CN111434681A

CN111434681A - Fully human monoclonal antibody 6J15 for resisting H7N9, and preparation method and application thereof

Info

Publication number: CN111434681A
Application number: CN201910035547.8A
Authority: CN
Inventors: 万晓春; 陈有海; 李俊鑫
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2019-01-15
Filing date: 2019-01-15
Publication date: 2020-07-21
Anticipated expiration: 2039-01-15
Also published as: CN111434681B

Abstract

The invention relates to an anti-H7N 9 fully human monoclonal antibody 6J15, a preparation method and application thereof. The fully human monoclonal antibody 6J15 is rapidly screened by a memory B cell PCR method and does not contain any murine components. The antibody can be combined with hemagglutinin HA of H7N9 virus in a targeted mode, and HAs the neutralizing activity of obviously resisting H7N9 virus infection; the antibody of the invention does not generate toxic and side effects such as anti-mouse anti-antibody and the like, has better biocompatibility, and is more suitable and has more potential to become a macromolecular drug for treating influenza virus.

Description

Fully human monoclonal antibody 6J15 for resisting H7N9, and preparation method and application thereof

Technical Field

The invention belongs to the field of immunology, and particularly relates to a fully human monoclonal antibody 6J15 for resisting H7N9, and a preparation method and application thereof.

Background

In 2015, 6 of ten popular drugs are all humanized 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 drugs (such as Orthoclone OKT3), chimeric monoclonal antibody drugs (Rituximab), humanized monoclonal antibody drugs (Herceptin), and fully human monoclonal antibody drugs (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 needs to overcome the fully humanized monoclonal antibody technology urgently.

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 no effective treatment means exists 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.

Disclosure of Invention

In one aspect, the invention relates to the fully human monoclonal antibody 6J15 against H7N9 or a biologically active fragment derived from the monoclonal antibody that is capable of neutralizing the H7N9 virus.

In another aspect, the invention relates to a gene encoding the anti-H7N 9 fully human monoclonal antibody 6J15 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.

In another aspect, the present invention relates to methods for producing said anti-H7N 9 fully human monoclonal antibody 6J 15.

In another aspect, the present invention relates to a pharmaceutical composition comprising said anti-H7N 9 fully human monoclonal antibody 6J15 or a biologically active fragment derived from said monoclonal antibody which is capable of specifically binding to H7N 9.

In another aspect, the invention relates to the application of the anti-H7N 9 fully human monoclonal antibody 6J15 or a bioactive fragment derived from the monoclonal antibody and capable of specifically binding to H7N9 or the pharmaceutical composition.

In another aspect, the invention relates to a kit for detecting H7N9 virus.

Specifically, in one aspect, the present invention provides an anti-H7N 9 fully human monoclonal antibody 6J15 or a biologically active fragment derived from the monoclonal antibody and capable of specifically binding to H7N9, wherein the amino acid sequences of the heavy and light chain CDR1, CDR2 and CDR3 regions of the antibody are as follows:

heavy chain CDR1: GYNIFTSYD;

heavy chain CDR2, MNPDSGDT;

heavy chain CDR3: ATGNADCSGSCYNWFDP;

light chain CDR1, R L RSTYY;

light chain CDR2: GKN;

light chain CDR3: NSRDTSGYH L V.

In some embodiments, the heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO. 2, or the amino acid sequence with the same function is formed by replacing, deleting or adding one or more amino acids in the sequence; and/or

The variable region amino acid sequence of the light chain of the antibody is shown as SEQ ID NO. 4, or the sequence is formed by replacing, deleting or adding one or more amino acids to form an amino acid sequence with the same function.

In some embodiments, the heavy chain amino acid sequence of the antibody is shown as SEQ ID NO. 6, or the amino acid sequence with equivalent functions formed by replacing, deleting or adding one or more amino acids in the sequence; and/or

The light chain amino acid sequence of the antibody is shown as SEQ ID NO. 8, or the amino acid sequence with the same function is formed by replacing, deleting or adding one or more amino acids in the sequence.

Proved by experiments of E L ISA, the anti-H7N 9 fully human monoclonal antibody 6J15 can be combined with hemagglutinin HA of H7N9 virus in a targeted mode, and the affinity is 2.06 × 10^-9M; in a model of virus-infected cells, their IC₅₀The value was only around 0.35 uM. Compared with a mouse antibody, the gene of the fully human antibody is completely derived from the human gene, has no other species of components, does not generate toxic and side effects such as anti-mouse anti-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.

In another aspect, the present invention provides a gene encoding the anti-H7N 9 fully human monoclonal antibody 6J15 of the present invention. In some embodiments, the gene comprises a nucleotide sequence encoding an amino acid having the amino acid sequence set forth in SEQ ID NO. 2, in some embodiments, the nucleotide sequence set forth in SEQ ID NO. 1; and/or

The gene comprises a nucleotide sequence encoding an amino acid having the amino acid sequence shown in SEQ ID NO. 4, and in some embodiments, the nucleotide sequence is shown in SEQ ID NO. 3.

In some embodiments, the gene comprises a nucleotide sequence encoding an amino acid having SEQ ID NO 6, in some embodiments, the nucleotide sequence is set forth in SEQ ID NO 5; and/or

The gene comprises a nucleotide sequence encoding an amino acid having the amino acid sequence shown in SEQ ID NO. 8, and in some embodiments, the nucleotide sequence is shown in SEQ ID NO. 7.

The sequence of SEQ ID NO 1-8 is shown in a sequence table, wherein:

(1) sequence 76 to 102 of SEQ ID NO. 1 and sequence 124 to 150 of SEQ ID NO. 5: sequences encoding the heavy chain CDR1 region;

(2) sequences 154 to 177 of SEQ ID NO. 1 and sequences 202 to 225 of SEQ ID NO. 5: sequences encoding the heavy chain CDR2 region;

(3) sequences 292 to 342 of SEQ ID NO. 1 and sequences 340 to 390 of SEQ ID NO. 5: sequences encoding the heavy chain CDR3 region;

(4) the sequence from 76 th to 96 th of SEQ ID NO. 3 and the sequence from 124 th to 144 th of SEQ ID NO. 7: sequences encoding the light chain CDR1 region;

(5) sequences 148 to 156 of SEQ ID NO. 3 and sequences 196 to 204 of SEQ ID NO. 7: sequences encoding the light chain CDR2 region;

(6) the 265 th to 297 th bit sequences in SEQ ID NO 3 and the 313 th to 345 th bit sequences in SEQ ID NO 7: sequences encoding the light chain CDR3 region;

(7) the sequence at positions 1 to 48 in SEQ ID NO 5 and SEQ ID NO 7 is a sequence for encoding a signal peptide.

(8) The 26 th to 34 th amino acid sequences in SEQ ID NO. 2 and SEQ ID NO. 6 are heavy chain CDR1 sequences; the amino acid sequences at positions 52-59 in SEQ ID NO. 2 and SEQ ID NO. 6 are heavy chain CDR2 sequences; the amino acid sequences from 98 th to 114 th in SEQ ID NO. 2 and SEQ ID NO. 6 are heavy chain CDR3 sequences.

(9) The 26 th-32 th amino acid sequences in SEQ ID NO. 4 and SEQ ID NO. 8 are light chain CDR1 sequences; the amino acid sequences at positions 50-52 in SEQ ID NO. 4 and SEQ ID NO. 8 are light chain CDR2 sequences; the 89-99 amino acid sequences in SEQ ID NO. 4 and SEQ ID NO. 8 are the light chain CDR3 sequences.

In another aspect, the present invention provides a vector comprising the gene as described above.

In a further aspect, the invention provides a cell comprising a gene as described above or a vector as described above.

In still another aspect, the present invention provides a method for producing the fully human monoclonal antibody 6J15 against H7N9 or a biologically active fragment derived from the monoclonal antibody and capable of specifically binding to H7N9, which comprises culturing genetically engineered cells containing the above gene encoding the heavy and light chains of the fully human monoclonal antibody 6J15 against H7N9 or the above vector or directly culturing the above cells, collecting and purifying to obtain the fully human monoclonal antibody 6J15 against H7N 9.

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 present invention separates B cell secreting functional antibody from patient's blood, extracts RNA and synthesizes cDNA, clones the gene secreting destination antibody, and recombines and expresses fully humanized monoclonal antibody. 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.

In another aspect, the present invention provides a pharmaceutical composition comprising the anti-H7N 9 fully human monoclonal antibody 6J15 of the present invention or a biologically active fragment derived from the monoclonal antibody that specifically binds to H7N 9.

In another aspect, the invention provides an application of the anti-H7N 9 fully human monoclonal antibody 6J15 or a biologically active fragment derived from the monoclonal antibody and capable of specifically binding to H7N9, or the pharmaceutical composition in preparation of a medicament for treating diseases caused by H7N9 virus.

In another aspect, the present invention provides a kit for detecting the level of H7N9 virus, comprising the anti-H7N 9 fully human monoclonal antibody 6J15 or a biologically active fragment derived from the monoclonal antibody and capable of specifically binding to H7N 9; in some embodiments, the kit further comprises a second antibody and an enzyme for detection or a fluorescent or radioactive label, and a buffer; the second antibody is, for example, an anti-antibody against the monoclonal antibody 6J15 of the present invention.

Compared with the prior art, the invention has the following beneficial effects:

(1) the anti-H7N 9 fully human monoclonal antibody 6J15 can be combined with hemagglutinin HA of H7N9 virus in a targeted mode, and HAs the neutralizing activity of obviously resisting H7N9 virus infection.

(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 H7N9 virus-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 CD 40L expression in example 1 by NTH-3T 3.

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 the ISA experiment of E L in example 1.

FIG. 4 is a graph showing the results of the neutralization experiment in example 3.

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 CD 40L (3T3-CD 40L)

Establishing 3T3-CD 40L feeder cells by using lentivirus, constructing a lentivirus expression vector p L VX-CD 40L, transfecting 293T cells, collecting virus supernatant on the fourth day of transfection, activating NIH-3T3 cells, infecting by using the lentivirus after culturing for 3 generations, continuously culturing and passaging for 3 times by using a flow cytometerSorting cells with FITC fluorescence intensity near MFI, adding into culture flask again, and culturing at 37 deg.C with 5% CO₂The cells were cultured in an incubator and tested as shown in FIG. 1, in which 3T3 cells expressing CD 40L and 3T3 cells transfected with the empty vector p L VX (with ZxGreen) were stained with anti-CD 40L with APC and then analyzed by an up-flow cytometer, respectively⁷Placing the cells in an irradiator for 5000rads irradiation, and resuspending the cells in a freezing medium at a concentration of 3.5 × 10 per 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

Separating and freezing PBMC of convalescent patients infected with H7N9 virus by using lymph separating medium, wherein each tube is 10-50 × 10⁶Cells, frozen in liquid nitrogen tank. PBMC flow staining solutions were prepared, and the components thereof are shown in Table 1 below

TABLE 1PBMC flow staining solution

Antibodies	Volume (mu 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

Thawing PBMC, adding the above PBMC flow staining solution and sorting on flow cytometer, and sorting out CD19 as shown in FIG. 2⁺IgM^–IgA^–IgD^–The 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 medium for activating B cells was prepared as 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-CD 40L obtained in step (1)	10mL

Adding memory B cells into mixed culture medium, mixing, diluting in 384-well plate with 1 cell per well and 50 μ l volume, standing at 37 deg.C and 5% CO₂Standing in incubator for 13 days, collecting supernatant, and performing E LISA。

(3) Obtaining human monoclonal antibody 6J15

In the invention, B cells capable of secreting antibody 6J15 which binds H7N9 virus are discovered through E L ISA, and the secreted human monoclonal antibody 6J15 can target hemagglutinin HA which binds H7N9 virus (figure 3).

E L ISA experiment details:

(1) 100 ng/100. mu.l of HA protein of H7N9 virus (available from ACROBIOSystems) was coated in a 96-well microplate, 100. mu.l per well;

(2) standing in a refrigerator at 4 deg.C overnight;

(3) washing with PBST solution for three times, adding 5% skimmed milk powder solution 200 μ l per well, and incubating at 37 deg.C for 1 hr;

(4) washing with PBST solution for three times, adding 100 μ l of normal human serum without virus infection (negative control) or patient serum with virus infection or anti-H7N 9 fully human monoclonal antibody 6J15, each in triplicate;

(5) incubation for 1 hour at 37 ℃ followed by three washes with PBST solution;

(6) adding 100 mul of HRP-carrying anti-human IgG antibody (abcam) diluted at a ratio of 1:5000 into an enzyme-labeled plate;

(7) incubation for 1 hour at 37 ℃ followed by three washes with PBST solution;

(8) add 100. mu.l TMB substrate solution (Thermo Scientific) to each well for 5min at 37 ℃;

(9) the result of the detection of the absorbance at 450nm wavelength in a microplate reader is shown in FIG. 3, and the result of the E L ISA experiment shows that the human monoclonal antibody 6J15 obtained by the invention can target and bind to hemagglutinin HA of H7N9 virus.

Example 2 cloning, recombination, expression and purification of humanized monoclonal antibody 6J15 Gene

The B cells capable of secreting 6J15 antibody that binds to H7N9 virus obtained in example 1 were lysed, and the lysates were subjected to reverse transcription of RNA to obtain PCR template cDNA of the human antibody gene. Designing and synthesizing primers for cloning antibody genes, cloning heavy chain and light chain genes of the antibody by taking cDNA as a template, and recombining to express and purify in a eukaryotic cell 293F or HEK 293. Specifically, the method comprises the following steps:

(1) the lysed B cell fluid was transferred to a 96-well plate (Eppendorf, 030133366).

(2) Reverse transcription system 150ng random primer (Invitrogen,48190-

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) Design and synthesis of primers:

forward Primer 5 '-3' sequence (Forward Primer 5 '-3' sequence)

Heavy chain variable region PCR primers:

5′VH1CTGCAACCGGTGTACATTCCCAGGTGCAGCTGGTGCAG(SEQ ID NO:9)

5′VH1/5CTGCAACCGGTGTACATTCCGAGGTGCAGCTGGTGCAG(SEQ ID NO:10)

5′VH3CTGCAACCGGTGTACATTCTGAGGTGCAGCTGGTGGAG(SEQ ID NO:11)

5′VH3-23CTGCAACCGGTGTACATTCTGAGGTGCAGCTGTTGGAG(SEQ ID NO:12)

5′VH4CTGCAACCGGTGTACATTCCCAGGTGCAGCTGCAGGAG(SEQ ID NO:13)

5′VH 4-34CTGCAACCGGTGTACATTCCCAGGTGCAGCTACAGCAGTG(SEQ ID NO:14)

5′VH 1-18CTGCAACCGGTGTACATTCCCAGGTTCAGCTGGTGCAG(SEQ ID NO:15)

5′VH 1-24CTGCAACCGGTGTACATTCCCAGGTCCAGCTGGTACAG(SEQ ID NO:16)

5′VH3-33CTGCAACCGGTGTACATTCTCAGGTGCAGCTGGTGGAG(SEQ ID NO:17)

5′VH 3-9CTGCAACCGGTGTACATTCTGAAGTGCAGCTGGTGGAG(SEQ ID NO:18)

5′VH4-39CTGCAACCGGTGTACATTCCCAGCTGCAGCTGCAGGAG(SEQ ID NO:19)

5′VH 6-1CTGCAACCGGTGTACATTCCCAGGTACAGCTGCAGCAG(SEQ ID NO:20)

3′JH 1/2/4/5TGCGAAGTCGACGCTGAGGAGACGGTGACCAG(SEQ ID NO:21)

3′JH 3TGCGAAGTCGACGCTGAAGAGACGGTGACCATTG(SEQ ID NO:22)

3′JH 6TGCGAAGTCGACGCTGAGGAGACGGTGACCGTG(SEQ ID NO:23)

kappa light chain variable region PCR product

5′Vκ1-5CTGCAACCGGTGTACATTCTGACATCCAGATGACCCAGTC(SEQ ID NO:24)

5′Vκ1-9TTGTGCTGCAACCGGTGTACATTCAGACATCCAGTTGACCCAGTCT(SEQ ID NO:25)

5′Vκ1D-43CTGCAACCGGTGTACATTGTGCCATCCGGATGACCCAGTC(SEQ ID NO:26)

5′Vκ2-24CTGCAACCGGTGTACATGGGGATATTGTGATGACCCAGAC(SEQ ID NO:27)

5′Vκ2-28CTGCAACCGGTGTACATGGGGATATTGTGATGACTCAGTC(SEQ ID NO:28)

5′Vκ2-30CTGCAACCGGTGTACATGGGGATGTTGTGATGACTCAGTC(SEQ ID NO:29)

5′Vκ3-11

TTGTGCTGCAACCGGTGTACATTCAGAAATTGTGTTGACACAGTC(SEQ ID NO:30)

5′Vκ3-15CTGCAACCGGTGTACATTCAGAAATAGTGATGACGCAGTC(SEQ ID NO:31)

5′Vκ3-20TTGTGCTGCAACCGGTGTACATTCAGAAATTGTGTTGACG CAGTCT(SEQ ID NO:32)

5′Vκ4-1CTGCAACCGGTGTACATTCGGACATCGTGATGACCCAGTC(SEQ ID NO:33)

3′Jκ1/4GCCACCGTACGTTTGATYTCCACCTTGGTC(SEQ ID NO:34)

3′Jκ2GCCACCGTACGTTTGATCTCCAGCTTGGTC(SEQ ID NO:35)

3′Jκ3GCCACCGTACGTTTGATATCCACTTTGGTC(SEQ ID NO:36)

3′Jκ5GCCACCGTACGTTTAATCTCCAGTCGTGTC(SEQ ID NO:37)

(6) Heavy and light chains of the antibody gene were amplified separately by PCR using KOD-Plus-Neo (TOYOBO, KOD401) kit, 40. mu. L system 3.5. mu. L cDNA, 20nM mixed primers, 4. mu. L buffer (buffer), 4. mu. L2 mM dNTPs, 2.4. mu. L MgSO 2₄，1μL KOD。

(7) Reaction procedure: 94 ℃ for 2 min; 45 cycles: 10s at 98 ℃; at 58 ℃ for 30 s; 68 ℃ for 28 s.

(8) The amplified products were subjected to agarose gel, and as a result, it was found that the size of the antibody light chain was 327bp and the size of the heavy chain was 375 bp.

(9) The sequencing result of the antibody gene heavy chain variable region PCR product is shown as the sequence shown in SEQ ID NO. 1, and the corresponding amino acid sequence is shown as the sequence shown in SEQ ID NO. 2. The sequencing result of the antibody gene light chain variable region PCR product is shown as the sequence shown in SEQ ID NO. 3, and the corresponding amino acid sequence is shown as the sequence shown in SEQ ID NO. 4.

Based on the obtained heavy and light chain variable region sequences, Invitrogen was designed and assigned to synthesize the full-length H gene of the heavy chain of the antibody gene, which had BamH1/EcoR1 double cleavage sites, and the full-length L gene of the light chain of the antibody gene, which had Not1/Xho1 double cleavage sites.

(10) The H gene and pcDNA3.1 are respectively subjected to BamH1/EcoR1 double enzyme digestion and then connected to form pcDNA3.1-H vector.

(11) The L gene and pcDNA3.1 were digested separately with Not1/Xho1 and ligated to form pcDNA3.1-L vector.

(12) 293F cells were cultured.

(13) Mu.g of pcDNA3.1-H (the nucleotide sequence of the H gene is shown in SEQ ID NO: 5) vector and 10. mu.g of pcDNA3.1-L (the nucleotide sequence of the L gene is shown in SEQ ID NO: 7) vector were co-transfected into 293F cells and cultured for 96 hours.

(14) Taking the supernatant, carrying out E L ISA (ABC is supernatant, DEF is positive control, GH is negative control), carrying out the specific experimental steps of E L ISA as described above, and carrying out the experimental results of E L ISA as shown in the following table 3 and figure 3:

TABLE 3

Data of	450nm	Data of	450nm
				A	2.245	E	1.185
B	1.852	F	1.201
				C	1.783	G	0.0675
D	1.114	H	0.0554

As can be seen from the data in table 3 and fig. 3: the supernatant contained antibodies capable of binding to H7N9 virus.

(15) Purification process, specifically, the purification process of the fully human monoclonal antibody 6J15 is as follows:

(a) 200. mu.g of pcDNA3.1-L vector and 100. mu.g of pcDNA3.1-H vector were co-transfected into 300ml of 293F cells and cultured for 96 hours.

(b) The supernatant was collected, applied to a proteinA affinity column, washed with 10-fold PBS, and added with 2ml of pH3.0, 0.1M glycine to collect the antibody, and 100 μ l of a neutralization buffer (1M Tri-HC L) was added to the collection tube to neutralize the pH of the antibody solution immediately after elution.

(c) Dialyzed against Phosphate Buffered Saline (PBS), and after dialysis, recently used was stored at 4 ℃ for a long period of time at-20 ℃. The total amount of 500. mu.g of pure 6J15 antibody was obtained, the amino acid sequence of the heavy chain is shown in SEQ ID NO. 6, and the amino acid sequence of the light chain is shown in SEQ ID NO. 8.

EXAMPLE 3 neutralization assay and antibody affinity assay of purified fully human monoclonal antibody 6J15

(1) Purpose of experiment

The inhibitory effect and effect of the 6J15 antibody on H7N9 influenza virus were evaluated by a micro-neutralization-E L ISA experiment using a virus-infected cell model (canine kidney cell MDCK), and the antibody activity against influenza virus was examined.

(2) Experimental procedure

(2.1) cell plating

Pancreatin digesting MDCK canine kidney cells in logarithmic growth phase, centrifuging and collecting after termination, blowing off uniformly to prepare single cell suspension, adjusting cell concentration to 5 × 10 with cell culture solution⁴Inoculating to 96-well cell culture plate, placing the cells at 37 deg.C and 5% CO₂The culture was carried out overnight in an incubator.

(2.2) pretreatment of 6J15 antibody and H7N9 virus (the virus A/Anhui/1/2013 is obtained from institute of microbiology, national academy of sciences)

10 concentration gradients of the 6J15 antibody are set, and the concentration gradients are 10-10 in sequence¹⁰Dilution was doubled with 3 parallel wells for each concentration in each group.

(2.3) viral infection

The cell culture supernatant cultured in step (2.1) was discarded and washed 3 times with PBS. The premixed antibody-virus mixture (at 10)²Mu.g/ml of 6J15 monoclonal antibody in turn at 10-10¹⁰Double dilution, each concentration of 6J15 antibody with an equal volume of 100TCID₅₀The viruses were mixed to obtain the mixture). Add 96-well cell culture plates, incubate at 37 ℃ for 1h, aspirate the mixture and wash with PBS 2 times.

(2.4) preparation of a maintenance liquid

TPCK-Trypsin (TPCK-Trypsin) (maintenance medium) was added to serum-free DMEM at a final concentration of 2. mu.g/ml. PBS in a 96-well plate was discarded, 100. mu.l of maintenance medium was added to each well, and the mixture was incubated at 37 ℃ with 5% CO₂The incubator is used for 20 h.

(2.5) neutralization experiment-E L ISA method

(2.5.1) discarding the maintenance solution in the microplate;

(2.5.2) washing the cells once with 100. mu.l PBS;

(2.5.3) discard PBS (not allow cells to dry) and add 50 μ l/well fixative (volume ratio acetone: absolute ethanol 2: 3);

(2.5.4) covering the microplate, and fixing the cells at room temperature for 10 min;

(2.5.5) discard the fixative, wash the cells with 100. mu.l PBS, repeat the wash 3 times (gently shaking to avoid vigorous washing) to remove residual acetone.

(2.5.6) blocking the cells with 5% skim milk powder at room temperature for 1h, washing the cells 1 time with 100. mu.l PBS;

(2.5.7) with PBS1: dilution 1 of the antibody (commercially available NP monoclonal antibody against H7N 9) was added to each well in 50. mu.l of the diluted antibody, and the mixture was allowed to act at room temperature for 1 hour.

(2.5.8) wash the plate 5 times with 100. mu.l PBST to remove 1 antibody;

(2.5.9) 2 anti (HRP-bearing anti-mouse IgG antibody) was diluted with PBS1:2000 and 50. mu.l was added to each well and allowed to act at room temperature for 1 hour.

(2.5.10) plates were washed 6 times with 100. mu.l PBST to remove 2 antibody.

(2.5.11) 50. mu.l of TMB developing solution was added to each well.

(2.5.12) after development of color at room temperature for about 10 minutes in the absence of light, 50. mu.l of 2M hydrochloric acid was added to each well to terminate the reaction.

(2.5.13) the OD of each well was read on an E L ISA reader (450 nm).

(3) Statistical analysis

Data were analyzed and dose-response curves were plotted using GraphPad Prism 6.0.1, and IC was calculated₅₀。

Inhibition rate calculation formula:

inhibition rate was 100% (OD well-OD negative cell control well) - (OD drug well-OD negative cell control well) ]/(OD well-OD negative cell control well) × 100%.

The resulting structure is shown in FIG. 4, from which FIG. 4 one can see an IC of 6J15₅₀＝0.35ug/mL。

As can be seen from example 3, the invention 6J15 has good IC for H7N9₅₀The value proves that 6J15 has good virus neutralizing capacity.

The invention compares the application number of 201610303416.X, the invention name of the monoclonal antibody 2L against H7N9 and the preparation method and application thereof which are submitted to the intellectual property administration of China in 2016 (05 year and 10 days), the monoclonal antibody 2L 11, the application number of the monoclonal antibody 2J17 against H7N9 and the preparation method and application thereof which are submitted to the intellectual property administration of China in 2016 (05 months and 03 days), and the monoclonal antibody 2J17, which is submitted to the intellectual property administration of China in 2016 (05 months and 03 days), the IC 6J15 neutralizing activity of the invention₅₀Research team 2018, month 07, month 24 and filed an application (application number: 201810818233.0) of "anti-H7N 9 fully human monoclonal antibody hIg311 and preparation and application thereof", which is not disclosed at present, on the basis of the content of the application, partial amino acid changes are carried out on amino acids in CDR1, CDR2 and CDR3 regions of a heavy light chain so as to research related functions of the antibody.

Antibody affinity detection:

the affinity detection instrument is Fortebio of PA LL, prepares 200 mu l of 50 mu g/ml 6J15 antibody, binds to proteinA A sensor for 120 seconds, prepares 100nM, 50nM, 2.5nM, 12.5nM, 6.25nM and 0nM concentration solution of HA antigen, binds to antibody for 120 seconds, HAs dissociation time of 5 minutes, shows that 6J15 HAs higher affinity to H7N9 virus, KD is 2.06 × 10^-9M。

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.

Sequence listing

<110> Shenzhen advanced technology research institute of Chinese academy of sciences

<120> H7N 9-resistant fully human monoclonal antibody 6J15, and preparation method and application thereof

<130>GAI18CN6779

<160>37

<170>SIPOSequenceListing 1.0

<210>1

<211>375

<212>DNA

<213> Artificial sequence ()

<400>1

caagtgcagc tggtggagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60

tcctgcaagg cttctggata caatatattc accagttatg atatcaactg ggtgcgacag 120

gccactggcc aagggcttga gtggatggga tggatgaacc ctgacagtgg tgacacaggc 180

tttgcacaga agttccaggg cagagtcacc atgaccagga acacctccat aaccacagcc 240

tacatggagc tgagcagcct gacttctgag gacacggccg tgtattactg tgcgacagga 300

aatgcggatt gtagtggtag ctgctacaat tggttcgacc cctggggcca gggaaccctg 360

gtcaccgtct cctca 375

<210>2

<211>125

<212>PRT

<213> Artificial sequence ()

<400>2

Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Asn Ile Phe Thr Ser

20 25 30

Tyr Asp Ile Asn Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp

35 40 45

Met Gly Trp Met Asn Pro Asp Ser Gly Asp Thr Gly Phe Ala Gln Lys

50 55 60

Phe Gln Gly Arg Val Thr Met Thr Arg Asn Thr Ser Ile Thr Thr Ala

65 70 75 80

Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Thr Gly Asn Ala Asp Cys Ser Gly Ser Cys Tyr Asn Trp Phe

100 105 110

Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210>3

<211>330

<212>DNA

<213> Artificial sequence ()

<400>3

tcgtctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60

acatgccaag gagacagact cagaagcact tattatgcaa gctggtacca gcagaagcca 120

ggacaggccc ctgtacttgt catctatggt aaaaacaacc ggccctcagg gatcccagac 180

cgattctctg gctccagctc aggaaacaca gcttccttga ccatcactgg ggctcaggcg 240

gaagatgagg ctgactatta ctgtaactcc cgggacacca gtggttacca tctggtgttc 300

ggcggaggga ccaagctgac cgtcctagta 330

<210>4

<211>110

<212>PRT

<213> Artificial sequence ()

<400>4

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Arg Leu Arg Ser Thr Tyr Tyr

20 25 30

Ala Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile

35 40 45

Tyr Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly

50 55 60

Ser Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala

65 70 75 80

Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Thr Ser Gly Tyr

85 90 95

His Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Val

100 105 110

<210>5

<211>1416

<212>DNA

<213> Artificial sequence ()

<400>5

atgggctggt cctgcatcat cctgttcctg gtggccaccg ccaccggcca agtgcagctg 60

gtggagtctg gggctgaggt gaagaagcct ggggcctcag tgaaggtctc ctgcaaggct 120

tctggataca atatattcac cagttatgat atcaactggg tgcgacaggc cactggccaa 180

gggcttgagt ggatgggatg gatgaaccct gacagtggtg acacaggctt tgcacagaag 240

ttccagggca gagtcaccat gaccaggaac acctccataa ccacagccta catggagctg 300

agcagcctga cttctgagga cacggccgtg tattactgtg cgacaggaaa tgcggattgt 360

agtggtagct gctacaattg gttcgacccc tggggccagg gaaccctggt caccgtctcc 420

tcagctagca ccaagggccc atcggtcttc cccctggcac cctcctccaa gagcacctct 480

gggggcacag cggccctggg ctgcctggtc aaggactact tccccgaacc ggtgacggtg 540

tcgtggaact caggcgccct gaccagcggc gtgcacacct tcccggccgt cctacagtcc 600

tcaggactct actccctcag cagcgtggtg accgtgccct ccagcagctt gggcacccag 660

acctacatct gcaacgtgaa tcacaagccc agcaacacca aggtggacaa gagagttgag 720

cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg 780

ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 840

cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 900

tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 960

aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 1020

aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 1080

tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 1140

gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1200

atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1260

gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1320

tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1380

acgcagaaga gcctctccct gtctccgggt aaatga 1416

<210>6

<211>455

<212>PRT

<213> Artificial sequence ()

<400>6

Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Asn Ile Phe Thr Ser

20 25 30

Tyr Asp Ile Asn Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp

35 40 45

Met Gly Trp Met Asn Pro Asp Ser Gly Asp Thr Gly Phe Ala Gln Lys

50 55 60

Phe Gln Gly Arg Val Thr Met Thr Arg Asn Thr Ser Ile Thr Thr Ala

65 70 75 80

Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Thr Gly Asn Ala Asp Cys Ser Gly Ser Cys Tyr Asn Trp Phe

100 105 110

Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr

115 120 125

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

130 135 140

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe ProAla Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

195 200 205

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu

210 215 220

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

245 250 255

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

260 265 270

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

275 280 285

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

290 295 300

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

305 310 315 320

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

325 330 335

Pro Ala Pro Ile GluLys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

340 345 350

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

355 360 365

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

370 375 380

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

385 390 395 400

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

405 410 415

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

420 425 430

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

435 440 445

Leu Ser Leu Ser Pro Gly Lys

450 455

<210>7

<211>699

<212>DNA

<213> Artificial sequence ()

<400>7

atgggctggt cctgcatcat cctgttcctg gtggccaccg ccaccggctc gtctgagctg 60

actcaggacc ctgctgtgtc tgtggccttg ggacagacag tcaggatcac atgccaagga 120

gacagactca gaagcactta ttatgcaagc tggtaccagc agaagccagg acaggcccct 180

gtacttgtca tctatggtaa aaacaaccgg ccctcaggga tcccagaccg attctctggc 240

tccagctcag gaaacacagc ttccttgacc atcactgggg ctcaggcgga agatgaggct 300

gactattact gtaactcccg ggacaccagt ggttaccatc tggtgttcgg cggagggacc 360

aagctgaccg tcctagtaac cgtggccgcc ccctccgtgt tcatcttccc cccctccgac 420

gagcagctga agtccggcac cgcctccgtg gtgtgcctgc tgaacaactt ctacccccgg 480

gaggccaagg tgcagtggaa ggtggacaac gccctgcagt ccggcaactc ccaggagtcc 540

gtgaccgagc aggactccaa ggactccacc tactccctgt cctccaccct gaccctgtcc 600

aaggccgact acgagaagca caaggtgtac gcctgcgagg ttacccacca gggcctgtcc 660

tcccccgtga ccaagtcctt caaccggggc gagtgctag 699

<210>8

<211>216

<212>PRT

<213> Artificial sequence ()

<400>8

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Arg Leu Arg Ser Thr Tyr Tyr

20 25 30

Ala Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile

35 40 45

Tyr Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly

50 55 60

Ser Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala

65 70 75 80

Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Thr Ser Gly Tyr

85 90 95

His Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Val Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210>9

<211>38

<212>DNA

<213> Artificial sequence ()

<400>9

ctgcaaccgg tgtacattcc caggtgcagc tggtgcag 38

<210>10

<211>38

<212>DNA

<213> Artificial sequence ()

<400>10

ctgcaaccgg tgtacattcc gaggtgcagc tggtgcag 38

<210>11

<211>38

<212>DNA

<213> Artificial sequence ()

<400>11

ctgcaaccgg tgtacattct gaggtgcagc tggtggag 38

<210>12

<211>38

<212>DNA

<213> Artificial sequence ()

<400>12

ctgcaaccgg tgtacattct gaggtgcagc tgttggag 38

<210>13

<211>38

<212>DNA

<213> Artificial sequence ()

<400>13

ctgcaaccgg tgtacattcc caggtgcagc tgcaggag 38

<210>14

<211>40

<212>DNA

<213> Artificial sequence ()

<400>14

ctgcaaccgg tgtacattcc caggtgcagc tacagcagtg 40

<210>15

<211>38

<212>DNA

<213> Artificial sequence ()

<400>15

ctgcaaccgg tgtacattcc caggttcagc tggtgcag 38

<210>16

<211>38

<212>DNA

<213> Artificial sequence ()

<400>16

ctgcaaccgg tgtacattcc caggtccagc tggtacag 38

<210>17

<211>38

<212>DNA

<213> Artificial sequence ()

<400>17

ctgcaaccgg tgtacattct caggtgcagc tggtggag 38

<210>18

<211>38

<212>DNA

<213> Artificial sequence ()

<400>18

ctgcaaccgg tgtacattct gaagtgcagc tggtggag 38

<210>19

<211>38

<212>DNA

<213> Artificial sequence ()

<400>19

ctgcaaccgg tgtacattcc cagctgcagc tgcaggag 38

<210>20

<211>38

<212>DNA

<213> Artificial sequence ()

<400>20

ctgcaaccgg tgtacattcc caggtacagc tgcagcag 38

<210>21

<211>32

<212>DNA

<213> Artificial sequence ()

<400>21

tgcgaagtcg acgctgagga gacggtgacc ag 32

<210>22

<211>34

<212>DNA

<213> Artificial sequence ()

<400>22

tgcgaagtcg acgctgaaga gacggtgacc attg 34

<210>23

<211>33

<212>DNA

<213> Artificial sequence ()

<400>23

tgcgaagtcg acgctgagga gacggtgacc gtg 33

<210>24

<211>40

<212>DNA

<213> Artificial sequence ()

<400>24

ctgcaaccgg tgtacattct gacatccaga tgacccagtc 40

<210>25

<211>46

<212>DNA

<213> Artificial sequence ()

<400>25

ttgtgctgca accggtgtac attcagacat ccagttgacc cagtct 46

<210>26

<211>40

<212>DNA

<213> Artificial sequence ()

<400>26

ctgcaaccgg tgtacattgt gccatccgga tgacccagtc 40

<210>27

<211>40

<212>DNA

<213> Artificial sequence ()

<400>27

ctgcaaccgg tgtacatggg gatattgtga tgacccagac 40

<210>28

<211>40

<212>DNA

<213> Artificial sequence ()

<400>28

ctgcaaccgg tgtacatggg gatattgtga tgactcagtc 40

<210>29

<211>40

<212>DNA

<213> Artificial sequence ()

<400>29

ctgcaaccgg tgtacatggg gatgttgtga tgactcagtc 40

<210>30

<211>45

<212>DNA

<213> Artificial sequence ()

<400>30

ttgtgctgca accggtgtac attcagaaat tgtgttgaca cagtc 45

<210>31

<211>40

<212>DNA

<213> Artificial sequence ()

<400>31

ctgcaaccgg tgtacattca gaaatagtga tgacgcagtc 40

<210>32

<211>46

<212>DNA

<213> Artificial sequence ()

<400>32

ttgtgctgca accggtgtac attcagaaat tgtgttgacg cagtct 46

<210>33

<211>40

<212>DNA

<213> Artificial sequence ()

<400>33

ctgcaaccgg tgtacattcg gacatcgtga tgacccagtc 40

<210>34

<211>30

<212>DNA

<213> Artificial sequence ()

<400>34

gccaccgtac gtttgatytc caccttggtc 30

<210>35

<211>30

<212>DNA

<213> Artificial sequence ()

<400>35

gccaccgtac gtttgatctc cagcttggtc 30

<210>36

<211>30

<212>DNA

<213> Artificial sequence ()

<400>36

gccaccgtac gtttgatatc cactttggtc 30

<210>37

<211>30

<212>DNA

<213> Artificial sequence ()

<400>37

gccaccgtac gtttaatctc cagtcgtgtc 30

Claims

1. An anti-H7N 9 fully human monoclonal antibody 6J15 or a biologically active fragment derived therefrom that specifically binds to H7N9, wherein the amino acid sequences of the heavy and light chain CDR1, CDR2 and CDR3 regions of said antibody are as follows:

heavy chain CDR1: GYNIFTSYD;

heavy chain CDR2, MNPDSGDT;

heavy chain CDR3: ATGNADCSGSCYNWFDP;

light chain CDR1, R L RSTYY;

light chain CDR2: GKN;

light chain CDR3: NSRDTSGYH L V.

2. The anti-H7N 9 fully human monoclonal antibody 6J15 or a biologically active fragment derived from the monoclonal antibody and capable of specifically binding to H7N9, according to claim 1, wherein the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO. 2, or the amino acid sequence with equivalent functions formed by replacing, deleting or adding one or more amino acids in the sequence shown in SEQ ID NO. 2; and/or

The amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 4, or the amino acid sequence with the same function is formed by replacing, deleting or adding one or more amino acids in the sequence shown as SEQ ID NO. 4;

preferably, the heavy chain amino acid sequence of the antibody is shown as SEQ ID NO. 6, or the amino acid sequence with the same function formed by replacing, deleting or adding one or more amino acids in the sequence shown as SEQ ID NO. 6; and/or

The light chain amino acid sequence of the antibody is shown as SEQ ID NO. 8, or the amino acid sequence with the same function is formed by replacing, deleting or adding one or more amino acids in the sequence shown as SEQ ID NO. 8.

3. A gene encoding the anti-H7N 9 fully human monoclonal antibody 6J15 of claim 1 or 2 or a biologically active fragment derived therefrom capable of specifically binding to H7N 9; preferably, the gene comprises a nucleotide sequence which codes for an amino acid with the amino acid sequence shown in SEQ ID NO. 2, more preferably, the nucleotide sequence of the gene is shown in SEQ ID NO. 1; and/or

The gene comprises a nucleotide sequence which codes amino acid shown as SEQ ID NO. 4, and preferably, the nucleotide sequence of the gene is shown as SEQ ID NO. 3.

4. The gene of claim 3, wherein the gene comprises a nucleotide sequence encoding an amino acid sequence shown as SEQ ID NO. 6, preferably the nucleotide sequence of the gene is shown as SEQ ID NO. 5; and/or

The gene comprises a nucleotide sequence which codes amino acid shown as SEQ ID NO. 8, and preferably, the nucleotide sequence of the gene is shown as SEQ ID NO. 7.

5. A vector comprising the gene of claim 3 or 4.

6. A cell containing the gene according to claim 3 or 4 or the vector according to claim 5.

7. A method of producing the anti-H7N 9 fully human monoclonal antibody 6J15 of claim 1 or 2, or a biologically active fragment derived therefrom that specifically binds H7N9, the method comprising:

culturing a genetically engineered cell comprising the gene of claim 3 or 4 or the vector of claim 5 encoding the heavy and light chain of fully human monoclonal antibody 6J15 against H7N9, or directly culturing the cell of claim 6; collecting and purifying to obtain the anti-H7N 9 fully human monoclonal antibody 6J 15.

8. A pharmaceutical composition comprising the anti-H7N 9 fully human monoclonal antibody 6J15 of claim 1 or 2 or a biologically active fragment derived therefrom that is capable of specifically binding H7N 9.

9. Use of the anti-H7N 9 fully human monoclonal antibody 6J15 of claim 1 or 2 or a biologically active fragment derived therefrom that specifically binds to H7N9, or the pharmaceutical composition of claim 8 in the manufacture of a medicament for the treatment of a disease caused by the H7N9 virus.

10. A kit for detecting the level of H7N9 virus, comprising the anti-H7N 9 fully human monoclonal antibody 6J15 of claim 1 or 2, or a biologically active fragment derived therefrom that specifically binds to H7N 9; preferably, the kit further comprises: a second antibody and an enzyme for detection or a fluorescent or radioactive label, and a buffer; preferably, the second antibody is an anti-antibody against the monoclonal antibody 6J15 of claim 1 or 2.