CN109957013B - Fully human monoclonal antibody 7O2 for resisting H7N9, and preparation method and application thereof - Google Patents

Fully human monoclonal antibody 7O2 for resisting H7N9, and preparation method and application thereof Download PDF

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CN109957013B
CN109957013B CN201711338976.XA CN201711338976A CN109957013B CN 109957013 B CN109957013 B CN 109957013B CN 201711338976 A CN201711338976 A CN 201711338976A CN 109957013 B CN109957013 B CN 109957013B
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antibody
variable region
monoclonal antibody
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CN109957013A (en
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万晓春
李俊鑫
刘绿艳
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Shenzhen Advanced Science And Technology Cci Capital Ltd
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Shenzhen Institute of Advanced Technology of CAS
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1018Orthomyxoviridae, e.g. influenza virus
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983Viruses
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577Immunoassay; 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
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    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/20Immunoglobulins specific features characterized by taxonomic origin
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    • C07K2317/00Immunoglobulins specific features
    • C07K2317/50Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565Complementarity determining region [CDR]
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    • C07K2317/00Immunoglobulins specific features
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    • C07ORGANIC CHEMISTRY
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    • C07K2317/92Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Abstract

The invention relates to a fully human monoclonal antibody 7O2 for resisting H7N9, a preparation method and application thereof. The heavy and light chain of this antibody 7O2 has 6 CDR regions. The antibody 7O2 can be combined with hemagglutinin HA of H7N9 virus in a targeted mode, and can be applied to virus detection of influenza patients.

Description

Fully human monoclonal antibody 7O2 for resisting H7N9, and preparation method and application thereof
Technical Field
The invention relates to a fully human monoclonal antibody against H7N9, a preparation method and application thereof, in particular to a fully human monoclonal antibody against H7N9, 7O2 and a preparation method and application thereof, belonging to the technical field of immunology.
Background
Six of the ten well-sold drugs worldwide in 2015 were all human or humanized monoclonal antibody drugs. The first one is Humira, a fully human monoclonal antibody, which is a monoclonal antibody and is sold in 100 hundred million over 3 years. Since the first monoclonal antibody drug was marketed in 1986, the monoclonal antibody drugs underwent the stages of murine monoclonal antibody drug (Orthoclone OKT3), chimeric monoclonal antibody drug (Rituximab), humanized monoclonal antibody drug (Herceptin), and fully human monoclonal antibody drug (Humira). Because human bodies have anti-mouse antibody reaction (HAMA), murine monoclonal antibody drugs and chimeric monoclonal antibody drugs are gradually eliminated, and the monoclonal antibody drugs occupying the market at present are all humanized monoclonal antibody drugs. Compared with the internationally advanced human antibody production technology, Shenzhen and even China have great gap, mainly manifested in the weak innovation ability of the human antibody drug field, few varieties of independent research and development, no report of the market of original humanized monoclonal antibody drug exists at present, and the huge antibody drug market is occupied by foreign drug enterprises. China changes the lagging situation and strives for antibody drug markets with huge consumption potential at home and abroad, and the humanized monoclonal antibody technology needs to be overcome urgently.
The humanized antibody refers to the antibody gene sequence completely derived from the human antibody gene sequence. The humanized antibody has high specificity, less side effect and high disease preventing and treating effect, and is the main development direction of monoclonal antibody medicine in future. The current common techniques for preparing human monoclonal antibodies mainly comprise phage display technology and single B cell PCR technology. The humanized monoclonal antibody prepared by the phage display technology has the advantages of low production cost and no complicated work such as immunization, cell fusion and the like. However, the disadvantage is also obvious, and the antibody obtained from the non-immune antibody library is often insufficient in affinity, limited by the conversion rate of foreign genes, insufficient in library capacity of the antibody library to cover the antibody diversity of animals, and the like. In recent years, the emerging single B cell PCR technology refers to the separation of B cells secreting functional antibodies from the blood of patients, then RNA extraction and cDNA synthesis, cloning of the genes secreting the target antibodies, and finally recombination and expression of fully human monoclonal antibodies. The technology is simple and quick to operate, the produced humanized antibody has high affinity and specificity, and the technology of separating the monoclonal antibody with the virus neutralizing function or the tumor killing function from the memory B cell which is improved recently greatly reduces the complicated operation and cost. The technology of preparing humanized monoclonal antibodies by single B cells is a development trend of developing humanized monoclonal antibodies.
The human monoclonal antibody has high specificity and obvious curative effect on inflammation, cancer, especially influenza. Influenza is an infectious disease caused by influenza virus, and seriously threatens human health. About 10 million people worldwide are infected with seasonal influenza virus each year, with 25-50 million people dying. The H7N9 virus is an influenza virus, has drug resistance to traditional antiviral drugs amantadine and rimantadine, and has no effective treatment means at present. The H7N9 virus needs to be bound with a receptor on a human cell by a specific molecule expressed by the virus itself when invading the cell, so that the cell can be infected and further amplified. The human antibody for neutralizing the virus is a certain specific antibody generated by human B lymphocytes, can be combined with the antigen on the surface of the virus, thereby preventing the virus from adhering to a target cell receptor, preventing the virus from invading cells and effectively preventing and treating the H7N9 influenza.
Therefore, it is of great significance to provide fully human monoclonal antibodies against H7N9 and methods for their preparation.
Disclosure of Invention
One of the purposes of the present invention is to provide an anti-H7N 9 fully human monoclonal antibody 7O2 or a biologically active fragment derived from the monoclonal antibody and capable of specifically binding to H7N 9.
Another objective of the invention is to provide a gene encoding the anti-H7N 9 fully human monoclonal antibody 7O2 or a biologically active fragment derived from the monoclonal antibody and capable of specifically binding to H7N9, and a vector or a cell containing the gene.
Another object of the present invention is to provide a method for producing said anti-H7N 9 fully human monoclonal antibody 7O 2.
Another objective of the invention is to provide a pharmaceutical composition comprising the anti-H7N 9 fully human monoclonal antibody 7O2 or a biologically active fragment derived from the monoclonal antibody and capable of specifically binding to H7N 9.
The invention also aims to provide application of the anti-H7N 9 fully human monoclonal antibody 7O2 or a bioactive fragment which is derived from the monoclonal antibody and can specifically bind to H7N9 or the pharmaceutical composition.
The invention also aims to provide a kit for detecting the H7N9 virus.
In order to achieve the above objects, the present invention provides an anti-H7N 9 fully human monoclonal antibody or a biologically active fragment derived from the monoclonal antibody and capable of specifically binding to H7N 9. The antibody is named 7O2 in the invention.
According to a specific embodiment of the present invention, the antibody of the present invention has a heavy chain variable region and a light chain variable region each having 3 Complementarity Determining Regions (CDRs), wherein:
the amino acid sequence of CDR1 of the heavy chain variable region is: GFTFSSYW (SEQ ID NO:5),
the amino acid sequence of CDR2 of the heavy chain variable region is: INSDGSST (SEQ ID NO:6),
the amino acid sequence of CDR3 of the heavy chain variable region is: ARRSKTGKFDP (SEQ ID NO:7),
the amino acid sequence of CDR1 of the light chain variable region is: ALPKQY (SEQ ID NO:8),
the amino acid sequence of CDR2 of the light chain variable region is: the KDS is a set of instructions for the KDS,
the amino acid sequence of CDR3 of the light chain variable region is: QSADSSGTLEV (SEQ ID NO: 9).
According to a particular embodiment of the invention, the heavy chain of the antibody of the invention is represented by SEQ ID NO 2.
According to a particular embodiment of the invention, the light chain of the antibody of the invention is represented by SEQ ID NO 4.
The invention also provides a polynucleotide encoding the heavy chain variable region and/or the light chain variable region of the antibody of the invention, or encoding the heavy chain and/or the light chain of the antibody. Preferably, the polynucleotide comprises at least one of the following sequences:
1, 3 and 10 to 14 SEQ ID NOs.
According to a particular embodiment of the invention, SEQ ID NO 1 is a polynucleotide encoding SEQ ID NO 2; SEQ ID NO. 3 is a polynucleotide encoding SEQ ID NO. 4; SEQ ID NO. 10 is a polynucleotide encoding SEQ ID NO. 5; SEQ ID NO. 11 is a polynucleotide encoding SEQ ID NO. 6; 12 is a polynucleotide encoding SEQ ID NO. 7; 13 is a polynucleotide encoding SEQ ID NO. 8; SEQ ID NO. 14 is a polynucleotide encoding SEQ ID NO. 9. The polynucleotide sequence encoding the amino acid sequence KDS of CDR2 of the light chain variable region may be: AAAGACAGT are provided.
The invention also provides vectors comprising the polynucleotides of the invention.
The invention also provides cells comprising a polynucleotide of the invention or a vector of the invention.
The invention also provides a method for producing the anti-H7N 9 fully human monoclonal antibody 7O2 or a bioactive fragment which is derived from the monoclonal antibody and can specifically bind to H7N9, wherein the method is used for preparing the anti-H7N 9 fully human monoclonal antibody 7O2 by adopting a single B cell method.
In the prior art, a method for preparing an anti-H7N 9 virus humanized monoclonal antibody by adopting a phage display technology exists, although the method has the advantages of low production cost and no complicated work such as immunization, cell fusion and the like, the method has obvious defects, and the antibody obtained from a non-immune antibody library is often insufficient in affinity, limited by the conversion rate of an exogenous gene, insufficient in library capacity of the antibody library to cover the antibody diversity of animals and the like. The invention adopts single B cell PCR technology to separate B cells secreting functional antibodies from blood of a patient, then extracts RNA and synthesizes cDNA, clones genes secreting target antibodies from the RNA and the cDNA, and finally recombines and expresses fully human monoclonal antibodies. The technology is simple and quick to operate, the produced humanized antibody has high affinity and specificity, and in addition, the technology of the improved monoclonal antibody with the virus neutralizing or tumor killing function separated from the memory B cells can be further adopted, so that the complicated operation and cost are greatly reduced.
The invention also provides a pharmaceutical composition, which comprises the anti-H7N 9 fully human monoclonal antibody 7O2 or a bioactive fragment which is derived from the monoclonal antibody and can specifically bind to H7N 9.
The invention also provides application of the anti-H7N 9 fully human monoclonal antibody 7O2 or a bioactive fragment which is derived from the monoclonal antibody and can specifically bind to H7N9, or application of the pharmaceutical composition in preparing a medicament for treating diseases caused by H7N9 virus.
The invention also provides a kit for detecting the H7N9 virus level, which contains the anti-H7N 9 fully human monoclonal antibody 7O2 or a bioactive fragment which is derived from the monoclonal antibody and can be specifically bound with H7N 9; preferably, the kit further comprises a second antibody and an enzyme or fluorescent or radioactive label for detection, and a buffer; preferably, the second antibody is an anti-antibody against monoclonal antibody 7O2 of the present invention.
Compared with the prior art, the invention has the following beneficial effects:
(1) the H7N 9-resistant fully-humanized monoclonal antibody 7O2 can be combined with hemagglutinin HA of H7N9 virus in a targeted mode, and can be applied to virus detection of influenza patients.
(2) Compared with the mouse antibody, the gene of the fully human antibody is completely derived from the human gene, has no components of other species, does not generate toxic and side effects such as anti-mouse antibody and the like in a human body, has better biocompatibility, and is more suitable and has more potential to become a macromolecular medicament for treating influenza virus.
(3) Compared with the method for preparing the H7N9 virus-resistant human monoclonal antibody by using the phage display technology provided by the prior art, the method for developing the H7N 9-resistant antibody by using the single B cell has the advantages of simple and rapid operation, high affinity and specificity of the produced human antibody and the like.
Drawings
FIG. 1 is a graph showing the results of flow-based assay of example 1 in which NTH-3T3 expresses CD 40L.
FIG. 2 is a graph showing the results of sorting memory B cells by flow cytometry in example 1.
FIG. 3 is a graph showing the results of ELISA experiments in example 1.
FIG. 4 is a graph showing the result of Western blot agarose gel electrophoresis in example 2.
FIG. 5 is a schematic sequence diagram of the heavy chain variable region and the light chain variable region of 7O 2.
Detailed Description
For a more clear understanding of the technical features, objects and advantages of the present invention, reference is now made to the following detailed description of the embodiments of the present invention taken in conjunction with the accompanying drawings, which are included to illustrate and not to limit the scope of the present invention. In the examples, each raw reagent material is commercially available, and the experimental method not specifying the specific conditions is a conventional method and a conventional condition well known in the art, or a condition recommended by an instrument manufacturer.
Example 1
(1) Construction of NTH-3T3 cell line stably expressing CD40L
Lentivirus was used to establish 3T3-CD40L feeder cells. Constructing a lentivirus expression vector pLVX-CD40L, transfecting 293T cells, and collecting virus supernatant on the fourth day of transfection. NIH-3T3 cells were activated, cultured for 3 passages, infected with lentivirus, cultured further and passaged 3 times. Sorting cells with FITC fluorescence intensity near MFI by using a flow cytometer, adding the cells into a culture bottle again at 37 ℃ and 5% CO2Culturing and detecting in an incubator, wherein the detection result is shown in figure 1,3T3 cells expressing CD40L and 3T3 cells transfected with the empty vector pLVX (with ZxGreen) were stained with anti-CD 40L with APC, respectively, and then analyzed by flow cytometry. As a result, all 3T3-CD40L feeder cells were found to express CD 40L. When the cells grow to 80% -90%, the cells are collected by digestion at a concentration of 1X 10 per ml7A cell. Placing in an irradiator for 5000rads irradiation, and resuspending the cells in the frozen stock solution at a concentration of 3.5 × 10/ml7The cells are packed in 1ml of freezing tubules and frozen in liquid nitrogen (can be stored for 2 years).
(2) Sorting and activation of memory B cells
Isolation and cryopreservation of PBMC from convalescent patients infected with H7N9 virus using lymph isolate, 10-50X 10 per tube6Cells, frozen in liquid nitrogen tank. A PBMC flow staining solution was prepared, the composition of which is shown in table 1 below:
table 1: PBMC (Poly-beta-phenylene PBMC) flow type staining solution
Figure GDA0003098626330000051
Figure GDA0003098626330000061
Thawing PBMC, adding the above PBMC flow staining solution and sorting on flow cytometer, and sorting out CD19 as shown in FIG. 2+IgMIgAIgDThe purity of the memory B cells is required to be more than 90%, and if the purity of the memory B cells is less than 90%, the sorting process is repeated.
A mixed B-cell activating medium was prepared, the composition of which is shown in table 2 below:
TABLE 2
Components Volume of
Complete IMDM Medium 336mL
IL-2(10,000U mL-1) 3.5mL
IL-21(100μg mL-1) 175μL
3T3-CD40L 10mL
Adding memory B cells into mixed culture medium, mixing, diluting in 384-well plate with 1 cell per well and 50ul volume, standing at 37 deg.C and 5% CO2And (5) standing and culturing in an incubator. After 13 days, the supernatant was subjected to ELISA.
(3) Obtaining human monoclonal antibody 7O2 against H7N9 virus
The influenza virus hemagglutinin HA is a virus envelope surface columnar antigen, can be combined with a plurality of erythrocyte receptors such as human, chicken, guinea pig and the like to cause erythrocyte agglutination, HAs immunogenicity, and can neutralize influenza virus by an anti-hemagglutinin antibody. In the present invention, B cells secreting antibody 7O2 binding to H7N9 virus were found by ELISA, and the secreted human monoclonal antibody 7O2 could target hemagglutinin HA binding to H7N9 virus (fig. 3).
The specific operation of the ELISA experiment is as follows:
(1) coating 100ng/100 ul of HA protein of H7N9 virus in a 96-well enzyme label plate, wherein each well is 100 ul;
(2) standing in a 4-degree refrigerator overnight;
(3) washing with PBST solution for three times, adding 5% skimmed milk powder solution 200 μ l to each well, and incubating at 37 deg.C for 1 hr;
(4) washing with PBST solution for three times, adding 100ul of normal human serum without virus infection (negative control) or patient serum infected with virus or anti-H7N 9 fully human monoclonal antibody, each repeating for three times;
(5) after incubation for 1 hour at 37 degrees, washed three times with PBST solution;
(6) diluting an anti-human IgG antibody with HRP at a ratio of 1:5000, and adding the diluted anti-human IgG antibody into an enzyme label plate, wherein each hole is 100 mu l;
(7) after incubation for 1 hour at 37 degrees, washed three times with PBST solution;
(8) adding 100 mul of TMB substrate solution into each hole, and keeping the temperature at 37 ℃ for 5 minutes;
(9) the stop solution 2M sulfuric acid 100. mu.l was added to each well, and the absorbance was immediately measured at a wavelength of 450nm in a microplate reader. As shown in FIG. 3, ELISA experiments showed that the human monoclonal antibody 7O2 obtained by the present invention can target hemagglutinin HA binding to H7N9 virus.
EXAMPLE 2 cloning, recombination and expression of humanized monoclonal antibody 7O2 Gene
The B cells capable of secreting antibody 7O2 that binds to H7N9 virus obtained in example 1 were lysed, and the lysate was subjected to reverse transcription of RNA to obtain PCR template cDNA of human antibody gene. The genes of the heavy chain and the light chain of the antibody are cloned by taking the cDNA as a template, and are recombined and expressed and purified in the eukaryotic cell 293F or HEK 293.
Specifically, the method comprises the following steps:
(1) the B cell fluid was transferred to a 96-well plate (Eppendorf, 030133366).
(2) Reverse transcription system: 150ng random primer (Invitrogen,48190-
Figure GDA0003098626330000071
III reverse transcriptase (Invitrogen,18080-044), DEPC water was supplied to 14. mu.l/well.
(3) Reverse transcription reaction procedure: 42 ℃ for 10 min; at 25 ℃ for 10 min; 50 ℃ for 60 min; 94 ℃ for 5 min.
(4) The cDNA was stored at-20 ℃.
(5) Using KOD-Plus-Neo (TOYOBO, KOD401) testKit PCR amplified heavy and light chains of antibody genes, respectively, 40 μ L system: 3.5. mu.L of cDNA, 20nM mixed primer, 4. mu.L of buffer (buffer), 4. mu.L of 2mM dNTPs, 2.4. mu.L of MgSO 24,1μL KOD。
(6) Reaction procedure: 94 ℃ for 2 min; 45 cycles: [98 ℃,10 s; 58 ℃ (IgH/Ig kappa) or 60 ℃ (Ig lambda), 30 s; 68 ℃ for 28s (1)stPCR) or 23s (2)nd PCR)]。
(7) The result of subjecting the amplification product to agarose gel analysis is shown in FIG. 4, which shows that the antibody light chain variable region is kappa and 354bp in size, and the heavy chain variable region is 327bp in size.
(8) The sequencing results of the antibody gene PCR products were as follows:
7O2 (SEQ ID NO: 1):
GAGGTGCAGCTGGTGGAGTCCGGGGGAGGCTTAGTTCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTACTGGATGCA
CTGGGTCCGCCAAGCTCCAGGGAAGGGGCTGGTGTGGGTCTCACGTATTAATAG
TGATGGGAGTAGCACAAGCTACGCGGACTCCGTGAAGGGCCGATTCACCATCTC
CAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAGAGCCG
AGGACACGGCTGTGTATTACTGTGCAAGAAGGAGTAAAACGGGGAAGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA
7O2 (SEQ ID NO: 3):
TCCTATGAGCTGACACAGCCACCCTCGGTGTCAGTGTCCCCAGGACAGACGGCCAGGATCACCTGCTCTGGAGATGCATTGCCAAAGCAATATGCTTATTGGTAC
CAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTGATATATAAAGACAGTGAGAG
GCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAGCTCAGGGACAACAGTCA
CGTTGACCATCAGTGGAGTCCAGGCAGAAGACGAGGCTGACTATTACTGTCAAT
CAGCAGACAGCAGTGGTACCCTAGAGGTGTTCGGCGGAGGGACCAAGCTGACC GTCCTAGTA
(9) the H gene and pcDNA3.1 are respectively subjected to BamH1/EcoR1 double enzyme digestion and then connected to form pcDNA3.1-H vector.
(10) The L gene and pcDNA3.1 were digested separately with Not1/Xho1 and ligated to form pcDNA3.1-L vector.
(11) 293F cells were cultured.
(12)20ug of pcDNA3.1-L vector and 10ug of pcDNA3.1-H vector were co-transfected into 293F cells and cultured for 96 hours.
(13) Taking the supernatant to perform ELISA (ABC is the supernatant, DEF is the positive control, GH is the negative control) and western blot; the results of the ELISA experiments are shown in Table 3 below:
TABLE 3
Data of 450 Data of 450
A 3.0025 E 1.2087
B 3.1215 F 1.1470
C 2.9562 G 0.0321
D 1.1463 H 0.1001
The above results show that the supernatant contains antibody 7O2 capable of binding H7N9 virus.
The Western blot experiment comprises the following specific processes:
running protein denaturation electrophoresis with supernatant, blocking with 5% skimmed milk powder solution for 1 hr after membrane conversion, then incubating with goat anti-human IgG antibody with HRP for 1 hr, and finally adding display substrate for exposure. The results of the experiment are shown in FIG. 4, and FIG. 4 shows the heavy and light chains of the fully human antibody, indicating that the supernatant contains the fully human monoclonal antibody 7O2 against H7N9 virus.
For monoclonal antibody 7O2 of this example, SEQ ID NO:2 and SEQ ID NO:4 are the VH and VL amino acid sequences of the heavy chain variable region and light chain variable region of 7O2, respectively, obtained in this example. Referring to fig. 5, the heavy chain variable region and the light chain variable region each have 3 Complementarity Determining Regions (CDRs) numbered CDR1, CDR2, CDR3, wherein: heavy chain: CDR 1: GFTFSSYW (SEQ ID NO: 5); CDR 2: INSDGSST (SEQ ID NO: 6); CDR 3: ARRSKTGKFDP (SEQ ID NO: 7). Light chain: CDR 1: ALPKQY (SEQ ID NO: 8); CDR 2: KDS; CDR 3: QSADSSGTLEV (SEQ ID NO: 9).
Finally, the description is as follows: although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that: modifications and equivalents may be made thereto without departing from the spirit and scope of the invention and it is intended to cover any modifications or equivalents as may fall within the scope of the invention.
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20 25 30
tat tgg tac cag cag aag cca ggc cag gcc cct gtg ctg gtg ata tat 144
Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr
35 40 45
aaa gac agt gag agg ccc tca ggg atc cct gag cga ttc tct ggc tcc 192
Lys Asp Ser Glu Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser
50 55 60
agc tca ggg aca aca gtc acg ttg acc atc agt gga gtc cag gca gaa 240
Ser Ser Gly Thr Thr Val Thr Leu Thr Ile Ser Gly Val Gln Ala Glu
65 70 75 80
gac gag gct gac tat tac tgt caa tca gca gac agc agt ggt acc cta 288
Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Ala Asp Ser Ser Gly Thr Leu
85 90 95
gag gtg ttc ggc gga ggg acc aag ctg acc gtc cta gta 327
Glu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Val
100 105
<210> 4
<211> 109
<212> PRT
<213> Artificial sequence ()
<220>
<223> Synthetic Construct
<400> 4
Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln
1 5 10 15
Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala Leu Pro Lys Gln Tyr Ala
20 25 30
Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr
35 40 45
Lys Asp Ser Glu Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser
50 55 60
Ser Ser Gly Thr Thr Val Thr Leu Thr Ile Ser Gly Val Gln Ala Glu
65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Ala Asp Ser Ser Gly Thr Leu
85 90 95
Glu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Val
100 105
<210> 5
<211> 8
<212> PRT
<213> Artificial sequence ()
<220>
<223> VH-CDR1
<400> 5
Gly Phe Thr Phe Ser Ser Tyr Trp
1 5
<210> 6
<211> 8
<212> PRT
<213> Artificial sequence ()
<220>
<223> VH-CDR2
<400> 6
Ile Asn Ser Asp Gly Ser Ser Thr
1 5
<210> 7
<211> 11
<212> PRT
<213> Artificial sequence ()
<220>
<223> VH-CNR3
<400> 7
Ala Arg Arg Ser Lys Thr Gly Lys Phe Asp Pro
1 5 10
<210> 8
<211> 6
<212> PRT
<213> Artificial sequence ()
<220>
<223> VL-CDR1
<400> 8
Ala Leu Pro Lys Gln Tyr
1 5
<210> 9
<211> 11
<212> PRT
<213> Artificial sequence ()
<220>
<223> VL-CDR3
<400> 9
Gln Ser Ala Asp Ser Ser Gly Thr Leu Glu Val
1 5 10
<210> 10
<211> 24
<212> DNA
<213> Artificial sequence ()
<220>
<223> gene encoding VH-CDR1
<400> 10
ggattcacct tcagtagcta ctgg 24
<210> 11
<211> 24
<212> DNA
<213> Artificial sequence ()
<220>
<223> gene encoding VH-CDR2
<400> 11
attaatagtg atgggagtag caca 24
<210> 12
<211> 33
<212> DNA
<213> Artificial sequence ()
<220>
<223> gene encoding VH-CDR3
<400> 12
gcaagaagga gtaaaacggg gaagttcgac ccc 33
<210> 13
<211> 18
<212> DNA
<213> Artificial sequence ()
<220>
<223> gene encoding VL-CDR1
<400> 13
gcattgccaa agcaatat 18
<210> 14
<211> 33
<212> DNA
<213> Artificial sequence ()
<220>
<223> gene encoding VL-CDR3
<400> 14
caatcagcag acagcagtgg taccctagag gtg 33

Claims (12)

1. An anti-H7N 9 fully human monoclonal antibody 7O2 or a biologically active fragment derived therefrom that specifically binds to H7N9, wherein the antibody has a heavy chain variable region and a light chain variable region each having 3 Complementarity Determining Regions (CDRs), wherein:
the amino acid sequence of CDR1 of the heavy chain variable region is: GFTFSSYW (SEQ ID NO:5),
the amino acid sequence of CDR2 of the heavy chain variable region is: INSDGSST (SEQ ID NO:6),
the amino acid sequence of CDR3 of the heavy chain variable region is: ARRSKTGKFDP (SEQ ID NO:7),
the amino acid sequence of CDR1 of the light chain variable region is: ALPKQY (SEQ ID NO:8),
the amino acid sequence of CDR2 of the light chain variable region is: the KDS is a set of instructions for the KDS,
the amino acid sequence of CDR3 of the light chain variable region is: QSADSSGTLEV (SEQ ID NO: 9).
2. The antibody of claim 1, wherein the heavy chain of the antibody is represented by SEQ ID NO 2.
3. The antibody of claim 1 or 2, wherein the light chain of the antibody is represented by SEQ ID NO. 4.
4. A polynucleotide encoding the variable region of the heavy chain and the variable region of the light chain of the antibody of any one of claims 1 to 3, or encoding the heavy chain and the light chain of the antibody of any one of claims 1 to 3.
5. The polynucleotide of claim 4, comprising at least one of the following sequences:
SEQ ID NOs 1 and 3.
6. The polynucleotide of claim 4, comprising at least one of the following sequences: 10-14, and a polynucleotide sequence encoding an amino acid sequence KDS: AAAGACAGT are provided.
7. A vector comprising the polynucleotide of any one of claims 4-6.
8. A cell comprising the polynucleotide of any one of claims 4 to 6 or comprising the vector of claim 7.
9. A kit for detecting the level of H7N9 virus, comprising the anti-H7N 9 fully human monoclonal antibody 7O2 of claim 1, or a biologically active fragment derived therefrom that specifically binds to H7N 9.
10. The kit according to claim 9, which comprises the anti-H7N 9 fully human monoclonal antibody 7O2 of any one of claims 2 to 3.
11. The kit for detecting the level of H7N9 virus of claim 9 or 10, further comprising a second antibody and an enzyme for detection or a fluorescent or radioactive label, and a buffer.
12. The kit for detecting the level of H7N9 virus of claim 9 or 10, wherein the second antibody is an anti-antibody against the monoclonal antibody 7O2 of any one of claims 1 to 3.
CN201711338976.XA 2017-12-14 2017-12-14 Fully human monoclonal antibody 7O2 for resisting H7N9, and preparation method and application thereof Active CN109957013B (en)

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CN107226861A (en) * 2017-05-26 2017-10-03 深圳市第三人民医院 The anti-H7N9 avian influenza virus neutrality antibody 1F7L in people source and its application
CN107337732A (en) * 2016-05-03 2017-11-10 中国科学院深圳先进技术研究院 The full human monoclonal antibody 2J17 of anti-H7N9 and its preparation method and application
CN107353340A (en) * 2016-05-10 2017-11-17 深圳先进技术研究院 The full human monoclonal antibody 2L11 of anti-H7N9 and its preparation method and application

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Publication number Priority date Publication date Assignee Title
CN107337732A (en) * 2016-05-03 2017-11-10 中国科学院深圳先进技术研究院 The full human monoclonal antibody 2J17 of anti-H7N9 and its preparation method and application
CN107353340A (en) * 2016-05-10 2017-11-17 深圳先进技术研究院 The full human monoclonal antibody 2L11 of anti-H7N9 and its preparation method and application
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