3, N5, N31, N83 and 3B7 antibodies with the N proteins of SARS virus, MERS virus, human coronavirus OC43 and HKU1 are measured by ELISA method, and the results show that the N3, N5, N31, N83 or 3B7 antibodies are specifically combined with the N protein antigen of SARS virus, but do not cross-react with the N protein antigens of MERS virus, human coronavirus OC43 and HKU1. Therefore, the antibody or the functional fragment thereof specifically binding to the N protein and the application thereof provided by the invention have important significance for developing a rapid diagnosis test based on an immune principle and designing an effective vaccine targeting SARS-CoV-2.
Drawings
FIG. 1 shows the sequence characteristics of 5N-protein specific monoclonal antibodies N3, N5, N31, N83 and 3B 7;
FIG. 2 shows the binding and dissociation curves of 5N-protein specific monoclonal antibodies N3, N5, N31, N83 and 3B7 with N protein;
FIG. 3 shows affinity KD values for 5 strains of N protein-specific monoclonal antibodies N3, N5, N31, N83 and 3B 7;
FIG. 4 is an epitope analysis chart of 5 strains of N protein specific monoclonal antibodies N3, N5, N31, N83 and 3B 7;
FIG. 5 is a graph showing the cross-reaction of 5 strains of N protein-specific monoclonal antibodies N3, N5, N31, N83 and 3B7 with N proteins of other coronaviruses.
Detailed Description
The above-described aspects of the present invention will be described in further detail with reference to the following embodiments. It should not be construed that the scope of the above subject matter of the present invention is limited to the following examples.
The flow instrument is a Beckmann coulter MoFlo Astrios EQ ultra-high-speed flow cell sorting system, and can be purchased from Shanghai right instrument and equipment limited company; the cDNA synthesis kit is SuperScript III First Strand Synthesis System, available from Invitrogen, inc., USA, numbered #18080051; the fluorescent labeled antibodies are a group of fluorescent antibodies, including IgD-FITC, CD19-ECD, CD27-PC7, CD38-APC A750, igM-PB and CD45-KO fluorescent antibodies, and are purchased from Beckmann Coulter; the cell growth factors CpG, IL21, IL2 and the healthy human PBMC irradiated by radiation are purchased from Guangzhou Haojin biotechnology Co., ltd; the Phusion super fidelity DNA polymerase is Phusion High-Fidelity PCR Master Mix with GC Buffer, available from NEB limited company under the number of # M0532s.
Example 1 identification and sorting of memory B cells
1.1 peripheral blood mononuclear cell isolation: taking novel peripheral vein EDTA anticoagulation of coronavirus infection rehabilitation person, separating Peripheral Blood Mononuclear Cells (PBMC) by density gradient centrifugation method, and subpackaging for 5×10 6 Tube, put in liquid nitrogen for freezing.
1.2 staining with fluorescently labeled antibody: the 1-2 tube PBMC were removed from the liquid nitrogen and placed in a 37℃water bath for solubilization. Washing 3 times with PBS buffer, adding antibody for staining, and incubating for 15min at room temperature in dark place. After washing again with PBS, 400. Mu.L of PBS was added to the suspension cell upflow meter. The flow meter is a Beckmann coulter (MoFloastros EQ) ultra-high-speed flow cell sorting system. Fluorescent-labeled antibody staining: 9 analysis tubes were set, 1-7 tubes were added with the corresponding single fluorescently labeled antibody, 9 th tube was added with 7 mixed fluorescently labeled antibodies, 8 th tube was a blank tube with only cells, and the sample tube was stained as 9 th tube. Wherein the fluorescent label comprises IgD-FITC, CD19-ECD, CD27-PC7, CD38-APC A750, igM-PB and CD45-KO fluorescent antibodies.
TABLE 1 memory of the information table of the combination of fluorescent-labeled antibodies in each tube of B cell sorting
Analysis tube group Group of Antibody and fluorescent label Volume of Fluorescent channel
1 Single dyeing IgD-FITC 10 FL1
2 Single dyeing CD19-ECD 5 FL3
3 Single dyeing 7-AAD 10 FL4
4 Single dyeing CD27-PC7 5 FL5
5 Single dyeing CD38-APC A750 2 FL8
6 Single dyeing IgM-PB 5 FL9
7 Single dyeing CD45-KO 5 FL10
8 Blank group No antibody is added - -
9 Sample group 7-antibody mixed solution - -
1.3 sorting of memory B cells: sample tubes were run on-machine to circl live CD45 positive leukocytes according to 7-AAD and CD45 markers and B cells according to CD 19. IgD-IgM-CD27+CD38low populations were defined as memory B cells in IgM and IgD dian B cell populations, and memory B cells were sorted into 96 well cell culture plates containing cell culture broth, 100 cells/well.
EXAMPLE 2 memory B cell culture and screening of culture supernatant antibody
Cell growth factors CpG, IL21, IL2, and B95.8 cell culture supernatant containing EBV were added to a 96-well cell culture plate containing memory B cells and cultured for 7-10 days. The B cell culture supernatants were screened for the presence of antibodies to SARS-CoV-2N protein by capture ELISA.
EXAMPLE 3 cloning of antibodies
3.1cDNA Synthesis: for screening out B cells in the supernatant in the presence of antibodies to SARS-CoV-2N protein, RNA was extracted and reverse transcribed into cDNA (cDNA Synthesis kit SuperScript III First Strand Synthesis System, invitrogen, # 18080051).
3.2 nested PCR amplification of antibody heavy and light chain variable region genes: the PCR primer sequences and specific PCR amplification processes are described in the references: j Immunol methods.20088 Jan 1;329 (1-2):112-24.. The reaction used Phusion super fidelity DNA polymerase (Phusion High-Fidelity PCR Master Mix with GC Buffer, NEB, #M0532 s).
3.3 construction of antibody plasmids: PCR products of the antibody heavy and light chain variable regions were cloned into antibody expression vectors (p 19eH-mIgG1-Zeo and p14B7K-Hygp, or A32L-Hyg) containing human IgG1 constant regions by cleavage (VH, ageI/SalI, VK, ageI/Xhol, VL, ageI/BsiWI). And (3) carrying out IMGT/V-Quest analysis on the detected heavy chain and light chain variable region gene sequences to obtain specific monoclonal antibody N3, N5, N31, N83 and 3B7 sequences of the targeted N protein.
Experimental results: FIG. 1 is a graph showing the genetic profile of five N-protein specific monoclonal antibodies N3, N5, N31, N83 and 3B7. The 3 antibodies of N3, N5 and N31 have the same heavy chain allele (IGHV 1-69), but the sequences and lengths of their complementarity determining regions (CDR 3 regions) are different; the heavy chain alleles of the 3-strain antibodies were paired with different light chains KV4-1, LV1-51 and KV3-15, respectively. N83 is paired with light chain allele KV2-28 by heavy chain allele IGHV4-61, and 3B7 is paired with light chain allele KV3-20 by heavy chain allele VH 1-24.
Example 4 characterization of antibodies
4.1 production of antibodies: cotransfecting 293F cells with heavy chain and light chain plasmids of the antibody subjected to sequence verification, and harvesting culture supernatant containing the antibody after 7 days;
the experimental procedure for cotransfecting 293F cells with the heavy and light chain plasmids of the sequence verified antibody is as follows: (1) cell count: 293F cell density was 3-6X10 6 cells/mL; (2) The transfection required the amount of heavy and light chain plasmids (. Mu.g) to be calculated as 1.5 times the transfection volume (mL) and the amount of transfection reagent PEI (. Mu.l) was 3 times the amount of plasmid used. PEI and 3mL 293T GE were incubatedMixing the base (Acro biosystems, #CF-116-12), standing for 5min, mixing with plasmid and 293T GE system, standing for 15-20min, dripping into 293F cells in the form of water drop, and standing for 37deg.C 5% CO 2 Shake cultivation in an incubator; (3) The next day, 1/10 of the transfection volume of CD feed X-supplemented solution (Acro biosystems, #CF-116-12) was added and the incubation was continued at 37℃with 5% CO 2 Shake cultivation in an incubator; on day seven, the cell suspension was collected, centrifuged at 3000rcf for 20min, filtered through a 0.22 μm filter cartridge and purified.
4.2 purification of antibodies: the antibody-containing culture supernatant was subjected to protein A affinity column purification, and the protein content was determined.
4.3 antibody affinity assay: affinity (KD) assays for binding of antibodies to the novel coronavirus (SARS-CoV-2) N protein employ the biological membrane interferometry technique (Bio-Layer Interferometry, BLI). The protein interactor used was Octet K2 (available from ForteBio Inc. of U.S.A.). The specific method comprises the following steps: streptavidin (Streptavidin Biosensor, SA) sensor was coupled to a biotin-labeled capture antibody, capturing SARS-CoV-2N protein in the supernatant. And adding the serial diluted antibody to be tested. Binding and dissociation curves for the antibodies were obtained and their KD values were calculated using instrument Analysis software (Data Analysis 11.0).
Experimental results: the experimental results of affinity analysis of the 5-strain N-protein specific monoclonal antibody are shown in fig. 2 and 3, wherein fig. 2 is a graph showing the binding and dissociation curves of the 5-strain N-protein specific monoclonal antibody and N protein, and antibodies N3, N5 and N31 all show high affinity to N protein, K D The value was below 1pM. Antibodies N83 and 3B7 also showed high affinity for the N protein, but were relatively low, K, compared to the three N3, N5 and N31 antibodies D The values were 1.76nM and 1.94nM, respectively.
4.4 epitope analysis of antibodies: a series of truncated SARS-CoV-2N protein expression plasmids were first constructed, including N (1-174), N (1-364), N (46-174, i.e., NTD), N (245-364, i.e., CTD), and N (245-419). The expression plasmid is transiently transfected into 293T cells to obtain the corresponding cell culture supernatant containing the target expression protein. ELISA was used to determine the binding of the 5 antibodies to the different truncated N proteins.
Experimental results: the results of epitope analysis experiments on 5N-protein specific monoclonal antibodies are shown in FIG. 4, where FIG. 4A shows a set of subdomains and fragments covering the entire N-protein sequence, and binding of antibodies to these truncated fragments was determined using ELISA, and FIG. 4B shows that these five N-specific antibodies are classified into two classes, N3, N5 and N31 binding to the CTD domain, and N83 and 3B7 binding selectively to the NTD domain.
4.5 antibody cross-reacts with other human coronavirus N protein: ELISA was used to determine the binding of the 5-strain antibody to other coronavirus N proteins. Other human coronavirus N proteins include SARS virus, MERS virus, human coronavirus OC43 and HKU1.
Experimental results: the cross reaction experimental result diagram of the 5 strain N protein specific monoclonal antibody and other human coronavirus N proteins is shown in the figure 5, and the experimental result shows that the obtained 5 strain antibodies are cross-combined with SARS virus N proteins, but do not cross-react with MERS virus, human coronavirus OC43 and HKU 1N proteins.
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.

Claims (10)

1. An isolated antibody or antigen binding fragment thereof, which specifically binds to SARS-CoV-2 coronavirus N protein, said antibody being one selected from the group consisting of N3, N5, N31, N83 and 3B 7; the antibody or antigen binding fragment thereof comprises a heavy chain hypervariable region and a light chain hypervariable region in an N3, N5, N31, N83, or 3B7 antibody;
wherein the N3, N5, N31, N83, or 3B7 antibody comprises heavy chain hypervariable regions CDR1, CDR2, and CDR3, and light chain hypervariable regions CDR1, CDR2, and CDR3;
the amino acid sequence of a heavy chain hypervariable region CDR1 of the N3 antibody is shown as SEQ ID NO.11, the amino acid sequence of a CDR2 is shown as SEQ ID NO.12, and the amino acid sequence of a CDR3 is shown as SEQ ID NO. 13; the amino acid sequence of a CDR1 of a light chain hypervariable region of the N3 antibody is shown as SEQ ID NO.14, the amino acid sequence of a CDR2 is shown as SEQ ID NO.15, and the amino acid sequence of a CDR3 is shown as SEQ ID NO. 16;
the amino acid sequence of a heavy chain hypervariable region CDR1 of the N5 antibody is shown as SEQ ID NO.17, the amino acid sequence of a CDR2 is shown as SEQ ID NO.18, and the amino acid sequence of a CDR3 is shown as SEQ ID NO. 19; the amino acid sequence of a CDR1 of a light chain hypervariable region of the N5 antibody is shown as SEQ ID NO.20, the amino acid sequence of a CDR2 is shown as SEQ ID NO.21, and the amino acid sequence of a CDR3 is shown as SEQ ID NO. 22;
the amino acid sequence of a heavy chain hypervariable region CDR1 of the N31 antibody is shown as SEQ ID NO.23, the amino acid sequence of a CDR2 is shown as SEQ ID NO.24, and the amino acid sequence of a CDR3 is shown as SEQ ID NO. 25; the amino acid sequence of a CDR1 of a light chain hypervariable region of the N31 antibody is shown as SEQ ID NO.26, the amino acid sequence of a CDR2 is shown as SEQ ID NO.27, and the amino acid sequence of a CDR3 is shown as SEQ ID NO. 28;
the amino acid sequence of a heavy chain hypervariable region CDR1 of the N83 antibody is shown as SEQ ID NO.29, the amino acid sequence of a CDR2 is shown as SEQ ID NO.30, and the amino acid sequence of a CDR3 is shown as SEQ ID NO. 31; the amino acid sequence of a CDR1 of a light chain hypervariable region of the N83 antibody is shown as SEQ ID NO.32, the amino acid sequence of a CDR2 is shown as SEQ ID NO.33, and the amino acid sequence of a CDR3 is shown as SEQ ID NO. 34;
the amino acid sequence of a CDR1 of a heavy chain hypervariable region of the 3B7 antibody is shown as SEQ ID NO.35, the amino acid sequence of a CDR2 is shown as SEQ ID NO.36, and the amino acid sequence of a CDR3 is shown as SEQ ID NO. 37; the amino acid sequence of CDR1 of the light chain hypervariable region of the 3B7 antibody is shown as SEQ ID NO.38, the amino acid sequence of CDR2 is shown as SEQ ID NO.39, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 40;
the amino acid sequence of the heavy chain variable region of the N3 antibody comprises a sequence shown as SEQ ID NO.1, and the amino acid sequence of the light chain variable region of the N3 antibody comprises a sequence shown as SEQ ID NO. 2;
the amino acid sequence of the heavy chain variable region of the N5 antibody comprises a sequence shown as SEQ ID NO.3, and the amino acid sequence of the light chain variable region of the N5 antibody comprises a sequence shown as SEQ ID NO. 4;
the amino acid sequence of the heavy chain variable region of the N31 antibody comprises a sequence shown as SEQ ID NO.5, and the amino acid sequence of the light chain variable region of the N31 antibody comprises a sequence shown as SEQ ID NO. 6;
the amino acid sequence of the heavy chain variable region of the N83 antibody comprises a sequence shown as SEQ ID NO.7, and the amino acid sequence of the light chain variable region of the N83 antibody comprises a sequence shown as SEQ ID NO. 8;
the amino acid sequence of the heavy chain variable region of the 3B7 antibody comprises a sequence shown as SEQ ID NO.9, and the amino acid sequence of the light chain variable region of the 3B7 antibody comprises a sequence shown as SEQ ID NO. 10.
2. The antibody or antigen-binding fragment thereof of claim 1, wherein the N3, N31, N83, or 3B7 antibody further comprises a heavy chain constant region comprising the amino acid sequence shown in SEQ ID NO41 and a light chain constant region comprising the amino acid sequence shown in SEQ ID No. 42.
3. The antibody or antigen-binding fragment thereof of claim 1, wherein the N5 antibody further comprises a heavy chain constant region comprising the amino acid sequence shown in SEQ ID NO41 and a light chain constant region comprising the amino acid sequence shown in SEQ ID NO 43.
4. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody.
5. The antibody or antigen-binding fragment thereof of claim 1, wherein the N3, N5, N31, N83 or 3B7 antibodies each specifically bind to the SARS virus N protein antigen but do not cross-react with MERS virus, human coronavirus OC43 and HKU 1N protein antigens.
6. An isolated polynucleotide encoding an antibody or antigen-binding fragment thereof according to any one of claims 1-5.
7. An isolated vector comprising the polynucleotide of claim 6.
8. A host cell comprising the polynucleotide of claim 6 or the vector of claim 7.
9. A method of expressing an antibody or antigen-binding fragment thereof according to any one of claims 1-5, comprising culturing the host cell of claim 8 under conditions suitable for expression of the antibody or antigen-binding fragment thereof, and optionally recovering the antibody or antigen-binding fragment thereof according to any one of claims 1-5 from the host cell or from the culture medium.
10. Use of the antibody or antigen binding fragment thereof according to any one of claims 1-5, the polynucleotide of claim 6, the vector of claim 7, the host cell of claim 8 or the method of claim 9 in the preparation of a reagent for detecting SARS virus N protein.
CN202310133633.9A 2023-02-16 2023-02-16 Antibodies or functional fragments thereof that specifically bind to N protein and uses thereof Pending CN116514962A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202310133633.9A CN116514962A (en) 2023-02-16 2023-02-16 Antibodies or functional fragments thereof that specifically bind to N protein and uses thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202310133633.9A CN116514962A (en) 2023-02-16 2023-02-16 Antibodies or functional fragments thereof that specifically bind to N protein and uses thereof

Publications (1)

Publication Number Publication Date
CN116514962A true CN116514962A (en) 2023-08-01

Family

ID=87389280

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202310133633.9A Pending CN116514962A (en) 2023-02-16 2023-02-16 Antibodies or functional fragments thereof that specifically bind to N protein and uses thereof

Country Status (1)

Country Link
CN (1) CN116514962A (en)

Similar Documents

Publication Publication Date Title
Guthmiller et al. An efficient method to generate monoclonal antibodies from human B cells
DK2021463T3 (en) Culture method to obtain a cloned population of antigen-specific B cells
RU2666915C2 (en) Antibodies to interleukin-1alpha, and application methods thereof
US10766949B2 (en) Method for preparing whole bovine-derived broadly neutralizing antibody against serotype O foot-and-mouth disease virus
CN113150129B (en) Single-chain antibody for resisting S2 protein on surface of new coronavirus SARS-CoV-2 and application thereof
US20160033504A1 (en) Protocol for identifying and isolating antigen-specific b cells and producing antibodies to desired antigens
WO2023216826A1 (en) Monoclonal antibody a38 against rift valley fever virus and use
TW201632621A (en) Method of sorting hybridoma cell having antigen specificity
Waltari et al. Functional enrichment and analysis of antigen-specific memory B cell antibody repertoires in PBMCs
JP2014528001A (en) Identification of antigen-specific adaptive immune responses using arm PCR and high-throughput sequencing
CN115386007A (en) anti-GPRC 5D antibody, preparation method and application thereof
Voigt et al. Therapeutic antibody discovery in infectious diseases using single-cell analysis
CN110256562B (en) PD-1 nano antibody, preparation method and application thereof
CN109503712B (en) Monoclonal antibody ZKns2E11 and application thereof
CN116514962A (en) Antibodies or functional fragments thereof that specifically bind to N protein and uses thereof
CN115947838A (en) Fully human-derived new coronavirus variant potent neutralizing antibody H4B6 and application thereof
CN109535249B (en) Monoclonal antibody ZKns3G2 and application thereof
CN109553680B (en) Monoclonal antibody ZKns4B8 and application thereof
US11946074B2 (en) Methods and compositions for inducible extracellular membrane capture of monoclonal immunoglobulins secreted by hybridomas
CN110702913A (en) Monoclonal antibody composition for quantitatively detecting coxiella burnetii I strain
Aartse et al. Primary antibody response after influenza virus infection is first dominated by low-mutated HA-stem antibodies followed by higher-mutated HA-head antibodies
CN107540747B (en) Anti-human DLL4 monoclonal antibody 6F12
CN116496395B (en) Monoclonal antibody combined with Dsg3 and application thereof
CN116003606B (en) TIGIT nano antibody and preparation method and application thereof
Zhai et al. Monoclonal Antibody Development Technology for Important Human Diseases

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination