CN109957013A - The full human monoclonal antibody 7O2 of anti-H7N9 and its preparation method and application - Google Patents
The full human monoclonal antibody 7O2 of anti-H7N9 and its preparation method and application Download PDFInfo
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- CN109957013A CN109957013A CN201711338976.XA CN201711338976A CN109957013A CN 109957013 A CN109957013 A CN 109957013A CN 201711338976 A CN201711338976 A CN 201711338976A CN 109957013 A CN109957013 A CN 109957013A
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- 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
- C07K16/1018—Orthomyxoviridae, e.g. influenza virus
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- 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
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- 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
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- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
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- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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- 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
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Abstract
The present invention relates to the full human monoclonal antibody 7O2 of anti-H7N9 and its preparation method and applications.The heavy and light chain of antibody 7O2 has 6 CDR regions.Antibody 7O2 of the present invention can target the hemagglutinin HA in conjunction with H7N9 virus, can be applied to influenza patient's viral diagnosis use.
Description
Technical field
The present invention relates to a kind of anti-full human monoclonal antibodies of H7N9 and the preparation method and application thereof, specifically, being
About the full human monoclonal antibody 7O2 of anti-H7N9 and its preparation method and application, belong to immunological technique field.
Background technique
In global ten big best-selling drugs in 2015, there are six be Quan Renyuan or Humanized monoclonal antibodies drug.Ranking
One be AbbVie Corp.'s treatment of arthritis anti-TNFa monoclonal antibody Humira, this is a full human monoclonal antibody,
It has been the king of medicine of continuous 10,000,000,000 or more 3 annual sales amount.Since first monoclonal antibody drug listing in 1986, monoclonal antibody
Drug experienced source of mouse monoclonal antibody medicine (Orthoclone OKT3), chimeric monoclonal antibody medicine (Rituximab), humanization monoclonal antibody medicine
The stages such as object (Herceptin) and full Human monoclonal antibody drug (Humira).Since there is anti-mouse antibody reaction (HAMA), mouse in human body
Source monoclonal antibody medicine, chimeric monoclonal antibody medicine are gradually eliminated, and the monoclonal antibody drug for occupying market at present is all source of people
Change monoclonal antibody drug.Compared with international advanced human antibody production technology, all there is a big difference for Shenzhen or even the whole of China,
The innovation ability for being mainly manifested in human antibody drug field is weak, and the less varieties of independent research, there is presently no original source of people
Change the report of monoclonal antibody drug listing, huge antibody drug market is captured by external medicine enterprise.China will change backward office
Face fights for the huge domestic and international antibody drug market of consumption potentiality, needs to capture Humanized monoclonal antibodies technology.
Human antibody refers to that antibody gene sequences are entirely derived from human immunoglobulin gene sequence.Human antibody specificity is high,
Few side effects, disease preventing and treating effect is good, is the main direction of development of monoclonal antibody medicine from now on.Preparation source of people monoclonal common at present
Antibody technique mainly has display technique of bacteriophage and single B cell round pcr.It is anti-that display technique of bacteriophage prepares source of people monoclonal
Body have the advantages that production cost it is low, without cumbersome work such as immune and cell fusions.But its disadvantage is also obvious, from
Often affinity is insufficient for the antibody obtained in nonimmune antibody library, is limited by gene transformation rate, the storage capacity of antibody library
It is not enough to cover the antibody diversity etc. of animal.And single B cell round pcr emerging in recent years refers to the blood from patient
The B cell of middle separation secreting function antibody, then extracts RNA and synthesis cDNA, therefrom the gene of clones secrete purpose antibody, most
After recombinate and express full human monoclonal antibody.The technical operation is simple and fast, the human antibody of production have high-affinity and
Specificity, and the recently improved separation from memory B cell has the monoclonal for neutralizing viral function or killing tumour function anti-
Body technique even more greatly reduces troublesome operation and cost.It is research and development people that single B cell, which prepares Humanized monoclonal antibodies technology,
The development trend of resource monoclonal antibody.
Human monoclonal antibody significant treatment with high specific in terms for the treatment of inflammation, cancer especially influenza
Effect.Influenza is the communicable disease as caused by influenza virus, seriously threatens human health.There are about 1,000,000,000 people every year in the whole world
By seasonal influenza virus infection, wherein there is ten thousand people of 25-50 dead.H7N9 virus is a kind of influenza virus, to traditional disease-resistant
Poison amantadine and rimantadine have drug resistance, at present still without effective treatment means.H7N9 virus is in invasion cell
When need the specific molecular of dependovirus oneself expression in conjunction with the receptor in people's cell, could infection cell, and further expand
Increase.The human antibody for neutralizing virus is certain specific antibodies of human B lymphocyte generation, can be with the antigen knot of virus surface
It closes, so that the virus be prevented to stick receptor in target cell, prevents Virus entry cell, can efficiently prevent and treat H7N9 influenza.
Therefore it provides the full human monoclonal antibody of anti-H7N9 and its preparation method are of great significance.
Summary of the invention
One of the objects of the present invention is to provide the full human monoclonal antibody 7O2 of anti-H7N9 or derive from the monoclonal antibody
The bioactive fragment that can specifically bind H7N9.
Another object of the present invention is to provide encode the full human monoclonal antibody 7O2 of the anti-H7N9 or derive from the list
The gene of the bioactive fragment that can specifically bind H7N9 of clonal antibody and carrier or cell containing the gene.
Another mesh of the invention is to provide the method for generating the anti-full human monoclonal antibody 7O2 of H7N9.
Another mesh of the invention is to provide the packet full human monoclonal antibody 7O2 of anti-H7N9 or from the Dan Ke
The pharmaceutical composition of the bioactive fragment that can specifically bind H7N9 of grand antibody.
Another object of the present invention is to provide the full human monoclonal antibody 7O2 of anti-H7N9 of the present invention or derive from
The bioactive fragment that can specifically bind H7N9 of the monoclonal antibody or the application of the pharmaceutical composition.
Another object of the present invention is to provide a kind of kits for detecting H7N9 virus.
To achieve the above object, the present invention provides a kind of full human monoclonal antibody of anti-H7N9 or the monoclonal is derived from
The bioactive fragment that can specifically bind H7N9 of antibody.It is 7O2 that the antibody is named in the present invention.
Specific embodiment according to the present invention, antibody of the invention has heavy chain variable region and light chain variable region, described
Heavy chain variable region and light chain variable region respectively have 3 complementary determining regions (CDR), in which:
The amino acid sequence of the CDR1 of the heavy chain variable region are as follows: GFTFSSYW (SEQ ID NO:5),
The amino acid sequence of the CDR2 of the heavy chain variable region are as follows: INSDGSST (SEQ ID NO:6),
The amino acid sequence of the CDR3 of the heavy chain variable region are as follows: ARRSKTGKFDP (SEQ ID NO:7),
The amino acid sequence of the CDR1 of the light chain variable region are as follows: ALPKQY (SEQ ID NO:8),
The amino acid sequence of the CDR2 of the light chain variable region are as follows: KDS,
The amino acid sequence of the CDR3 of the light chain variable region are as follows: QSADSSGTLEV (SEQ ID NO:9).
Specific embodiment according to the present invention, the heavy chain of antibody of the invention is as shown in SEQ ID NO:2.
Specific embodiment according to the present invention, the light chain of antibody of the invention is as shown in SEQ ID NO:4.
The present invention also provides a kind of polynucleotides, the weight chain variable of the polynucleotide encoding antibody of the present invention
Area and/or light chain variable region, or the heavy chain and/or light chain of antibody described in coding.Preferably, the polynucleotides include with
At least one lower sequence:
SEQ ID NOs:1,3 and 10~14.
Specific embodiment according to the present invention, SEQ ID NO:1 are a kind of polynucleotides for encoding SEQ ID NO:2；
SEQ ID NO:3 is a kind of polynucleotides for encoding SEQ ID NO:4；SEQ ID NO:10 is encode SEQ ID NO:5 one
Kind polynucleotides；SEQ ID NO:11 is a kind of polynucleotides for encoding SEQ ID NO:6；SEQ ID NO:12 is coding SEQ
A kind of polynucleotides of ID NO:7；SEQ ID NO:13 is a kind of polynucleotides for encoding SEQ ID NO:8；SEQ ID NO:
14 be a kind of polynucleotides for encoding SEQ ID NO:9.The multicore glycosides of the amino acid sequence EVT of the CDR2 of coding light chain variable region
Acid sequence can are as follows: AAAGACAGT.
The present invention also provides the carriers comprising polynucleotides of the present invention.
The present invention also provides the cells containing polynucleotides of the present invention or containing carrier of the present invention.
The full human monoclonal antibody 7O2 of anti-H7N9 of the present invention is generated or from the list the present invention also provides a kind of
The method of the bioactive fragment that can specifically bind H7N9 of clonal antibody, this method are prepared using single B cell method
The full human monoclonal antibody 7O2 of anti-H7N9.
The method that anti-H7N9 virus human monoclonal antibody is prepared using display technique of bacteriophage exists in the prior art, to the greatest extent
Pipe this method have the advantages that production cost it is low, without cumbersome work such as immune and cell fusions, but its disadvantage also compares
Obviously, often affinity is insufficient for the antibody obtained from nonimmune antibody library, limited by gene transformation rate, antibody library
Storage capacity is not enough to cover the antibody diversity etc. of animal.The present invention uses single B cell round pcr, from the blood of patient
The B cell of secreting function antibody is separated, then extracts RNA and synthesis cDNA, therefrom the gene of clones secrete purpose antibody, finally
Recombinate and express full human monoclonal antibody.The technical operation is simple and fast, and the human antibody of production has high-affinity and spy
The opposite sex, in addition, can further be separated from memory B cell using improved with neutralization viral function or kill tumour function
Monoclonal antibody technique of the present invention, even more greatly reduces troublesome operation and cost.
The present invention also provides a kind of pharmaceutical compositions, and it includes the anti-full human monoclonal antibodies of H7N9 of the present invention
7O2 or the bioactive fragment that can specifically bind H7N9 from the monoclonal antibody.
The present invention also provides the full human monoclonal antibody 7O2 of the anti-H7N9 or from the energy of the monoclonal antibody
The bioactive fragment or the pharmaceutical composition for enough specifically binding H7N9 are caused for treating by H7N9 virus in preparation
Disease drug in application.
It is complete containing anti-H7N9 of the present invention the present invention also provides a kind of kit for detecting H7N9 virus levels
Human monoclonal antibody 7O2 or the bioactive fragment that can specifically bind H7N9 from the monoclonal antibody；It is preferred that
Enzyme or fluorescence or radio-labeled object and buffer of the kit also containing secondary antibody and for detection；It is preferred that institute
Stating secondary antibody is the antiantibody for resisting monoclonal antibody 7O2 of the present invention.
Compared with prior art, the invention has the following beneficial effects:
(1) the full human monoclonal antibody 7O2 of anti-H7N9 of the present invention can target the hemagglutinin HA in conjunction with H7N9 virus,
It can be applied to influenza patient's viral diagnosis use.
(2) source of mouse antibody is compared, the gene of human antibody of the present invention is entirely derived from the gene of people, without other kinds
Ingredient, the toxic side effects such as anti-mouse antiantibody do not occur in human body, have better biocompatibility, be more suitable for and more have it is latent
Power becomes the macromolecular drug for the treatment of influenza virus.
(3) display technique of bacteriophage provided compared to the prior art prepares the side of anti-H7N9 virus human monoclonal antibody
Method, the antibody that the single B cell that the present invention uses develops anti-H7N9 have simple and quick, and the human antibody of production has height
The advantages that affinity and specificity.
Detailed description of the invention
Fig. 1 is the flow cytometer detection result figure that embodiment 1NTH-3T3 expresses CD40L.
Fig. 2 is 1 selected by flow cytometry apoptosis memory B cell result figure of embodiment.
Fig. 3 is embodiment 1ELISA experimental result picture.
Fig. 4 is embodiment 2Western blot agarose gel electrophoresis results figure.
Fig. 5 is the sequence diagram of 7O2 heavy chain variable region and light chain variable region.
Specific embodiment
In order to which technical characteristic of the invention, purpose and beneficial effect are more clearly understood, now in conjunction with specific implementation
Example and attached drawing carry out technical solution of the present invention described further below, it should be understood that these examples be merely to illustrate the present invention without
For limiting the scope of the invention.In embodiment, each Starting reagents material is commercially available, and the experiment of actual conditions is not specified
Method is conventional method and normal condition known to fields, or according to condition proposed by apparatus manufacturer.
Embodiment 1
(1) the NTH-3T3 cell line of expression CD40L is stablized in building
3T3-CD40L feeder cells are established using slow virus.Lentiviral pLVX-CD40L is constructed, 293T is transfected
Cell transfects the 4th day collection vial supernatant.NIH-3T3 cell is activated, 3 Dai Houyong slow-virus infections is cultivated, continues to cultivate
And it passes on 3 times.Cell of the sorting FITC fluorescence intensity near MFI is carried out using flow cytometer, is rejoined to culture bottle
In, 37 DEG C, 5%CO2In incubator cultivate and detect, testing result as shown in Figure 1, its be will express CD40L 3T3 cell and
The 3T3 cell of empty carrier pLVX (having ZxGreen) transfection is dyed with the anti-CD 40 L with APC respectively, then up flow type cell
Instrument analysis.As a result, it has been found that all 3T3-CD40L feeder cells all express CD40L.When cell grows to 80%~90%, digestion
Cell is collected, concentration is every milliliter 1 × 107Cell.It is placed in progress 5000rads radiation in radiation gauge, cell is resuspended in frozen stock solution,
Concentration is every milliliter 3.5 × 107Cell, packing 1ml (can be saved 2 years) in freezing tubule, liquid nitrogen cryopreservation.
(2) sorting and activation of memory B cell
Separate and freeze the PBMC for once infecting the rehabilitation patient of H7N9 virus with lymph separating liquid, every pipe 10~50 ×
106Cell freezes in liquid nitrogen container.PBMC streaming dyeing liquor is prepared, ingredient is as shown in table 1 below:
Table 1:PBMC streaming dyeing liquor
| Antibody | Volume (μ L) |
| CD19-PE-Cy7 | 0.5 |
| IgM-PE | 1.0 |
| IgA-APC | 2.5 |
| IgD-FITC | 2.5 |
| PBS-1% (wt/vol) BSA | 43.5 |
Defrosting PBMC is added above-mentioned PBMC streaming dyeing liquor and sorts on flow cytometer, as a result as shown in Fig. 2, dividing
Select CD19+IgM–IgA–IgD–Memory B cell, cell purity need to 90% or more, if be lower than 90%, repeat assorting room.
The mixed culture medium of activation B cell is prepared, component is as shown in table 2 below:
Table 2
| Component | Volume |
| Complete IMDM culture medium | 336mL |
| IL-2(10,000U mL-1) | 3.5mL |
| IL-21(100μg mL-1) | 175μL |
| 3T3-CD40L | 10mL |
Memory B cell is added in mixed culture medium, limiting dilution is in 384 orifice plates, 1, every hole cell, body after mixing
Product is 50ul, is placed in 37 DEG C, 5%CO2Stationary culture in incubator.After 13 days, supernatant is taken to carry out ELISA.
(3) the human monoclonal antibody 7O2 of anti-H7N9 virus is obtained
Influenza virus hemagglutinin HA is peplos surface column antigen, can be with a variety of erythrocyte receptors such as people, chicken, cavy
In conjunction with erythrocyte agglutination is caused, there is immunogenicity, antihemagglutinin antibody can neutralize influenza virus.In the present invention, pass through
ELISA has found the B cell that can secrete the antibody 7O2 in conjunction with H7N9 virus, and the human monoclonal antibody 7O2 of secretion can be with
Targeting combines the hemagglutinin HA (Fig. 3) of H7N9 virus.
ELISA tests concrete operations:
(1) the HA albumen of the H7N9 virus of 100ng/100 μ l is coated in 96 hole elisa Plates, every 100 μ l of hole；
(2) 4 degree of refrigerator overnights are placed；
(3) it is washed three times with PBST solution, every hole adds 5% 200 μ l of skimmed milk power solution, and 37 degree are incubated for 1 hour；
(4) it is washed three times with PBST solution, 100ul is added not have the normal human serum (negative control) of virus infection or loading
The patients serum for catching an illness malicious or the full human monoclonal antibody of anti-H7N9, each three repetitions；
(5) 37 degree be incubated for 1 hour after washed three times with PBST solution；
(6) anti-human IgG antibodies with HRP are diluted with 1:5000, be added in enzyme mark version, every 100 μ l of hole；
(7) 37 degree be incubated for 1 hour after washed three times with PBST solution；
(8) every hole adds 100 μ l tmb substrate solution, 37 degree 5 minutes；
(9) every hole adds 100 μ l of stop bath 2M sulfuric acid, at once the 450nm wavelength detecting light absorption value in microplate reader.Such as Fig. 3
Shown, the human monoclonal antibody 7O2 that ELISA experiment shows that the present invention obtains can target the hemagglutinin in conjunction with H7N9 virus
HA。
Clone, recombination and the expression of 2 Humanized monoclonal antibodies 7O2 gene of embodiment
B cell by capable of the secreting of obtaining of embodiment 1 in conjunction with the antibody 7O2 of H7N9 virus cracks, and takes lysate
The reverse transcription for carrying out RNA, obtains the pcr template cDNA of humanized antibody gene.Using cDNA as the heavy chain of template clonal antibody and gently
The gene of chain, and be binned in eukaryocyte 293F or HEK293 and expressed and purified.
Specifically:
(1) B cell liquid is transferred to 96 orifice plates (Eppendorf, 030133366).
(2) reverse transcription system: 150ng random primer (invitrogen, 48190-011), 0.5ul 10mM dNTP
(Invitrogen, 18427-088), 1 μ l 0.1M DTT (Invitrogen, 18080-044), 0.5%v/v Igepal CA-
630(Sigma,I3021-50ML),4U RNAsin(Promega),6U Prime RNAse Inhibitor(Eppendorf)
and 50UIII reverse transcriptase (Invitrogen, 18080-044) mends DEPC water extremely
14μl/well。
(3) reverse transcription reaction program: 42 DEG C, 10min；25 DEG C, 10min；50 DEG C, 60min；94 DEG C, 5min.
(4) cDNA is stored in -20 DEG C.
(5) heavy chain of antibody gene and light is expanded respectively with KOD-Plus-Neo (TOYOBO, KOD401) kit PCR
Chain, 40 μ L systems: 3.5 μ L cDNA, 20nM mix primers, 4 μ L buffers (buffer), 4 μ L 2mM dNTPs, 2.4 μ L
MgSO4, 1 μ L KOD.
(6) response procedures: 94 DEG C, 2min；45 circulations: [98 DEG C, 10s；58 DEG C (IgH/Ig κ) or 60 DEG C (Ig λ),
30s；68℃,28s(1st) or 23s (2 PCRnd PCR)]。
(7) Ago-Gel is carried out to amplified production, result is as shown in figure 4, the results show that antibody's light chain variable region is
κ, size 354bp, heavy chain variable region size are 327bp.
(8) antibody gene PCR product sequencing result is as follows:
The heavy chain variable region gene (SEQ ID NO:1) of 7O2:
GAGGTGCAGCTGGTGGAGTCCGGGGGAGGCTTAGTTCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCA
GCCTCTGGATTCACCTTCAGTAGCTACTGGATGCACTGGGTCCGCCAAGCTCCAGGGAAGGGGCTGGTGTGGGTCTC
ACGTATTAATAGTGATGGGAGTAGCACAAGCTACGCGGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACG
CCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCAAGAAGGAGT
AAAACGGGGAAGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA
The light-chain variable region gene (SEQ ID NO:3) of 7O2:
TCCTATGAGCTGACACAGCCACCCTCGGTGTCAGTGTCCCCAGGACAGACGGCCAGGATCACCTGCTCT
GGAGATGCATTGCCAAAGCAATATGCTTATTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTGATATATAA
AGACAGTGAGAGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAGCTCAGGGACAACAGTCACGTTGACCATCA
GTGGAGTCCAGGCAGAAGACGAGGCTGACTATTACTGTCAATCAGCAGACAGCAGTGGTACCCTAGAGGTGTTCGGC
GGAGGGACCAAGCTGACCGTCCTAGTA
(9) it is connected after H gene being carried out BamH1/EcoR1 double digestion respectively with pcDNA3.1, forms pcDNA3.1-H and carry
Body.
(10) it is connected after L gene being carried out Not1/Xho1 double digestion respectively with pcDNA3.1, forms pcDNA3.1-L and carry
Body.
(11) 293F cell is cultivated.
(12) 20ug pcDNA3.1-L carrier and 10ug pcDNA3.1-H carrier cotransfection 293F cell, culture 96 are small
When.
(13) take supernatant carry out ELISA (ABC is supernatant, and DEF is positive control, and GH is negative control) and
western blot；ELISA experimental result is as shown in table 3 below:
Table 3
| Data | 450 | Data | 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 are shown in supernatant containing the antibody 7O2 that can combine H7N9 virus.
Western blot tests specific process are as follows:
Albuminous degeneration electrophoresis is run with supernatant, is closed 1 hour after transferring film with 5% skimmed milk power solution, then with band HRP
Goat anti-human IgG antibodies be incubated for 1 hour, finally plus display substrate is exposed.Experimental result is as shown in figure 4, Fig. 4 is shown entirely
The heavy chain and light chain of human antibody show in supernatant containing the anti-full human monoclonal antibody 7O2 of H7N9 virus.
For the monoclonal antibody 7O2 of the present embodiment, SEQ ID NO:2 and SEQID NO:4 are the present embodiment respectively
The heavy chain variable region of the 7O2 of acquisition and VH the and VL amino acid sequence of light chain variable region.Referring to Fig. 5, the heavy chain variable region and
Light chain variable region respectively has 3 complementary determining regions (CDR), and number is CDR1, CDR2, CDR3 respectively, in which: heavy chain: CDR1:
GFTFSSYW(SEQ ID NO:5)；CDR2:INSDGSST (SEQ ID NO:6)；CDR3:ARRSKTGKFDP (SEQ ID NO:
7).Light chain: CDR1:ALPKQY (SEQ ID NO:8)；CDR2:KDS；CDR3:QSADSSGTLEV (SEQ ID NO:9).
Finally, it is stated that: above embodiments are merely to illustrate implementation process and feature of the invention, rather than limit this hair
Bright technical solution, although the present invention has been described in detail with reference to the above embodiments, those skilled in the art answer
Work as understanding: it is still possible to modify or equivalently replace the present invention, without departing from the spirit and scope of the present invention any
Modification or part replacement, should all cover in protection scope of the present invention.
Sequence table
<110>Shenzhen Institutes of Advanced Technology, Chinese Academy of Science
<120>the full human monoclonal antibody 7O2 of anti-H7N9 and its preparation method and application
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20 25 30
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Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Val Trp Val
35 40 45
tca cgt att aat agt gat ggg agt agc aca agc tac gcg gac tcc gtg 192
Ser Arg Ile Asn Ser Asp Gly Ser Ser Thr Ser Tyr Ala Asp Ser Val
50 55 60
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65 70 75 80
ctg caa atg aac agt ctg aga gcc gag gac acg gct gtg tat tac tgt 288
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85 90 95
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Ala Arg Arg Ser Lys Thr Gly Lys Phe Asp Pro Trp Gly Gln Gly Thr
100 105 110
ctg gtc acc gtc tcc tca 354
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aaa gac agt gag agg ccc tca ggg atc cct gag cga ttc tct ggc tcc 192
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50 55 60
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Claims (10)
1. the full human monoclonal antibody 7O2 of anti-H7N9 or the biology that can specifically bind H7N9 from the monoclonal antibody
Active fragment, wherein the antibody has heavy chain variable region and light chain variable region, and the heavy chain variable region and light chain variable region are respectively
With 3 complementary determining regions (CDR), in which:
The amino acid sequence of the CDR1 of the heavy chain variable region are as follows: GFTFSSYW (SEQ ID NO:5),
The amino acid sequence of the CDR2 of the heavy chain variable region are as follows: INSDGSST (SEQ ID NO:6),
The amino acid sequence of the CDR3 of the heavy chain variable region are as follows: ARRSKTGKFDP (SEQ ID NO:7),
The amino acid sequence of the CDR1 of the light chain variable region are as follows: ALPKQY (SEQ ID NO:8),
The amino acid sequence of the CDR2 of the light chain variable region are as follows: KDS,
The amino acid sequence of the CDR3 of the light chain variable region are as follows: QSADSSGTLEV (SEQ ID NO:9).
2. antibody according to claim 1, the heavy chain of the antibody is as shown in SEQ ID NO:2.
3. antibody according to claim 1 or 2, the light chain of the antibody is as shown in SEQ ID NO:4.
4. a kind of polynucleotides, the heavy chain variable region of the described in any item antibody of the polynucleotide encoding claim 1~3
And/or light chain variable region, or the heavy chain and/or light chain of the coding described in any item antibody of claims 1 to 3；Preferably, institute
Stating polynucleotides includes at least one following sequence:
SEQ ID NOs:1,3 and 10~14.
5. the carrier containing polynucleotides described in claim 4.
6. containing polynucleotides described in claim 4 or the cell containing carrier described in claim 5.
7. a kind of full human monoclonal antibody 7O2 of any one of generation claims 1 to 3 anti-H7N9 derives from the monoclonal
The method of the bioactive fragment that can specifically bind H7N9 of antibody, this method are using described in the preparation of single B cell method
The anti-full human monoclonal antibody 7O2 of H7N9.
8. a kind of pharmaceutical composition, it includes the described in any item anti-full human monoclonal antibody 7O2 of H7N9 of claims 1 to 3
Or the bioactive fragment that can specifically bind H7N9 from the monoclonal antibody.
9. the full human monoclonal antibody 7O2 of the described in any item anti-H7N9 of claims 1 to 3 or from the monoclonal antibody
Can specifically bind H7N9 bioactive fragment or pharmaceutical composition according to any one of claims 8 preparation for treat by
Application in the drug of disease caused by H7N9 virus.
10. a kind of kit for detecting H7N9 virus levels, contains the described in any item anti-full people of H7N9 of claims 1 to 3
Resource monoclonal antibody 7O2 or the bioactive fragment that can specifically bind H7N9 from the monoclonal antibody；It is preferred that institute
Enzyme or fluorescence or radio-labeled object and buffer of the kit stated also containing secondary antibody and for detection；It is preferred that described
Secondary antibody is the antiantibody of any one of the anti-claims 1 to 3 monoclonal antibody 7O2.
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| CN106519027A (en) * | 2016-11-11 | 2017-03-22 | 深圳先进技术研究院 | Anti-H7N9 full-human-derived monoclonal antibody 5J13 and preparation method and application thereof |
| 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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|---|---|---|---|---|
| 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 |
| CN106519027A (en) * | 2016-11-11 | 2017-03-22 | 深圳先进技术研究院 | Anti-H7N9 full-human-derived monoclonal antibody 5J13 and preparation method and application thereof |
| 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 |
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