CN103665155A - Anti-influenza-virus broad-spectrum-neutrality neutralizing molecule 1F2 - Google Patents
Anti-influenza-virus broad-spectrum-neutrality neutralizing molecule 1F2 Download PDFInfo
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- CN103665155A CN103665155A CN201210342658.1A CN201210342658A CN103665155A CN 103665155 A CN103665155 A CN 103665155A CN 201210342658 A CN201210342658 A CN 201210342658A CN 103665155 A CN103665155 A CN 103665155A
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Abstract
The invention relates to an anti-influenza-virus broad-spectrum-neutrality neutralizing molecule 1F2 capable of neutralizing multiple influenza virus subtypes. The functions of the antibody provided by the invention are determined by specific gene sequences of genes in light chain and heavy chain variable regions; and the antibody can be combined with HA2 subunit of influenza virus hemagglutinin (HA) with native conformation, and can prevent multiple influenza virus subtypes from infecting permissive cells. By utilizing the variable region genes or complementary determining region (CDR) genes, different forms of gene engineering antibodies can be modified and produced in any expression system using prokaryotic and eukaryotic cells.
Description
Technical field
The invention provides total man's monoclonal antibody 1F2 that a strain can neutralize multiple subtype influenza virus, there are the potentiality of preventing clinically or treating multiple influenza infection.
Background technology
From in April, 2009, Influenza A H1N1 (A/H1N) epidemic situation worldwide breaks out.This epidemic situation is diffused into 5 200Duo Ge countries and regions, continent from North America bamboo telegraph.According to the World Health Organization, end on April 9th, 2010, A type A/H1N1 Influenza epidemic situation is surpassing 213 country's outbursts, and global Influenza A H1N1 death has surpassed 1.77 ten thousand examples.In China, according to CDC, by March 31st, 2010,31, whole nation province accumulative total reported Influenza A H1N1 confirmed cases 12.7 more than ten thousand people, has cured 12.2 ten thousand, dead 800 examples.
Influenza virus belongs to orthomyxoviridae family, has coating, is ball-type, ellipse or thread, and peplos is from the BLM of host cell.The RNA viruses of Influenza Virus strand.According to the difference of nucleoprotein (NP) and cytoplasm protein (M1), influenza virus can be divided into A, B, three kinds of hypotypes of C (first, second, third).To A type influenza virus, according to hemagglutinin (HA) and the antigenic difference of neuraminidase (NA) surface glycoprotein, can be further divided into different hypotypes again.At present, A type influenza virus has 16 kinds of HA hypotypes (H1-H16) and 9 kinds of NA hypotypes (N1-N9).
The genome of A type influenza virus forms (seeing Fig. 1) by 8 independent single stranded RNA fragments, each one or two albumen of RNA fragment coding.Therefore A type influenza virus has 8 genes, the 10 kinds of albumen of encoding.Wherein, HA albumen is for playing an important role with the film fusion of cell in conjunction with cell surface receptor and virus, before cells infected, HA can be hydrolyzed into HA1 and HA2, wherein be positioned at receptor binding domain (the receptor binding domain on HA1, RBD) near zone, in different strains of influenza viruses camber variations (RBD itself is more conservative), important neutrality epi-position region, the multiple neutrality antibody of finding at present for epi-position be mostly positioned at the RBD near zone of HA1 albumen.During but these antibody scarcely possess with the abilities of different subtype or the different strain influenza viruses of same hypotype.NA can remove sialic acid, helps the release of progeny virus and avoids the gathering of virus self; Tri-kinds of polysaccharases of PA, PB1 and PB2 and NP nucleoprotein are mainly used in copying and transcribing of viral RNA; Matrix prote m1 is responsible for core and is carried outward, and M2 is responsible for forming ionic channel; Non-structural protein NS 1 is the inhibitor of Interferon, rabbit, and NS2 is responsible for core and carries outward.
At present, country permits the medicine that is used for the treatment of influenza to be mainly following two large classes: 1) alkanamine class (amantadine and Rimantadine); 2) influenza virus neuraminidase inhibitor (Oseltamivir and zanamivir).To alkane amine drug, there is a large amount of resistance influenza strains, therefore the World Health Organization does not advise using alkane amine drug as the medicine for the treatment of influenza at present; Current most of influenza virus (comprising Influenza A H1N1) is still responsive to Oseltamivir and zanamivir, but in areas such as Japan, has also occurred Drug resistance strain, and the appearance of Drug resistance strain has limited the extensive use of these chemical small-molecule drugs.
Therapeutic antibodies early has report for the treatment that waits disease of viral infection of influenza, antiserum(antisera) be used for the treatment of SARS and severe H5N1 avian influenza person's case verified the vital role brought into play in treatment virus infection of antibody.Have in wide spectrum and the therapeutic antibodies of active resisiting influenza virus has following potential advantages, on the one hand it can blocking virus and the combination of target cell, by the effector cells' such as complement and T cell, NK cell effect, will be killed by the cell of virus infection on the other hand.
Therefore, this area is necessary develops for influenza virus the therapeutic antibodies that affinity is good and side effect is low, as the antibody in humanization or total man source, to meet the demand of clinical treatment.
Summary of the invention
The object of the present invention is to provide a kind of resisiting influenza virus wide spectrum neutrality total man's monoclonal antibody and application thereof.
In one aspect of the invention, provide a kind of binding molecule of separation, the epi-position in described binding molecule identification (being preferably specific recognition) influenza virus HA albumen between HA2 protein protomer sequence 345-504 position.
In a preference, described binding molecule comprises heavy chain CDR3 district shown in heavy chain CDR2 shown in heavy chain CDR1 district shown in SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10; It can be identified and in conjunction with the epi-position in the HA2 subunit of influenza hemagglutinin protein (HA).
In another preference, described binding molecule comprises light chain CDR3 district shown in light chain CDR2 shown in light chain CDR1 district shown in SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16; It can be identified and in conjunction with the epi-position in the HA2 subunit of influenza hemagglutinin protein (HA).
In another preference, described binding molecule comprises following aminoacid sequence: the heavy chain CDR1 shown in SEQ ID NO:8, heavy chain CDR3 shown in heavy chain CDR2 shown in SEQ ID NO:9 and SEQ ID NO:10, the light chain CDR3 shown in the light chain CDR2 shown in the light chain CDR1 shown in SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16; It can be identified and in conjunction with the epi-position in the HA2 subunit of influenza hemagglutinin protein (HA).
In another preference, described binding molecule comprises variable region of heavy chain, and described variable region of heavy chain has the aminoacid sequence shown in SEQ ID NO:2.
In another preference, described binding molecule comprises variable region of light chain, and described variable region of light chain has the aminoacid sequence shown in SEQ ID NO:4.
In another preference, described binding molecule comprises variable region of heavy chain and variable region of light chain, and its variable region of heavy chain and variable region of light chain have respectively the aminoacid sequence shown in SEQ ID NO:2 and SEQ ID NO:4.
In another preference, the CH that described binding molecule comprises antibody and or constant region of light chain.
In another preference, described binding molecule is Fab, F (ab '), F (ab ') 2, Fv, dAb, Fd, complementary determining region (CDR) fragment, single-chain antibody (scFv), divalence single-chain antibody, single chain variable fragment phage antibody, two special double-chain antibody, three chain antibodies, four chain antibodies.
Preferably, described binding molecule is human monoclonal antibodies.
Preferred, its variable region of heavy chain of described human monoclonal antibodies and variable region of light chain have respectively the aminoacid sequence shown in SEQ ID NO:2 and SEQ ID NO:4, its CH is selected the constant region of one of heavy chain type in lower group: IgG1, IgG2a, IgG2b and IgG3, and its constant region of light chain is selected one of constant region of lower group light chain type: κ chain and λ chain.
Preferred, its variable region of heavy chain of described human monoclonal antibodies and variable region of light chain have respectively the aminoacid sequence shown in SEQ ID NO:2 and SEQ ID NO:4, and its CH and constant region of light chain have respectively the aminoacid sequence shown in Genebank ACK87036 and ACK87038.
In another preference, the epi-position on described monoclonal antibody identification influenza virus HA albumen between linearity and space conformation.
In another preference, the epi-position (the HA2 albumen of the calculating of sequence and figure place based on GenBank accession number ACP41105.1) in described monoclonal antibody identification influenza virus HA albumen between HA2 protein protomer sequence 345-504 position.
The nucleic acid molecule of the described binding molecule of coding is provided in one aspect of the invention.
In one aspect of the invention, provide described binding molecule for the preparation of diagnosis, treat and/or prevent the purposes in the medicine of the influenza infection due to influenza virus.
In another preference, the strain of described influenza virus is selected from as next group: H1 subtype influenza virus (as H1N1), H3 subtype influenza virus (as H3N2) and H9 subtype influenza virus (H9N2).
In one aspect of the invention, provide a kind of expression vector, in described expression vector, contain the DNA of the described binding molecule of coding.
In one aspect of the invention, provide a kind of host cell, in described host cell, contain described expression vector.
In one aspect of the invention, provide a kind of composition, the described monoclonal antibody that it contains significant quantity, and pharmaceutically acceptable carrier.
In one aspect of the invention, provide a kind of test kit that detects influenza virus, it comprises described binding molecule.
In another preference, described test kit also comprises: antigen or antibody are coated with reagent, washing reagent, second antibody, marker (as horseradish peroxidase, alkaline phosphatase, glucose oxidase, β – D-tilactase, urase, catalase or glucoamylase), developer enzyme substrates etc.
In one aspect of the invention, provide a kind of (preferably non-therapeutic ground) to suppress the method for influenza virus, described method comprises the described binding molecule that gives patient's significant quantity.
In one aspect of the invention, provide a kind of (preferably diagnostically non-) to detect the method for influenza virus, utilize described binding molecule to contact with testing sample, by detecting described binding molecule and the combination situation of given the test agent, acquisition influenza virus there is situation and amount.
Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.
Accompanying drawing explanation
Fig. 1, A type influenza virus structural representation.
The electrophoresis result of the pcr amplification product of Fig. 2, β-actin internal reference.
The electrophoresis result of the pcr amplification product of Fig. 3, heavy chain gene.
The electrophoresis result of the pcr amplification product of Fig. 4, light chain gene.
Fig. 5, total man's monoclonal antibody (1F2) concentration.
Fig. 6, total man's monoclonal antibody (1F2) are identified the recognition capability of HA/HA1 albumen.
Fig. 7, total man's monoclonal antibody (1F2) identification HA2 Protein G 345 are to P504 peptide section.
The broad spectrum of Fig. 8, total man's monoclonal antibody (1F2) identification influenza virus.
In the wide spectrum of Fig. 9, total man's monoclonal antibody 1F2 and active.A：A/Sichuan/1/2009(H1N1)；B：A/Jiangxi-Donghu/312/2006(H3N2)；C：A/Guangzhou/333/99(H9N2)。
The plasmid map of Figure 10, carrier A bVec-hIgG.
The plasmid map of Figure 11, AbVec-hIgKappa.
Embodiment
The inventor is through extensive and deep research, obtain a kind of binding molecule of the resisiting influenza virus wide spectrum neutrality that contains unique complementary determining region (CDR district), preferred total man's monoclonal antibody, this binding molecule has wide spectrum neutralizing effect for influenza virus.Completed on this basis the present invention.
Binding molecule
The invention provides the binding molecule of energy specific binding influenza virus.Preferably, described binding molecule is human binding molecules.Preferably, binding molecule of the present invention presents for the neutralization of influenza virus active.
Binding molecule of the present invention can be complete immunoglobulin molecules, described binding molecule can be Fab, includes but not limited to Fab, F (ab '), F (ab ') 2, Fv, dAb, Fd, complementary determining region (CDR) fragment, single-chain antibody (scFv), divalence single-chain antibody, single chain variable fragment phage antibody, two special double-chain antibody, three chain antibodies, four chain antibodies and at least contains (many) peptides or its fragment that is enough to give with the fragment of the specific antigens binding domain-immunoglobulin of fowl influenza virus strain.
Binding molecule of the present invention also can specific binding influenza virus one or more fragment.For treating and/or preventing the method for influenza virus, the surface that described binding molecule preferably can specific binding influenza virus can and protein.In specific embodiment, the HA2 molecule of binding molecule energy specific binding influenza virus of the present invention.
The application of binding molecule described in the present invention also provides in preparing the medicine of diagnosing, prevent and/or treat influenza infection.This infection can occur in microcommunity, but also can at world wide, propagate in prevailing disease mode in season, or more seriously at global spread, millions of individualities are in danger.The invention provides to neutralize and cause prevailing disease and the binding molecule of the infection of potential global epiphytotics influenza virus strain in this season.Importantly, can expect at present and utilize binding molecule of the present invention to play protection and therapeutic action for multiple influenza virus strain, because disclosed due to and the combination of shared epi-position between the HA albumen of various flows Influenza Virus strain, therefore can use at present binding molecule of the present invention between these strains, to carry out cross-neutralization.Binding molecule of the present invention can be prepared on a large scale and store, because it provides the provide protection for different popular strains, and is favourable for preparing for contingent flu outbreak in the future.
CDR district is the sequence of the interested protein of immunology.In embodiments of the invention, binding molecule can comprise two, three, four, five or all Liu Ge CDR district disclosing herein.Preferably, binding molecule of the present invention comprises at least two CDR that disclose herein.
Another aspect of the present invention comprises the functional variant of binding molecule described herein.If variant can with parental generation binding molecule competition specific binding influenza virus or its protein fragments, think the functional variant that this variant molecule is binding molecule of the present invention.In other words, described functional variant still can be in conjunction with influenza virus or its fragment.Preferably, the competitive specific binding of described functional variant energy is by the different influenza virus strain of at least two (or more) of parental generation binding molecule specific binding or its fragment.In addition,, if certain molecule has the active influenza virus of neutralization, preferably has neutralization activity at least two (or a plurality of) influenza virus strain it for parental generation binding molecule, think that this molecule is the functional variant of binding molecule of the present invention.Functional variant include but not limited to primary structure sequence substantially similar, but for example contain in parental generation binding molecule the derivative of chemistry in undiscovered external or body and/or biochemical modification.This modification comprise covalent attachment, lipid or the lipid derivate of second phthalein, phthalein, Nucleotide or nucleotide derivative covalent attachment, crosslinked, disulfide linkage formation, glycosylation, hydroxylation, methylate, oxidation, Pegylation, proteolysis processing, phosphorylation etc.In other words, the amino acid of parental generation binding molecule and/or the not remarkably influenced of modification in nucleotide sequence or change the binding characteristic by described described binding molecule nucleotide sequence coded or that contain described aminoacid sequence, described binding molecule still can be identified and in conjunction with its target position.
Described functional variant can have conserved sequence to be modified, and comprises Nucleotide and aminoacid replacement, interpolation and disappearance.These modifications can import by standard technique known in the art, for example mutagenesis of directed mutagenesis and random PCR mediation, and can comprise natural and non-natural nucleotide and amino acid.
Conserved amino acid replaces and comprises that wherein amino-acid residue is by the replacement with another radical amino acid replacement of analog structure or chemical property.The family with the amino-acid residue of similar side chain limits in the art.These families comprise the amino acid (Methionin for example with basic side chain, arginine, Histidine), acid side-chain amino acid (aspartic acid for example, L-glutamic acid), for example, without charge polarity side chain amino acid (asparagus fern phthalein amine, paddy ammonia phthalein amine, Serine, Threonine, tyrosine, half skin propylhomoserin, tryptophane), non-polar sidechain amino acid (glycine for example, L-Ala, α-amino-isovaleric acid, leucine, Isoleucine, proline(Pro), phenylalanine, methionine(Met)), branched building block amino acid (Threonine for example, α-amino-isovaleric acid, Isoleucine) and aromatic side chain amino acid (tyrosine for example, phenylalanine, tryptophane).Those skilled in the art understand other amino-acid residue family classification mode except above-mentioned family of also can using.In addition, variant can have nonconservative aminoacid replacement, and for example amino acid is by another radical amino acid replacement with different structure or chemical property.Similar little variation also can comprise aminoacid deletion or insertion, or these two.Use computer program well known in the art can find to determine which amino-acid residue can be substituted, inserts or lack and do not eliminate the guidance of immunologic competence.
In addition, functional variant can comprise aminoacid sequence at the truncate at N-terminal or C-terminal or these two ends.Functional variant of the present invention is compared and can be had identical or different, higher or lower binding affinity with parental generation binding molecule, but still can be in conjunction with influenza virus or its fragment.For example, functional variant of the present invention is compared with parental generation binding molecule for influenza virus H1N1 or its fragment and can be had the binding affinity that increases or reduce.Preferably, variable region includes but not limited to that the aminoacid sequence in framework region, HuoCDR district, hypervariable region is modified.Conventionally, light chain and variable region of heavy chain comprise three hypervariable regions, comprise three CDR, and more conservative region, i.e. so-called framework region ((FR).Hypervariable region comprises from the amino-acid residue of CDR with from the amino-acid residue of hypermutation ring.Functional variant within the scope of the present invention and parental generation binding molecule described herein have at least about 50% to about 99%, preferably at least about 60% to about 99%, more preferably at least about 70% to about 99%, even more preferably at least about 80% to about 99%, most preferably at least about 90% to about 99%, particularly at least about 95% to about 99%, and at least about 97% to about 99% amino acid sequence homology particularly.Computerized algorithm well known by persons skilled in the art can be used for best arranged amido acid sequence to contrast and precisely similar or identical amino-acid residue as Gap or Bestfit.Functional variant can be by being used common molecular biology method known in the art to change parental generation binding molecule or its a part of acquisition, and described method includes but not limited to mutagenesis, site-directed mutagenesis and heavy chain and/or the light chain reorganization method that fallibility PCR, oligonucleotide instruct.In one embodiment, functional variant of the present invention has neutralization activity for influenza virus.Active the comparing with parental generation binding molecule of described neutralization can be identical or higher or lower.After this, when using term (people) binding molecule, it also contains the functional variant of described (people) binding molecule.
As optimal way of the present invention, described binding molecule is monoclonal antibody, and preferably it comprises the constant region (as people source constant region IgH sequence and IgKappa sequence) in people source.Variable region of heavy chain, the variable region of light chain of described resisiting influenza virus monoclonal antibody and be positioned at variable region of heavy chain and the complementary determining region of variable region of light chain (CDR) all has unique structure that is different from prior art, and they are total man sources.
The present invention includes: there is the monoclonal antibody of the corresponding aminoacid sequence of described monoclonal antibody, there is the monoclonal antibody of described variable region of mab chain.The present invention also comprises having containing the described light chain of complementary determining region (CDR) and any antibody of heavy chain, and the CDR of CDR district and monoclonal antibody of the present invention has any antibody of more than 90% homology of (preferably more than 95%).
The antigenic binding property of antibody can be described by 3 specific regions that are positioned at heavy chain and variable region of light chain, be called complementary determining region (complementarity determining region, CDR), described CDR district is partitioned into 4 frame areas (FR) by variable region, the aminoacid sequence of 4 FR is relatively conservative, does not participate in association reaction directly.These CDR form ring texturees, and the β-pleated sheet structure that the FR by therebetween forms is mutually close on space structure, and the CDR on the CDR on heavy chain and corresponding light chain has formed the antigen binding site of antibody.Can determine be which Amino acid profile FR or CDR region by the aminoacid sequence of antibody more of the same type.
For monoclonal antibody heavy chain of the present invention and sequence of light chain, can measure by ordinary method.
Empirical tests, resisiting influenza virus monoclonal antibody CDR of the present invention district is brand-new, its for be an epitope on unique influenza virus HA albumen, technical conceive is different from existing antibodies against influenza virus.The epi-position that resisiting influenza virus monoclonal antibody of the present invention is identified is between linearity and space conformation; More preferably described monoclonal antibody is identified the epi-position between HA2 protein protomer sequence 345-504 position in influenza virus HA albumen.
Monoclonal antibody of the present invention is total man source, and its heavy chain, variable region of light chain and constant region all derive from people's antibody.Therefore, it,, when having the effect of excellent especially identification and neutralized stream Influenza Virus, also has low, the safe feature of immunogenicity.
In an embodiment of the present invention, the present invention obtains peripheral blood mononuclear cell (PBMC) from inoculating in H1N1virus split vaccine (2009/Cal) volunteer body in 2009, uses CD19
+/ IgG
+/ HA is Specific marker, through selected by flow cytometry apoptosis (FACS), obtains identification HA protein-specific B cell.The single-cell RT-PCR technology (Journal of Immunological Methods 329 (2008) 112-124) of using document to report, has obtained antibody gene and in 293T cell, has expressed acquisition total man monoclonal antibody 1F2.ELISA epitope analysis is learnt the HA2 subunit of this strain antibody identification hemagglutinin HA albumen, and HA2 has conservative property in different genera influenza virus, shows the identification broad spectrum that this strain antibody is possible.Micro-neutralization experimental results show that this antibody can in and A/Sichuan/1/2009 (H1N1), A/Jiangxi-Donghu/312/2006 (H3N2), A/Guangzhou/333/99 (H9N2) influenza virus, show that this strain antibody has in wide spectrum and activity, has the potential value of preventing and treating multiple subtype influenza virus infection clinically.
On the other hand, the present invention includes immunoconjugates, comprise at least one binding molecule described herein and further comprise at least one mark as the molecule of detectable part/material.The invention still further relates to the mixture of immunoconjugates of the present invention or the mixture of at least one immunoconjugates of the present invention and another molecule, described another molecule is as therapeutical agent or another binding molecule or immunoconjugates.Immunoconjugates of the present invention can comprise more than one mark.These marks can be same to each other or different to each other, and can noncovalently be combined/put together with binding molecule.Described mark also can by covalent linkage and human binding molecules directly in conjunction with/put together.Or described mark can connect compound by one or more and be combined/put together with described binding molecule.The conjugation techniques of mark and binding molecule is well known to those skilled in the art.
The mark of immunoconjugates of the present invention can be therapeutical agent, but they can be also detectable part/materials.The mark that is suitable for treating and/or preventing can be other binding molecule of toxin or its funtion part, microbiotic, enzyme, enhancing phagolysis or immunostimulation.The immunoconjugates diagnosticability that comprises detectable substance for for example evaluate object whether influenza virus infection strain or as the generation of the part monitoring influenza infection of clinical experiment program or progress for example to determine the effect of TA scheme.Yet they also can be for other detection and/or analysis and/or diagnostic purpose.Detectable part/material includes but not limited to enzyme, prothetic group, fluorescent material, luminescent material, bioluminescent material, radio active material, positron emitting metal and on-radiation paramagnetic metal ion.In order to detect and/or analysis and/or diagnostic purpose depend on the particular detection/analysis/diagnostic techniques of use and/or method such as immunohistochemical staining (tissue) sample, flow cytometry, the detection of laser scanning cytometry, fluorescence immunoassay, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), biological assay (such as phagolysis mensuration), western blotting application etc. for the mark of mark binding molecule.For detection/analysis/diagnostic techniques known in the art and/or the suitable mark of method, be well known to those skilled in the art.
In addition, human binding molecules of the present invention or immunoconjugates also can be attached on solid support, and it is used in particular in vitroimmunoassay or the purifying of influenza virus HA albumen or its fragment.This solid support can be porous or atresia, plane or nonplanar.Binding molecule of the present invention can be with flag sequence if skin fusion be so that purifying.The example of described flag sequence includes but not limited to six histidine marks, hemagglutinin (HA) mark, myc mark or flag mark.Or a kind of antibody can be puted together and form antibody allos conjugate (heteroconjugate) with another kind of antibody.
On the other hand, binding molecule of the present invention can be puted together/adhere to one or more antigen.Preferably, these antigens are by the antigen of immune system recognition that has given the object of binding molecule-antigen conjugate.Described antigen can be mutually the same, but can be also different.The conjugation methods that makes to adhere to antigen and binding molecule is known in the art, includes but not limited to use linking agent.Binding molecule of the present invention is attacked in conjunction with influenza virus and the antigen that is attached to binding molecule the strong T cell causing for described conjugate, finally causes the destruction of influenza virus.
Except for example, puting together by direct or indirect (passing through joint), chemistry produces immunoconjugates, and described immunoconjugates can be used as fusion rotein and produces, and described fusion rotein comprises binding molecule of the present invention and suitable mark.Fusion rotein can produce by means known in the art, for example, by building nucleic acid molecule and expressing subsequently generations of recombinating of described nucleic acid molecule, the nucleotide sequence of the nucleotide sequence that described nucleic acid molecule comprises in-frame coding binding molecule and the appropriate flags of encoding.
The present invention provides the nucleic acid molecule of encode at least one binding molecule of the present invention, its functional variant or immunoconjugates on the other hand.This nucleic acid molecule can be as intermediate to clone, for example, for affinity maturation method described above.In a preferred embodiment, described nucleic acid molecule is isolated or purified.The sequence of DNA molecular can be used routine techniques, or utilizes hybridoma technology to obtain.
The functional variant that those skilled in the art will recognize that these nucleic acid molecule is also a part of the present invention.Functional variant is such nucleotide sequence, by using standard genetic code it directly can be translated so that the aminoacid sequence identical with the sequence of translating from parental generation nucleic acid molecule to be provided.
Once obtain relevant sequence, just can obtain in large quantity relevant sequence with recombination method.This is normally cloned into carrier, then proceeds to cell, then by ordinary method separation from the host cell propagation, obtains relevant sequence.
In addition, also can synthesize relevant sequence by the method for synthetic, especially fragment length more in short-term.Conventionally, by first synthetic a plurality of small segments, and then connect and can obtain the fragment that sequence is very long.
At present, can be completely by chemosynthesis obtain the encoding DNA sequence dna of binding molecule of the present invention (or its fragment, or derivatives thereof).Then this DNA sequence dna can be introduced in various existing DNA moleculars as known in the art (or as carrier) and cell.In addition also can sudden change be introduced in the sequence of binding molecule of the present invention by chemosynthesis.
The invention still further relates to the carrier that comprises above-mentioned suitable DNA sequence dna and suitable promotor or control sequence.These carriers can be for transforming suitable host cell, with can marking protein.Preferably, carrier of the present invention is for example to contain the plasmid expression vector of viral promotors, and in described expression vector, has inserted respectively IgH (from the constant region of people source IgH) fusion sequence and variable region of light chain VL and human body Igkappa (from the constant region of the people source Igkappa) fusion sequence of resisiting influenza virus monoclonal antibody variable region of heavy chain (VH) with constant region.
Host cell can be prokaryotic cell prokaryocyte, as bacterial cell; Or the eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: bacterial cell is as intestinal bacteria, streptomyces; Salmonella typhimurium; Fungal cell is as yeast; Vegetable cell; Insect cell is as fruit bat S2 or Sf9; Zooblast is as CHO, COS7, NSO or Bowes melanoma cells etc.Being specially adapted to host cell of the present invention is eukaryotic host cell, and mammalian cell especially, as 293 cells.
With recombinant DNA transformed host cell, can carry out with routine techniques well known to those skilled in the art.When host is prokaryotic organism during as intestinal bacteria, the competent cell that can absorb DNA can, in exponential growth after date results, be used CaCl
2method is processed, and step used is well-known in this area.Another kind method is to use MgCl
2.If needed, the also method of available electroporation that transforms is carried out.When host is eukaryote, can select following DNA transfection method: calcium phosphate precipitation, or conventional mechanical method is as microinjection, electroporation, liposome packing etc.
The transformant obtaining can be cultivated by ordinary method, expresses binding molecule of the present invention.According to host cell used, substratum used in cultivation can be selected from various conventional mediums.Under the condition that is suitable for host cell growth, cultivate.When host cell grows into after suitable cell density, the promotor of selecting with suitable method (as temperature transition or chemical induction) induction, cultivates cell for some time again.
Binding molecule of the present invention preferably adopts mammalian cell to produce, and mammalian cell need to cultivated containing in the substratum of serum conventionally.Need to carry out after the adaptive process of serum-free cell, can allow cell grow normally in serum free medium.
If needed, can utilize the albumen of and purification of Recombinant separated by various separation methods with other characteristic its physics, chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation processes, with protein precipitant, process the combination of (salt analysis method), centrifugal, the broken bacterium of infiltration, supersound process, ultracentrifugation, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.
Binding molecule of the present invention also can produce in as rabbit, goat or ox at transgenic nonhuman mammal, and is secreted into for example its Ruzhong.
Pharmaceutical composition
Binding molecule of the present invention can be used for the composition that preparation suppresses influenza virus.
Based on new discovery of the present invention, a kind of composition that suppresses influenza virus or influenza infection relative disease is also provided, it comprises: the binding molecule of the present invention of significant quantity; And pharmaceutically acceptable carrier.
Term used herein " pharmaceutically acceptable " refers to when molecule body and composition suitably give animal or human, and they can not produce disadvantageous, irritated or other untoward reaction." pharmaceutically acceptable carrier " used herein should be compatible with binding molecule of the present invention, can significantly not reduce under normal conditions the effect of composition with its blend.
The object lesson that can be used as some materials of pharmaceutically acceptable carrier or its component is carbohydrate, as lactose, dextrose plus saccharose; Starch, as W-Gum and potato starch; Mierocrystalline cellulose and derivative thereof, as Xylo-Mucine, ethyl cellulose and methylcellulose gum; Tragakanta powder; Fructus Hordei Germinatus; Gelatin; Talcum; Solid lubricant, as stearic acid and Magnesium Stearate; Calcium sulfate; Vegetables oil, as peanut oil, Oleum Gossypii semen, sesame oil, sweet oil, Semen Maydis oil and theobroma oil; Polyvalent alcohol, as propylene glycol, glycerine, Sorbitol Powder, mannitol and polyoxyethylene glycol; Lalgine; Emulsifying agent, as
wetting agent, as Sodium Lauryl Sulphate BP/USP; Tinting material; Seasonings; Tablet agent, stablizer; Antioxidant; Sanitas; Apirogen water; Deng oozing salts solution; With phosphate buffered saline buffer etc.
Composition of the present invention can be made various formulations as required, and can by doctor according to patient's kind, age, body weight and roughly the factor such as disease condition, administering mode determine the useful dosage of patient used.Administering mode for example can adopt injection or other therapeutic modality.
Binding molecule of the present invention can be used with unsegregated or separated form.In addition, binding molecule of the present invention can be applied separately or apply in the mixture that comprises at least one binding molecule of the present invention (or its variant or fragment).In other words, described binding molecule can applied in any combination, for example, as comprising two or the pharmaceutical composition of more kinds of binding molecule of the present invention, its variant or fragment.For example, there is difference but the binding molecule of complementary activity can be combined in a treatment plan to reach prevention, treatment or the diagnostic effect of hope, but or also the binding molecule with identical activity can be combined in a treatment plan to reach prevention, treatment or the diagnostic effect of hope.Optionally, described mixture further comprises at least one other therapeutical agent.Preferably, for example, as MZ inhibitor (amantidine, Rimantadine (rimantadine)) and/or neuraminidase inhibitor, (for example zanamivir ((zanamivir), Oseltamivir (oseltamivir)) can be used for preventing and/or treating influenza infection to described therapeutical agent.
Described pharmaceutical composition can comprise two or more and have the active binding molecule of neutralization for influenza virus.In one embodiment, when applied in any combination, it is active that described binding molecule presents collaborative neutralization.In other words, described composition comprises at least two kinds and has the active binding molecule of neutralization, is characterised in that described binding molecule plays synergy in neutralized stream Influenza Virus.As used herein, term " is worked in coordination with " and is referred to when applied in any combination, the adduction of the compound action of binding molecule during higher than independent application.Described synergistic binding molecule can be in conjunction with the different structure in the identical or different fragment of influenza virus.Calculating synergistic mode is to calculate by combinatorial index.The concept of combinatorial index (CI) is described by Chou and Talalay (1984).
Binding molecule of the present invention or drug regimen can detect at suitable animal model system before for human body.This animal model system includes but not limited to mouse, ferret (ferret) and monkey.In Influenza Virus influenza virus, also can act synergistically.
For example can adjust dosage regimen, so that best required replying (treatment is replied) to be provided.Suitable dosage range can be for example 0.01-100mg/kg body weight, preferably 0.1-15mg/kg body weight.In addition, for example can give once to inject, give in time repeatedly separate doses or can reduce in proportion or increase dosage according to the emergency for the treatment of situation.Molecule of the present invention and composition are preferably aseptic.The method that makes these molecules and composition sterile is known in the art.For other molecule of diagnosing, preventing and/or treating, can give with the dosage regimen similar to binding molecule of the present invention.If give separately other molecule, can be before giving one or more human binding molecules of the present invention or pharmaceutical composition, simultaneously or give afterwards patient.Accurate dosage regimen for people patient is picked out conventionally during clinical experiment.
Detection reagent and test kit
Binding molecule of the present invention can be used for reagent or the test kit that preparation detects influenza virus.
As used herein, several samples type contained in term " testing sample ", comprises blood and other humoral sample of biological origin, and solid tissue sample is as biopsy sample or tissue culture, or derived from cell or its offspring wherein.This term is also included in the sample of having processed by any mode after acquisition, for example, use some composition of agent treated, dissolving or enrichment as protein or polynucleotide.The various clinical samples that derive from any species contained in this term, also comprises cultured cells, cell conditioned medium and cell lysates.
Take described binding molecule as basis, can easy to prepare, fast and accurately detect influenza virus (as influenza A virus; More particularly as H1N1, H3N2, H9N2 virus) test kit.
Therefore, the invention provides a kind ofly for detection of whether there is the detection kit of influenza virus in sample, in this test kit, contain the binding molecule of resisiting influenza virus of the present invention.
After having obtained binding molecule provided by the invention, can prepare easily the detection kit for specific detection influenza virus.
As a kind of detection mode of the present invention, adopt indirect elisa method, by be measured antigen coated on solid phase carrier, utilize binding molecule of the present invention to detect.
As a kind of optimal way of the present invention, described binding molecule is antibody, can detect according to double antibodies sandwich ratio juris.The way of double antibodies sandwich method routine is that primary antibodie (as monoclonal antibody of the present invention) is fixed on to carrier, then make primary antibodie and antigen-reactive, after washing again with two anti-reactions (the described two anti-detectable signals that carry, or can be combined with the material that carries detectable signal), finally carry out chemoluminescence or enzyme connection color reaction detection signal.Double antibodies sandwich method is specially adapted to have the detection of the antigen of two or more epi-positions.
For more convenient when detecting, in described test kit except containing binding molecule of the present invention, can also comprise other detection reagent or auxiliary reagent, described auxiliary reagent is for example conventional some reagent that use in ELISA test kit, the characteristic of these reagent and their compound method are all well-known to those skilled in the art, as developer, marker, two anti-, anti-antibody, sensitizer etc.Those skilled in the art should be understood that the detection kit of various versions is all included in the present invention, as long as utilized therein binding molecule of the present invention as the reagent of identification influenza virus.
In addition, in described test kit, also can comprise working instructions, for the using method of the reagent wherein loading is described.
After having obtained binding molecule provided by the invention and/or test kit, can utilize panimmunity methods involving to detect HA albumen or its content in sample, thereby whether the donor of learning testing sample influenza virus infection, and these methods are all in the present invention involved.Preferably, described method be take non-medical diagnosis on disease as object.
As a kind of optimal way, the invention provides the method that a kind of external (non-diagnosis or therapeutic ground) detects influenza virus, comprise the following steps:
(a1) testing sample is coated in to solid phase carrier;
(a2) by binding molecule application of sample of the present invention in the solid phase carrier of (a1), thereby the influenza virus in testing sample is combined with binding molecule, form the solid phase carrier with " influenza virus-binding molecule of the present invention " binary complex;
(a3) by the detection thing application of sample of specific binding binding molecule of the present invention in the solid phase carrier of (a2), form the solid phase carrier with " influenza virus-binding molecule-detection thing of the present invention " ternary complex; On described detection thing, carry a marker;
(a4) detect the marker in ternary complex, the existence of determining influenza virus in detected sample whether with or the amount that exists.
According to the method described above, as long as the antigen control of concentration known is set, make concentration standard curve, by just drawing the influenza virus content in testing sample according to concentration standard curve.
Major advantage of the present invention is:
(1) provide a kind of brand-new binding molecule that has, it is total man source, and the molecule of animal derived with other (as mouse) resisiting influenza virus is compared, and immunogenicity greatly reduces, and affinity is good.Not only result for the treatment of is good, and side effect is low.
(2) binding molecule of the present invention can, in conjunction with the HA2 subunit with the influenza virus hemagglutinin albumen (HA) of native conformation, can stop multiple subtype influenza virus infection permissive cell.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, writes molecular cloning experiment guide, the condition described in Science Press, or the condition of advising according to manufacturer conventionally as J. Pehanorm Brooker etc. according to normal condition.
I. materials and methods
Below illustrate that the i.e. strain of the present invention can neutralize multiple influenza virus wide spectrum neutrality total man monoclonal antibody preparation and antibody specificity analysis process.Be divided into two portions:
(1) single-cell RT-PCR method obtains antibody gene and antibody expression;
(2) antibody specificity analysis.
Detailed process is as follows:
1, the acquisition of peripheral blood lymphocytes (PBMC)
From according to extracting peripheral blood in volunteer's body of blood clotting inhibition experiment screening, adopt conventional Ficoll-Paque density gradient centrifugation, obtain 10
7with last peripheral blood lymphocytes (PBMC).
2, HA specificity memory B cell sorting
(FITC-CD 19/APC-IgG is purchased from BD to use FITC-CD 19/APC-IgG/Cy3-HA, Cy3-HA preparation method is: by baculovirus insect cell protein expression system (Invitrogen), express preparation HA albumen, and with after biotin protein labelling kit (Thermo Scientific) mark, with Cy3-Streptavidin (Sigma) coupling) be mark, through flow cytometer, obtain specific b cells to 96 hole RT-PCR plate, one, every hole cell, obtains HA specificity memory B cell.
3, antibody gene
Use RT-PCR and Nested-PCR technology, obtain antibody gene (referring to Nat Protoc.2009; 4 (3): 372-84).Connect pGEM-T Easy (Promega) carrier, check order, ordinary method checking antibody gene.As document Kenneth Smith et al.Rapid generation of fully human monoclonal antibodies specific to a vaccinating antige.Nat Protoc.2009; Carrier construction AbVec-hIgG and AbVec-hIgKappa described in 4 (3): 372 – 384, carrier collection of illustrative plates is respectively as Figure 10 and 11, sequence is respectively described in genebank FJ475055 and FJ475056, wherein comprise respectively the base sequence of encoding human IgG1 heavy chain and constant region of light chain, the aminoacid sequence of encoding human IgG1 heavy chain and constant region of light chain is described in Genebank ACK87036 and ACK87038.Then weight, light chain gene are connected respectively and express carrier A bVec-hIgG (insertion point is AgeI and SalI) and AbVec-hIgKappa (insertion point is AgeI and BsiwI).
4, antibody expression
Liposome method transient transfection 293T cell, carries out human antibody expression.
1) first 1 day of transfection by 1.0 * 10
6cell is inoculated in 6 porocyte culture plates;
2) A pipe: 500 μ l Opti-MEM (Invitrogen)+4 μ g IgG+4 μ g IgL
3) B pipe: 500 μ l Opti-MEM+20 μ l Lipofectamine Reagent (Invitrogen)
4), after standing 5 minutes, A pipe is mixed with B pipe, at the standing 20min of room temperature;
5) A, B pipe mixture adds in cell cultures dish, hatches 6h for 37 ℃;
6) after 6h, absorb the nutrient solution that contains Lipofectamine Reagent, every hole adds 2mlFreeStyle
tM293Expression Medium (Invitrogen);
7), after 72h, collect the cell conditioned medium containing human antibody, 4 ℃, 3000rpm, 5min, saves backup.
5, antibody concentration is measured
ELISA method is measured human antibody concentration.
1) be coated with Goat Anti-Human IgG (Fab Specific) Antibody (purchased from S igma) in elisa plate, 10 μ g/ml, every hole 100 μ l, 4 ℃ are spent the night;
2) PBST washes plate, 3 times;
3) sealing: 1g/mlBSA, every hole 200 μ l, 37 ℃, 2h;
4) PBST washes plate, 3 times;
5) use human IgG (purchased from Sigma) the production standard curve of doubling dilution, concentration 400ng/ml, 200ng/ml, 100ng/ml, 50ng/ml, 25ng/ml, 12.5ng/ml, 6.25ng/ml, 0ng/ml, 100 μ l/ holes; 5000 times of the human antibody cell conditioned medium dilutions of aforementioned preparation, every hole 100 μ l, 37 ℃, 2h;
6) PBST washes plate, 3 times;
7) add Goat Anti-Human IgG (Fc specific)-Peroxidase antibody (purchased from Sigma), 1:10000 dilution, every hole 100 μ l, 37 ℃, 1h;
8) PBST washes plate, 3 times;
9) tmb substrate (purchased from Sigma), every hole 100 μ l, 37 ℃, 15min, lucifuge reaction;
10) add 2M H
2sO
4, every hole 50 μ l;
11) measure OD450, and carry out data processing.
6, antigen-specific detects
Detect the human antibody of expressing and whether identify HA albumen, HA1 albumen, 2009 H1N1virus vaccine lysates (2009/Cal) and 2008-2009 seasonal current influenza virus vaccine lysate (A/Brisbane/59/2007 (H1N1)).ELISA detects, respectively coated HA-His (baculovirus insect cell protein expression system (Invitrogen) expression), 100 ℃ of 5 minutes thermally denature HA-His, HA1-Fc (baculovirus insect cell protein expression system (Invitrogen) expression), 2009 Influenza A H1N1 vaccine lysates (2009/Cal) (blue biological purchased from China) and 2008-2009 seasonal current influenza vaccine lysate (A/Brisbane/59/2007 (H1N1)) (blue biological purchased from China).
1) be coated with respectively HA-His, thermally denature HA-His, HA1-Fc, 2009 Influenza A H1N1 vaccine lysates (2009/Cal) and 2008-2009 seasonal current influenza vaccine lysate (A/Brisbane/59/2007 (H1N1)) in elisa plate, 10 μ g/ml, two multiple holes of each sample, every hole 100 μ l, 4 ℃ are spent the night;
2) PBST washes plate, 3 times;
3) sealing: 1%BSA, every hole 200 μ l, 37 ℃, 2h;
4) PBST washes plate, 3 times;
5) according to measuring concentration, adjust human antibody to 300 μ g/ml of the present invention, every hole 100 μ l, (conformation that identification is positioned on HA1 relies on epi-position to mouse resource monoclonal antibody S-95-7, it is preserved in Chinese Typical Representative culture collection center, preserving number CCTCC NO:C201024) positive contrast, 37 ℃, 2h;
6) PBST washes plate, 3 times;
7) Goat Anti-Human IgG (Fc specific)-Peroxidase antibody, dilution in 1: 10000, every hole 100 μ l, are added to HA-His, thermally denature HA-His, 2009 H1N1virus vaccine lysates and 2008-2009 seasonal current influenza virus vaccine lysate elisa plate; GoatAnti-Human IgG (Fab specific)-Peroxidase antibody, dilution in 1: 10000, every hole 100 μ l, are added to HA1-Fc elisa plate, and 37 ℃, 1h;
8) PBST washes plate, 3 times;
9) tmb substrate (purchased from Sigma), every hole 100 μ l, 37 ℃, 15min, lucifuge reaction;
10) add 2M H
2sO
4, every hole 50 μ l;
11) measure OD450, and carry out data processing.
7, antibody neutralization is measured
(1) micro-neutralization test
1) mdck cell (purchased from ATCC) paving is to 96 orifice plates, and DMEM+10% (v/v) FBS training liquid is cultivated, and grows to 90% after 12h, standby.
2) 96 orifice plates, DMEM+0.2%BSA 2 * gradient dilution for antibody, final volume 50 μ l/ holes.
3) every hole adds 50 μ l containing 100 TCID
50virus (is diluted to 100TCID with DMEM+0.2%BSA in advance
50/ 50 μ l).Virus is: A/Sichuan/1/2009 (H1N1), A/Jiangxi-Donghu/312/2006 (H3N2) or A/Guangzhou/333/99 (H9N2).
4) every block of plate also comprises contrast:
Virus control (PC does not add antibody)---50 μ l DMEM 0.2%BSA+50 μ l viruses (viral species is as 4) are listed);
Mdck cell contrast (NC)---100 μ l DMEM 0.2%BSA;
5) 37 ℃ of 5% (v/v) CO
2, 1h, makes antibody and fully effect of virus.
6) MDCK monolayer cell sucks training liquid, and PBS washes twice, adds 100 μ l DMEM+0.2%BSA+2 μ g/ml TPCK-trypsin.
7) the whole correspondences of antibody-viral mixtures incubated 100 μ l are moved in mdck cell flat board.
8)37℃，5%CO
2，20h。
10) PBS washes one time.80% (v/v) acetone-PBS solution of precooling is 10min fixedly;
(2)ELISA
1) cell plate PBS+0.05% (v/v) Tween 20 (rinsing liquids) that fix are washed 3 times.
2) by PBS+1%BSA 0.1%Tween 20 (diluent) 1:10000 dilution anti-NP (HRP) antibody, 50 μ l/ holes, room temperature 1 hour.
3) rinsing liquid is washed 6 times.
4) the substrate 2mg OPD (o-phenylenediamine, O-Phenylene Diamine) of new preparation joins in 4ml 0.05M citrate buffer solution (containing 0.03g/ml Sodium peroxoborate), 50 μ l/ holes, room temperature 5min.
5) 2M H
2sO
4termination reaction, 50 μ l/ holes.
6) spectrophotometer 490nm reads plate.
7) at least do independent experiment twice.
II. embodiment
Use FITC-CD19/APC-IgG/Cy3-HA is Specific marker, has obtained several HA specific b cells.
RT-PCR and Nested-PCR method acquisition antibody are heavy, chain variable region gene, and molecular weight is 400bp left and right, and β-actin is as internal reference (343bp), and electrophoretogram is shown in Fig. 2, Fig. 3 and Fig. 4.To derive from same B cell antibody heavy and light chain gene variable region and connect T carrier, and check order and carry out expression vector establishment.
1F2 heavy chain variable region gene sequence following (SEQ ID NO:1):
GAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGC
TGGGTCCGCCAGGCTCGAGGGAAGGGGCTGGAGTGGGTCTCA
CGCTTCACCATCTCCAGAGACAATTCCAAGAACACGGTGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCCCTATATTACTGTGCGAAA
TGGTTCGACCCCTGGGGCCAGGGAACCCGGGTCACCGTCTCCTC
Remarks: that wherein uses double underline mark is followed successively by heavy chain gene CD R1 (SEQ ID NO:5), CDR2 (SEQ ID NO:6), CDR3 (SEQ ID NO:7) sequence.
1F2 weight chain variable region amino acid sequence following (SEQ ID NO:2):
EVQLLESGGGLVQPGGSLRLSCAASGFTFS
WVRQARGKGLEWVS
RFTISRDNSKNTVYLQMNSLRAEDTALYYCAK
WFDPWGQGTLVTVSS
Remarks: that wherein uses double underline mark is followed successively by heavy chain amino acid CD R1 (SEQ ID NO:8), CDR2 (SEQ ID NO:9), CDR3 (SEQ ID NO:10) sequence.
1F2 chain variable region gene sequence following (SEQ ID NO:3):
GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGC
TGGTACCAGCAGAAACCTGGCCAGGGTCCCAGGCTCCTCATCTAT
GGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGT
ATCACCTTCGGGCAAGGGACACGACTGGAGATTAAACGAACTGTGGCTGCACCATCTGTCAATCACTAG
Remarks: that wherein uses double underline mark is followed successively by light chain gene CD R1 (SEQ ID NO:11), CDR2 (SEQ ID NO:12), CDR3 (SEQ ID NO:13) sequence.
1F2 light chain variable region amino acid sequence following (SEQ ID NO:4):
EIVMTQSPATLSVSPGERATLSC
WYQQKPGQGPRLLIY
GIPARFSGSGSGTEFTLTISSLQSEDFAVYYC
ITFGQGTRLEIK
Remarks: that wherein uses double underline mark is followed successively by light chain amino acid CDR1 (SEQ ID NO:14), CDR2 (SEQ ID NO:15), CDR3 (SEQ ID NO:16) sequence.
ELISA result has shown successful expression human antibody, because having contained the constant region of heavy and light chain in expression vector, so the 293T cell of empty carrier transfection also can express the heavy constant region of light chain of antibody, sees Fig. 5.
ELISA shows, 1F2 can identify HA albumen, and after HA thermally denature, 1F2 can partly identify HA, shows that the epi-position of 1F2 identification is between linearity and space conformation.But 1F2 nonrecognition HA1 albumen, and HA albumen is only comprised of HA1 and HA2 two portions, illustrates that this this strain antibody binding site is positioned at HA2, sees Fig. 6.HA2 has very high conservative property in different strains of influenza viruses, therefore prompting, and this strain antibody has the broad spectrum of identification influenza virus.S-95-7 is mouse source specific recognition H1N1virus HA1 conformational epitope monoclonal antibody in 2009, as positive control, and the negative contrast of Vector (supernatant of the 293T emiocytosis of empty carrier transfection).
The inventor has further obtained the fragment of HA2 albumen, carries out ELISA experiment and detects the fragment that this antibody is identified.ELISA shows, one section of peptide section (G345-P504) between this human antibody identification HA2 albumen (the HA2 albumen of the calculating of sequence and figure place based on GenBank accession number ACP41105.1) the 345th to the 504th (A/California/07/2009 (H1N1) sequence), is shown in Fig. 7; Non-peptide in figure (no peptide) is not coated any peptide section in finger-hole, and vaccine refers to H1N1virus lysate.
Embodiment 5, antibody recognition influenza virus broad spectrum
ELISA result shows, 1F2 to 2009 influenza A virus (A/California/07/2009 (H1N1)) there is very high identity, 2008-2009 annual seasons influenza virus (A/Brisbane/59/2007 (H1N1)) is also had to very strong binding characteristic simultaneously, see Fig. 8.Show that this strain total man monoclonal antibody has identification broad spectrum.S-95-7 is mouse source specific recognition H1N1virus HA1 conformational epitope monoclonal antibody in 2009, as positive control, and the negative contrast of Vector (supernatant of the 293T emiocytosis of empty carrier transfection).
Embodiment 6, antibody have wide spectrum neutrality
Total man's monoclonal antibody (1F2) is used 293T cell to express, and its concentration is adjusted into 1000 μ g/ml, contrasts dilution.Use micro-neutralization test to measure it to A/Sichuan/1/2009 (H1N1) (being called for short SC09), the neutralization of A/Jiangxi-Donghu/312/2006 (H3N2) and A/Guangzhou/333/99 (H9N2) (available from CDC) influenza virus is active.Experiment shows, 1F2 not only can in and H1 subtype influenza virus, and to H3, it is active that H9 subtype influenza virus all has neutralization, illustrated in the wide spectrum of this strain antibody and active, sees Fig. 9.1F2 presents concentration gradient dependency to the neutralization activity of these viruses.
All documents of mentioning in the present invention are all quoted as a reference in this application, just as each piece of document, are quoted as a reference separately.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (17)
1. a separated binding molecule, is characterized in that, the epi-position in described binding molecule identification influenza virus HA albumen between HA2 protein protomer sequence 345-504 position.
2. binding molecule as claimed in claim 1, is characterized in that, described binding molecule comprises heavy chain CDR3 district shown in heavy chain CDR2 shown in heavy chain CDR1 district shown in SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10.
3. binding molecule as claimed in claim 1, is characterized in that, described binding molecule comprises light chain CDR3 district shown in light chain CDR2 shown in light chain CDR1 district shown in SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16.
4. binding molecule as claimed in claim 1, it is characterized in that, described binding molecule comprises following aminoacid sequence: the heavy chain CDR1 shown in SEQ ID NO:8, heavy chain CDR3 shown in heavy chain CDR2 shown in SEQ ID NO:9 and SEQ ID NO:10, light chain CDR1 shown in SEQ ID NO:14, the light chain CDR3 shown in the light chain CDR2 shown in SEQ ID NO:15 and SEQ ID NO:16.
5. the binding molecule as described in as arbitrary in claim 1-4, is characterized in that, comprise variable region of heavy chain, described variable region of heavy chain has the aminoacid sequence shown in SEQ ID NO:2.
6. the binding molecule as described in as arbitrary in claim 1-4, is characterized in that, comprise variable region of light chain, described variable region of light chain has the aminoacid sequence shown in SEQ ID NO:4.
7. the binding molecule as described in as arbitrary in claim 1-4, is characterized in that, comprise variable region of heavy chain and variable region of light chain, its variable region of heavy chain and variable region of light chain have respectively the aminoacid sequence shown in SEQ ID NO:2 and SEQ ID NO:4.
8. the binding molecule as described in as arbitrary in claim 1-4, it is characterized in that, described binding molecule is Fab, F (ab '), F (ab ') 2, Fv, dAb, Fd, complementary determining region (CDR) fragment, single-chain antibody (scFv), divalence single-chain antibody, single chain variable fragment phage antibody, two special double-chain antibody, three chain antibodies, four chain antibodies;
Preferably, described binding molecule is human monoclonal antibodies;
Preferred, its variable region of heavy chain of described human monoclonal antibodies and variable region of light chain have respectively the aminoacid sequence shown in SEQ ID NO:2 and SEQ ID NO:4, its CH is selected the constant region of one of heavy chain type in lower group: IgG1, IgG2a, IgG2b and IgG3, and its constant region of light chain is selected one of constant region of lower group light chain type: κ chain and λ chain;
Preferred, its variable region of heavy chain of described human monoclonal antibodies and variable region of light chain have respectively the aminoacid sequence shown in SEQID NO:2 and SEQ ID NO:4, and its CH and constant region of light chain have respectively the aminoacid sequence shown in Genebank ACK87036 and ACK87038.
9. the nucleic acid molecule of the binding molecule described in the claim 1-8 that encodes is arbitrary.
The arbitrary described binding molecule of claim 1-8 for the preparation of diagnosis, treat and/or prevent the purposes in the medicine of the influenza infection due to influenza virus.
11. purposes as claimed in claim 10, is characterized in that, the strain of described influenza virus is selected from as next group: H1 subtype influenza virus, H3 subtype influenza virus and H9 subtype influenza virus.
12. 1 kinds of expression vectors, is characterized in that, contain the DNA of the arbitrary described binding molecule of coding claim 1-7 in described expression vector.
13. 1 kinds of host cells, is characterized in that, contain the expression vector described in claim 12 in described host cell.
14. 1 kinds of compositions, is characterized in that, the arbitrary described monoclonal antibody of claim 1-8 that it contains significant quantity, and pharmaceutically acceptable carrier.
15. 1 kinds of test kits that detect influenza virus, it comprises the arbitrary described binding molecule of claim 1-8.
16. suppress a method for influenza virus, it is characterized in that, described method comprises the arbitrary described binding molecule of claim 1-8 that gives patient's significant quantity.
17. 1 kinds of methods that detect influenza virus, it is characterized in that, utilize the arbitrary described binding molecule of claim 1-8 to contact with testing sample, by detecting described binding molecule and the combination situation of given the test agent, acquisition influenza virus there is situation and amount.
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