CN103665153B - With molecule 1 E1 in a kind of resisiting influenza virus wide spectrum neutrality - Google Patents
With molecule 1 E1 in a kind of resisiting influenza virus wide spectrum neutrality Download PDFInfo
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- CN103665153B CN103665153B CN201210342406.9A CN201210342406A CN103665153B CN 103665153 B CN103665153 B CN 103665153B CN 201210342406 A CN201210342406 A CN 201210342406A CN 103665153 B CN103665153 B CN 103665153B
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Abstract
The present invention relates in a kind of resisiting influenza virus wide spectrum neutrality and molecule 1 E1, multiple subtype influenza virus can be neutralized.The function of antibody of the present invention is determined by its light chain and heavy chain variable region gene specific gene sequences, can, in conjunction with the HA2 subunit of influenza virus hemagglutinin albumen (HA) with native conformation, multiple subtype influenza virus can be stoped to infect permissive cell.Utilize antibody variable gene of the present invention or complementary determining region (CDR) gene, can transformation utilized in protokaryon and eukaryotic any expression system and produce multi-form genetic engineering antibody.
Description
Technical field
The invention provides the human monoclonal antibodies 1E1 that a strain can neutralize multiple subtype influenza virus, there are the potentiality of preventing or treating multiple influenza infection clinically.
Background technology
From in April, 2009, Influenza A H1N1 (A/H1N) epidemic situation worldwide breaks out.This epidemic situation is diffused into countries and regions, more than 200,5 continents from North America bamboo telegraph.According to World Health Organization, end on April 9th, 2010, A type A/H1N1 Influenza epidemic situation is in country's outburst more than 213, and global Influenza A H1N1 death is more than 1.77 ten thousand examples.In China, according to CDC, by March 31st, 2010, the accumulative report in province, 31, whole nation 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, and in ball-type, ellipse or thread, 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, C (first, second, third) three kinds of hypotypes.To influenza A, according to hemagglutinin (HA) and the antigenic difference of neuraminidase (NA) surface glycoprotein, different hypotypes can be further divided into again.At present, influenza A has 16 kinds of HA hypotypes (H1-H16) and 9 kinds of NA hypotypes (N1-N9).
The genome of influenza A is made up of (see Fig. 1) 8 independent single stranded RNA fragments, each one or two albumen of RNA fragment coding.Therefore influenza A has 8 genes, 10 kinds of albumen of encoding.Wherein, HA albumen plays an important role for merging in conjunction with cell surface receptor and film that is viral and cell, before cells infected, HA can be hydrolyzed into HA1 and HA2, wherein be positioned at the receptor binding domain (receptorbindingdomain on HA1, RBD) near zone, different strains of influenza viruses camber variation (RBD itself is more conservative), important neutrality epi-position region, the multiple neutrality antibody found at present for epi-position be mostly positioned at the RBD near zone of HA1 albumen.The ability of strain influenza virus different from different subtype or same hypotype during but these antibody scarcely possess.NA can remove sialic acid, helps the release of progeny virus and avoids self gathering of virus; PA, PB1 and PB2 tri-kinds of polysaccharases and NP nucleoprotein are mainly used in copying of viral RNA and transcribe; 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 that the medicine being used for the treatment of influenza is mainly following two large classes: 1) alkanamine class (amantadine and Rimantadine); 2) influenza virus neuraminidase inhibitor (Oseltamivir and zanamivir).To alkane amine drug, occurred a large amount of resistance influenza strain, 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 to Oseltamivir and zanamivir sensitivity, but also occurred Drug resistance strain in areas such as Japan, the appearance of Drug resistance strain limits the extensive use of these chemical small molecule medicines.
The treatment of disease of viral infection that waits that therapeutic antibodies is used for influenza early has report, and the case that antiserum(antisera) is used for the treatment of SARS and severe H5N1 avian influenza person has demonstrated the vital role that antibody plays in treatment virus infection.The therapeutic antibodies with the resisiting influenza virus of wide spectrum Neutralization effect has following potential advantages, on the one hand it can the combination of blocking virus and target cell, on the other hand by the effect of complement and the effector cell such as T cell, NK cell, kill by the cell of virus infection.
Therefore, this area necessary for influenza virus exploitation affinity the good and therapeutic antibodies that 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 human monoclonal antibodies 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 protein between HA2 protein protomer sequence 345-504 position.
In a preference, described binding molecule comprises heavy chain CDR3 district shown in heavy chain CDR2 and SEQIDNO:10 shown in the CDR1 of heavy chain shown in SEQIDNO:8 district, SEQIDNO:9; It can identify 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 and SEQIDNO:16 shown in the CDR1 of light chain shown in SEQIDNO:14 district, SEQIDNO:15; It can identify 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 SEQIDNO:8, the heavy chain CDR3 shown in heavy chain CDR2 and SEQIDNO:10 shown in SEQIDNO:9, the light chain CDR3 shown in light chain CDR2 and SEQIDNO:16 shown in light chain CDR1, SEQIDNO:15 shown in SEQIDNO:14; It can identify 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 SEQIDNO: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 SEQIDNO: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 the aminoacid sequence shown in SEQIDNO:2 and SEQIDNO:4 respectively.
In another preference, described binding molecule comprises CH and or the constant region of light chain of antibody.
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), bivalent single-chain antibodies, single chain variable fragment phage antibody, two specific duplex 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 the aminoacid sequence shown in SEQIDNO:2 and SEQIDNO:4 respectively, its CH selects the constant region of one of heavy chain type in lower group: IgGl, IgG2a, IgG2b and IgG3, and its constant region of light chain selects 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 the aminoacid sequence shown in SEQIDNO:2 and SEQIDNO:4 respectively, and its CH and constant region of light chain have the aminoacid sequence shown in Genebank ACK87036 and ACK87038 respectively.
In another preference, the linear epitope on described monoclonal antibody identification Influenza virus HA protein.
In another preference, epi-position in described monoclonal antibody identification Influenza virus HA protein between HA2 protein protomer sequence 345-504 position (calculating of sequence and figure place is based on the HA2 albumen of GenBank accession number ACP41105.1).
In one aspect of the invention, the nucleic acid molecule of the binding molecule described in coding is provided.
In one aspect of the invention, provide described binding molecule for the preparation of diagnosis, the purposes treated and/or prevented in the medicine of the influenza infection caused by 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, the DNA containing the binding molecule described in coding in described expression vector.
In one aspect of the invention, provide a kind of host cell, containing described expression vector in described host cell.
In one aspect of the invention, provide a kind of composition, it contains the described monoclonal antibody of significant quantity, and pharmaceutically acceptable carrier.
In one aspect of the invention, provide a kind of test kit detecting influenza virus, it comprises described binding molecule.
In another preference, described test kit also comprises: antigen or antibody bag are by 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 (preferred non-therapeutic ground) to suppress the method for influenza virus, described method comprises the described binding molecule giving patient effective amounts.
In one aspect of the invention, a kind of (preferred nondiagnostic ground) is provided to detect the method for influenza virus, binding molecule described in utilization contacts with testing sample, by detect described binding molecule and given the test agent in conjunction with situation, there is situation and amount in what obtain influenza virus.
Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.
Accompanying drawing explanation
Fig. 1, influenza A 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, human monoclonal antibodies (1E1) concentration.
Fig. 6, human monoclonal antibodies (1E1) identify the recognition capability of HA/HA1 albumen.
Fig. 7, human monoclonal antibodies (1E1) identify that HA2 Protein G 345 is to P504 peptide section.
Fig. 8, human monoclonal antibodies (1E1) identify the broad spectrum of H1 subtype influenza virus.
The wide spectrum Neutralization effect of Fig. 9, human monoclonal antibodies 1E1.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 present inventor is through extensive and deep research, obtain a kind of binding molecule of the resisiting influenza virus wide spectrum neutrality containing unique complementary determining region (CDR district), preferred human monoclonal antibodies, this binding molecule has wide spectrum neutralizing effect for influenza virus.Complete the present invention on this basis.
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 the Neutralization effect for influenza virus.
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), bivalent single-chain antibodies, single chain variable fragment phage antibody, two specific duplex antibody, three chain antibodies, four chain antibodies and at least gives and (many) peptides of the fragment of the specific antigens binding domain-immunoglobulin of fowl influenza virus strain or its fragment containing being enough to.
Binding molecule of the present invention also can one or more fragment of specific binding influenza virus.For the method treating and/or preventing influenza virus, described binding molecule preferably can specific binding influenza virus surface can and protein.In certain embodiments, the HA2 molecule of binding molecule energy specific binding influenza virus of the present invention.
Present invention also offers described binding molecule in preparation diagnosis, the application prevented and/or treated in the medicine of influenza infection.This infection can occur in microcommunity, but also can with season prevailing disease mode at world's range propagation, or more seriously at global spread, millions of individuality is in danger.The invention provides to neutralize and cause prevailing disease and the binding molecule of infection of Influenza virus strain of potential global prevalence disease in this season.Importantly, it is expected at present utilize binding molecule of the present invention to play protection and therapeutic action for multiple Influenza virus strain, because oneself, through disclosing due to the combination from the epi-position shared between the HA albumen of different Influenza virus strain, therefore can use binding molecule of the present invention to carry out cross-neutralization between these strains at present.Binding molecule of the present invention can be prepared on a large scale and store, because which provide 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 six CDR districts that disclose herein.Preferably, binding molecule of the present invention comprises at least two CDR disclosed herein.
Another aspect of the present invention comprises the functional variant of binding molecule described herein.If variant can compete specific binding influenza virus or its protein fragments with parent binding molecule, then think that this Variant molecules is the functional variant of binding molecule of the present invention.In other words, described functional variant still can in conjunction with influenza virus or its fragment.Preferably, described functional variant can competitive specific binding by least two (or more) the different Influenza virus strain of parent binding molecule specific binding or its fragment.In addition, if certain molecule for parent binding molecule to its have Neutralization effect influenza virus, preferably at least two (or multiple) Influenza virus strain, there is Neutralization effect, then think that this molecule is the functional variant of binding molecule of the present invention.But functional variant includes but not limited to that primary structural sequence basic simlarity is containing the such as derivative of chemistry and/or biochemical modification in undiscovered external or body in parent binding molecule.This modification comprise the covalent attachment of second phthalein, phthalein, Nucleotide or nucleotide derivative, lipid or lipid derivate covalent attachment, crosslinked, disulfide formation, glycosylation, hydroxylation, methylate, be oxidized, the processing of Pegylation, proteolysis, phosphorylation etc.In other words, modification not remarkably influenced in the amino acid of parent binding molecule and/or nucleotide sequence or change by the binding characteristic of described nucleotide sequence coded or containing described aminoacid sequence described binding molecule, namely described binding molecule still can identify and in conjunction with its target position.
Described functional variant can have conserved sequence and modify, and comprises Nucleotide and aminoacid replacement, interpolation and disappearance.These modifications can oneself knows by this area standard technique import, the mutagenesis of such as directed mutagenesis and random PCR mediation, and can comprise natural and non-natural nucleotide and amino acid.
Conserved amino acid replaces and comprises wherein amino-acid residue by the replacement of another radical amino acid replacement with analog structure or chemical property.The family with the amino-acid residue of similar side chain is own through limiting in the art.These families comprise amino acid (the such as Methionin with basic side chain, arginine, Histidine), acidic side chains (such as aspartic acid, L-glutamic acid), without charge polarity side chain amino acid (such as asparagus fern phthalein amine, paddy ammonia phthalein amine, Serine, Threonine, tyrosine, half skin propylhomoserin, tryptophane), nonpolar side chains (such as glycine, L-Ala, α-amino-isovaleric acid, leucine, Isoleucine, proline(Pro), phenylalanine, methionine(Met)), branched side chains (such as Threonine, α-amino-isovaleric acid, Isoleucine) and aromatic side chain amino acid (such as tyrosine, phenylalanine, tryptophane).Those skilled in the art understand other amino acid residue families mode classification that also can use except above-mentioned family.In addition, variant can have nonconservative aminoacid replacement, and such as 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 both.Use computer program well known in the art can find to determine which amino-acid residue can be substituted, inserts or lack and not eliminate the guidance of immunologic competence.
In addition, functional variant can comprise the truncate of aminoacid sequence at N-terminal or C-terminal or these two ends.Functional variant of the present invention can have identical or different, higher or lower binding affinity compared with parent binding molecule, but still can in conjunction with influenza virus or its fragment.Such as, functional variant of the present invention can have the binding affinity increasing or reduce compared with parent binding molecule for influenza virus H1N1 or its fragment.Preferably, variable region includes but not limited to framework region, the aminoacid sequence in hypervariable region or CDR district modified.Usually, 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 the amino-acid residue from CDR and the amino-acid residue from Gao Bianhuan.Functional variant within the scope of the present invention and parent 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 the amino acid sequence homology of particularly at least about 97% to about 99%.Computerized algorithm well known by persons skilled in the art as Gap or Bestfit can be used for best arranged amido acid sequence to carry out contrasting and precisely similar or identical amino-acid residue.Functional variant can obtain by using oneself the common molecular biology method known of this area to change parent binding molecule or its part, and described method includes but not limited to that mutagenesis that fallibility PCR, oligonucleotide instruct, site-directed mutagenesis and heavy chain and/or light chain reorganize method.In one embodiment, functional variant of the present invention has Neutralization effect for influenza virus.Described Neutralization effect can be identical or higher or lower compared with parent binding molecule.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.The variable region of heavy chain of described resisiting influenza virus monoclonal antibody, variable region of light chain and the complementary determining region (CDR) being positioned at variable region of heavy chain and variable region of light chain all have unique structure being different from prior art, and they are total man sources.
The present invention includes: the monoclonal antibody with the corresponding aminoacid sequence of described monoclonal antibody, has the monoclonal antibody of described variable region of mab chain.The present invention also comprises and 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 the homology of more than 90% (preferably more than 95%).
The antigenic binding property of antibody can be described by 3 the specific regions being positioned at heavy chain and variable region of light chain, be called complementary determining region (complementaritydeterminingregion, CDR), variable region is partitioned into 4 frame areas (FR) by described CDR district, the aminoacid sequence of 4 FR is relatively conservative, does not participate in association reaction directly.These CDR form ring texture, and the β-pleated sheet structure formed by FR is therebetween close to each other on space structure, and the CDR on the CDR on heavy chain and corresponding light chain constitutes the antigen binding site of antibody.FR or the CDR region that has been which Amino acid profile can be determined by the aminoacid sequence of antibody more of the same type.
For monoclonal antibody heavy of the present invention and sequence of light chain, can measure by ordinary method.
Empirical tests, the CDR district of resisiting influenza virus monoclonal antibody of the present invention is brand-new, its for be epitope on a unique Influenza virus HA protein, technical conceive is different from existing antibodies against influenza virus.The epi-position that resisiting influenza virus monoclonal antibody of the present invention identifies is linear epitope; Epi-position in more preferably described monoclonal antibody identification Influenza virus HA protein between HA2 protein protomer sequence 345-504 position.
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, while the effect with identification excellent especially and neutralized stream Influenza Virus, also has the feature that immunogenicity is low, security is high.
In an embodiment of the present invention, the present invention obtains peripheral blood mononuclear cell (PBMC) in inoculation H1N1virus split vaccine (2009/Cal) volunteer body in 2009, uses CD19
+/ IgG
+/ HA is Specific marker, obtains identify HA protein-specific B cell through selected by flow cytometry apoptosis (FACS).The single-cell RT-PCR technology (JournalofImmunologicalMethods329 (2008) 112-124) using document to report, obtains antibody gene and in 293T cell, carries out expression acquisition human monoclonal antibodies 1E1.ELISA epitope analysis learns 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-Neutralizing test prove 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 wide spectrum Neutralization effect, there is potential value that the multiple subtype influenza virus of prevention and therapy infects clinically.
On the other hand, the present invention includes immunoconjugates, namely comprise at least one binding molecule described herein and comprise at least one mark further 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, another molecule described 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 with binding molecule/put together.Described mark also directly can be combined with human binding molecules/be puted together by covalent linkage.Or described mark can connect compound by one or more and be combined with described binding molecule/put together.Mark is well known to those skilled in the art with the conjugation techniques of binding molecule.
The mark of immunoconjugates of the present invention can be therapeutical agent, but they also can be detectable part/materials.The mark being suitable for treating and/or preventing can be other binding molecule of toxin or its funtion part, microbiotic, enzyme, enhancing phagolysis or immunostimulation.With comprising the immunoconjugates diagnosticability of detectable substance for such as evaluate object whether oneself through influenza virus infection strain or as the generation of the part monitoring influenza infection of clinical experiment program or progress with the effect such as determining TA scheme.But they also may be used for other and detect and/or analyze 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 analyze and/or diagnostic purpose depending on particular detection/analysis/diagnostic techniques and/or method such as immunohistochemical staining (tissue) sample, flow cytometry, laser scanning Cytometry detection, fluorescence immunoassay, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), biological assay (such as phagolysis mensuration), the western blotting application etc. of use for the mark marking binding molecule.Detection/analysis/diagnostic techniques known in the art and/or the suitable mark of method are 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 protein or its fragment.This solid support can be porous or atresia, plane or nonplanar.Binding molecule of the present invention can with flag sequence as skin merges 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 form antibody heteroconjugate (heteroconjugate) with another kind of antibody conjugate.
On the other hand, binding molecule of the present invention can be puted together with one or more antigen/adhere to.Preferably, these antigens are by the antigen of the immune system recognition of the object giving binding molecule-antigen conjugate.Described antigen can be mutually the same, but also can be different.Make the conjugation methods of attachment antigen and binding molecule be known in the art, include but not limited to use linking agent.Binding molecule of the present invention in conjunction with influenza virus and the antigen being attached to binding molecule the strong T cell caused for described conjugate is attacked, finally cause the destruction of influenza virus.
Except being puted together by direct or indirect (such as passing through joint), chemistry produces except immunoconjugates, and described immunoconjugates can produce as fusion rotein, and described fusion rotein comprises binding molecule of the present invention and suitable mark.Fusion rotein can be produced by means known in the art, such as by building nucleic acid molecule and expressing described nucleic acid molecule subsequently and generation of recombinating, described nucleic acid molecule comprises the nucleotide sequence of in-frame encoding binding molecules and the nucleotide sequence of coding appropriate flags.
The present invention provides the nucleic acid molecule of coding at least one binding molecule of the present invention, its functional variant or immunoconjugates on the other hand.This nucleic acid molecule can be used as intermediate to clone, such as, in affinity maturation method described above.In a preferred embodiment, described nucleic acid molecule is isolated or purified.The sequence of DNA molecular can use routine techniques, or utilizes hybridoma technology to obtain.
Those skilled in the art will recognize that the functional variant of these nucleic acid molecule is also a part of the present invention.Functional variant is such nucleotide sequence, it directly can be translated with the identical aminoacid sequence of the sequence provided with translate from parent nucleic acid molecules by using standard genetic code.
Once obtain relevant sequence, just relevant sequence can be obtained in large quantity with recombination method.This is normally cloned into carrier, then proceeds to cell, is then separated from the host cell after propagation by ordinary method and obtains relevant sequence.
In addition, also relevant sequence can be synthesized, when especially fragment length is shorter by the method for synthetic.Usually, by first synthesizing multiple small segment, and then carry out connect can obtain the very long fragment of sequence.
At present, can obtain encoding by chemosynthesis the DNA sequence dna of binding molecule of the present invention (or its fragment, or derivatives thereof) completely.Then this DNA sequence dna can be introduced in various existing DNA molecular (or as carrier) as known in the art and cell.In addition, also by chemosynthesis, sudden change is introduced in the sequence of binding molecule of the present invention.
The invention still further relates to the carrier comprising above-mentioned suitable DNA sequence dna and suitable promotor or control sequence.These carriers may be used for transforming suitable host cell, with can marking protein.Preferably, carrier of the present invention is such as containing the plasmid expression vector of viral promotors, and in described expression vector, insert IgH (constant region from people source IgH) fusion sequence and variable region of light chain VL and human body Igkappa (constant region from the people source Igkappa) fusion sequence of resisiting influenza virus monoclonal antibody heavy variable region (VH) and constant region respectively.
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 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, especially mammalian cell, as 293 cells.
Can carry out with routine techniques well known to those skilled in the art with recombinant DNA transformed host cell.When host be prokaryotic organism as intestinal bacteria time, the competent cell that can absorb DNA can be gathered in the crops at exponential growth after date, uses CaCl
2method process, step used is well-known in this area.Another kind method uses MgCl
2.If needed, transform and also can be undertaken by the method for electroporation.When host is eukaryote, following DNA transfection method can be selected: calcium phosphate precipitation, or conventional mechanical methods is as microinjection, electroporation, liposome packaging etc.
The transformant obtained 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 medium.Cultivate under the condition being suitable for host cell growth.When after host cell growth to suitable cell density, the promotor selected with the induction of suitable method (as temperature transition or chemical induction), cultivates for some time again by cell.
Binding molecule of the present invention preferably adopts mammalian cell to produce, and mammalian cell needs to cultivate in containing the substratum of serum usually.After needing to carry out the adaptive process of serum-free to cell, cell can be allowed to grow normally in serum free medium.
If needed, can utilize its physics, the albumen of being recombinated by various separation method abstraction and purification with other characteristic of 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 process, combination by protein precipitant process (salting-out 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 transgenic nonhuman mammal is as rabbit, goat or ox, and is secreted into such as its Ruzhong.
Pharmaceutical composition
Binding molecule of the present invention can be used for preparing the composition suppressing influenza virus.
Based on new discovery of the present invention, additionally provide a kind of composition suppressing influenza virus or influenza infection relative disease, it comprises: the binding molecule of the present invention of significant quantity; And pharmaceutically acceptable carrier.
Term used herein " pharmaceutically acceptable " refers to that, when biomolecule ontology and composition suitably give animal or human, 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 be blended with it and significantly can not reduce the effect of composition under normal conditions.
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; Cellulose and its derivates, 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; Isotonic salts solution; With phosphate buffered saline buffer etc.
Composition of the present invention can make various formulation as required, and can by doctor according to patient category, the age, body weight and roughly the factor such as disease condition, administering mode determine that the dosage useful to patient is used.Administering mode such as can adopt injection or other therapeutic modality.
Binding molecule of the present invention can use with form that is unsegregated or that be separated.In addition, binding molecule of the present invention can be applied separately or apply in the mixture comprising at least one binding molecule of the present invention (or its variant or fragment).In other words, described binding molecule can Combination application, such as, as comprising the pharmaceutical composition that two or more plant binding molecule of the present invention, its variant or fragment.Such as, there is difference but the binding molecule of complementary activity can be combined in reach the prevention of hope, treatment or diagnostic effect in a treatment plan, but or also the binding molecule with identical activity can be combined in a treatment plan to reach the prevention of hope, treatment or diagnostic effect.Optionally, described mixture comprises other therapeutical agent of at least one further.Preferably, (such as zanamivir ((zanamivir), Oseltamivir (oseltamivir)) can be used for preventing and/or treating influenza infection described therapeutical agent such as MZ inhibitor (such as amantidine, Rimantadine (rimantadine)) and/or neuraminidase inhibitor.
Described pharmaceutical composition can comprise the Neutralization effect of binding molecule two or more to have to(for) influenza virus.In one embodiment, when Combination application, described binding molecule presents collaborative Neutralization effect.In other words, described composition comprises the binding molecule that at least two kinds have Neutralization effect, 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 Combination application, and the compound action of binding molecule is higher than adduction when applying separately.Described synergistic binding molecule can in conjunction with the different structure in the identical or different fragment of influenza virus.Calculating synergistic mode is calculated by combinatorial index.The concept of combinatorial index (CI) is own to be described via ChouandTalalay (1984).
Binding molecule of the present invention or drug regimen can detect before for human body in suitable animal model system.This animal model system includes but not limited to mouse, ferret (ferret) and monkey.Also can act synergistically in Influenza Virus influenza virus.
Dosage regimen can be adjusted to provide response (such as treating response) needed for the best.Suitable dosage range can be such as 0.01-100mg/kg body weight, preferred 0.1-15mg/kg body weight.In addition, such as can give once to inject, give repeatedly separate doses in time or can reduce according to the emergency for the treatment of situation in proportion or increase dosage.Molecule of the present invention and composition are preferably aseptic.The method of these molecules and composition sterile is made to be known in the art.Other molecule for diagnosing, preventing and/or treating can give with the dosage regimen similar to binding molecule of the present invention.If give separately other molecule, then can before giving one or more human binding molecules of the present invention or pharmaceutical composition, simultaneously or give patient afterwards.Accurate dosage regimen for people patient is picked out usually during clinical experiment.
Detection reagent and test kit
Binding molecule of the present invention can be used for reagent or the test kit of preparing detection influenza virus.
As used herein, term " testing sample " covers several samples type, comprises blood and other humoral sample of biological origin, solid tissue sample as tissue biopsy sample or tissue culture, or derived from cell wherein or its offspring.This term to be also included in after acquisition by the sample of any mode process, such as, use some composition of agent treated, dissolving or enrichment as protein or polynucleotide.This term covers the various clinical samples deriving from any species, also comprises cultured cells, cell conditioned medium and cell lysates.
Based on described binding molecule, easy to prepare, fast and accurately can detect influenza virus (as influenza A virus; More particularly as H1N1, H3N2, H9N2 virus) test kit.
Therefore, the invention provides a kind of for detecting the detection kit that whether there is influenza virus in sample, the binding molecule containing resisiting influenza virus of the present invention in this test kit.
After obtaining binding molecule provided by the invention, the detection kit for specific detection influenza virus can be prepared easily.
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-antibody method routine is that primary antibodie (as monoclonal antibody of the present invention) is fixed on carrier, then primary antibodie and antigen-reactive is made, again with two anti-reflective should (described two anti-carry detectable signal after washing, or can be combined with the material carrying detectable signal), finally carry out chemoluminescence or enzyme connection color reaction detection signal.Double-antibody method is specially adapted to the detection of the antigen with two or more epi-positions.
In order to more convenient when detecting, except containing except binding molecule of the present invention in described test kit, other detection reagent or auxiliary reagent can also be comprised, described auxiliary reagent is such as conventional some reagent used in ELISA 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, sensitizers etc.Those skilled in the art should be understood that the detection kit of various version is all included in the present invention, as long as make use of binding molecule of the present invention wherein as the reagent identifying influenza virus.
In addition, in described test kit, also working instructions can be comprised, for illustration of the using method of the reagent wherein loaded.
After obtaining binding molecule provided by the invention and/or test kit, panimmunity methods involving can be utilized to detect HA albumen or its content in sample, thus learn the donor whether influenza virus infection of testing sample, these methods are all in the present invention involved.Preferably, described method is diagnosed as object with non-diseases.
As a kind of optimal way, the invention provides the method that a kind of external (non-diagnostic or therapeutic ground) detects influenza virus, comprise the following steps:
(a1) testing sample is coated in solid phase carrier;
(a2) by binding molecule application of sample of the present invention in the solid phase carrier of (a1), thus the influenza virus in testing sample is combined with binding molecule, forms the solid phase carrier with " influenza virus-binding molecule of the present invention " binary complex;
(a3) by the detection thing application of sample of binding molecule of the present invention for specific binding in the solid phase carrier of (a2), form the solid phase carrier with " influenza virus-binding molecule of the present invention-detection thing " ternary complex; Described detection thing carries a marker;
(a4) detect the marker in ternary complex, the existence determining influenza virus in detected sample whether with or the amount that exists.
According to the method described above, as long as arrange the antigen control of concentration known, make concentration standard curve, by just can draw the influenza virus content in testing sample according to concentration standard curve.
Major advantage of the present invention is:
(1) provide and a kind ofly have brand-new binding molecule, it is total man source, and compared with the molecule of animal derived with other (as mouse) resisiting influenza virus, 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 of influenza virus hemagglutinin albumen (HA) with native conformation, can stop multiple subtype influenza virus to infect permissive cell.
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, conveniently condition such as J. Pehanorm Brooker etc. is write usually, Molecular Cloning: A Laboratory guide, the condition described in Science Press, or according to the condition that manufacturer advises.
I. materials and methods
Below illustrate that a present invention i.e. strain can neutralize multiple influenza virus wide spectrum neutrality human monoclonal antibodies preparation and antibody characteristic analytic process.Be divided into two portions:
(1) single-cell RT-PCR method obtains antibody gene and antibody expression;
(2) antibody characteristic analysis.
Detailed process is as follows:
1, the acquisition of peripheral blood lymphocytes (PBMC)
In the volunteer's body screened according to blood clotting Inhibition test, extract peripheral blood, adopt conventional Ficoll-Paque density gradient centrifugation, obtain 10
7with last peripheral blood lymphocytes (PBMC).
2, HA specific memory B-cell sorting
(FITC-CD19/APC-IgG is purchased from BD to use FITC-CD19/APC-IgG/Cy3-HA, Cy3-HA preparation method is: express preparation HA albumen by baculovirus insect cell protein expression system (Invitrogen), and with after biotin protein labelling kit (ThermoScientific) mark, with Cy3-Streptavidin (Sigma) coupling) be mark, specific b cells to 96 hole RT-PCR plate is obtained through flow cytometer, one, every hole cell, obtains HA specific memory B-cell.
3, antibody gene
Use RT-PCR and Nested-PCR technology, obtain antibody gene (see NatProtoc.2009; 4 (3): 372-84).Connect pGEM-TEasy (Promega) carrier, check order, ordinary method checking antibody gene.As document KennethSmithetal.Rapidgenerationoffullyhumanmonoclonalan tibodiesspecifictoavaccinatingantige.NatProtoc.2009; Carrier construction AbVec-hIgG and AbVec-hIgKappa described in 4 (3): 372-384, Vector map is respectively as Figure 10 and 11, sequence is respectively described in genebank FJ475055 and FJ475056, wherein comprise the base sequence of encoding human IgG1 heavy chain and constant region of light chain respectively, the aminoacid sequence of encoding human IgG1 heavy chain and constant region of light chain is for described in Genebank ACK87036 and ACK87038.Then heavy, 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 lOpti-MEM (Invitrogen)+4 μ gIgG+4 μ gIgL
3) B pipe: 500 μ lOpti-MEM+20 μ lLipofectamineReagent (Invitrogen)
4) leave standstill after 5 minutes, A pipe is mixed with B pipe, leaves standstill 20min in room temperature;
5) A, B pipe mixture adds in cell culture, hatches 6h for 37 DEG C;
6), after 6h, absorb the nutrient solution containing LipofectamineReagent, every hole adds 2mlFreeStyle
tM293ExpressionMedium (Invitrogen);
7), after 72h, collect the cell conditioned medium containing human antibody, 4 DEG C, 3000rpm, 5min, save backup.
5, antibody concentration measures
ELISA method measures human antibody concentration.
1) wrap by GoatAnti-HumanIgG (FabSpecific) Antibody (purchased from Sigma) in elisa plate, 10 μ g/ml, every hole 100 μ l, 4 DEG C are spent the night;
2) PBST washes plate, 3 times;
3) close: 1g/mlBSA, every hole 200 μ l, 37 DEG C, 2h;
4) PBST washes plate, 3 times;
5) human IgG (purchased from Sigma) the production standard curve of doubling dilution is used, concentration 400ng/ml, 200ng/ml, 100ng/ml, 50ng/ml, 25ng/ml, 12.5ng/ml, 6.25ng/ml, 0ng/ml, 100 μ l/ holes; The human antibody cell conditioned medium of aforementioned preparation dilutes 5000 times, every hole 100 μ l, 37 DEG C, 2h;
6) PBST washes plate, 3 times;
7) add GoatAnti-HumanIgG (Fcspecific)-Peroxidaseantibody (purchased from Sigma), 1:10000 dilutes, every hole 100 μ l, 37 DEG C, 1h;
8) PBST washes plate, 3 times;
9) tmb substrate (purchased from Sigma), every hole 100 μ l, 37 DEG C, 15min, lucifuge is reacted;
10) 2MH is added
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, and wraps respectively by HA-His (baculovirus insect cell protein expression system (Invitrogen) expression), 100 DEG C 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 lysates (A/Brisbane/59/2007 (H1N1)) (blue biological purchased from China).
1) wrap respectively by 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, the multiple hole of two, each sample, every hole 100 μ l, 4 DEG C are spent the night;
2) PBST washes plate, 3 times;
3) close: 1%BSA, every hole 200 μ l, 37 DEG C, 2h;
4) PBST washes plate, 3 times;
5) according to mensuration 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 China typical culture collection center, preserving number CCTCCNO:C201024) be positive control, 37 DEG C, 2h;
6) PBST washes plate, 3 times;
7) GoatAnti-HumanIgG (Fcspecific)-Peroxidaseantibody, 1: 10000 dilution, every hole 100 μ l, is added to HA-His, thermally denature HA-His, 2009 H1N1virus vaccine lysates and 2008-2009 seasonal current influenza virus vaccine lysate elisa plate; GoatAnti-HumanIgG (Fabspecific)-Peroxidaseantibody, 1: 10000 dilution, every hole 100 μ l, is added to HA1-FcELISA plate, 37 DEG C, 1h;
8) PBST washes plate, 3 times;
9) tmb substrate (purchased from Sigma), every hole 100 μ l, 37 DEG C, 15min, lucifuge is reacted;
10) 2MH is added
2sO
4, every hole 50 μ l;
11) measure OD450, and carry out data processing.
7, antibody neutralization measures
(1) micro-neutralization test
1) mdck cell (purchased from ATCC) paving is to 96 orifice plates, and DMEM+10% (v/v) FBS trains liquid and cultivates, and grows to 90% after 12h, for subsequent use.
2) 96 orifice plates, antibody DMEM+0.2%BSA2 × gradient dilution, final volume 50 μ l/ hole.
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 plate also comprises contrast:
Virus control (PC does not add antibody)---50 μ lDMEM0.2%BSA+50 μ l virus (viral species is as 4) are listed);
Mdck cell contrast (NC)---100 μ lDMEM0.2%BSA;
5) 37 DEG C of 5% (v/v) CO
2, 1h, makes antibody and virus fully act on.
6) MDCK monolayer cell sucks training liquid, and PBS washes twice, adds 100 μ lDMEM+0.2%BSA+2 μ g/mlTPCK-trypsin.
7) the whole correspondence of antibody-viral mixtures incubated 100 μ l is 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 fixes 10min;
(2)ELISA
1) cell plate PBS+0.05% (v/v) Tween20 (rinsing liquid) fixed washes 3 times.
2) anti-NP (HRP) antibody is diluted, 50 μ l/ holes, room temperature 1 hour with PBS+1%BSA0.1%Tween20 (diluent) 1:10000.
3) rinsing liquid washes 6 times.
4) the substrate 2mgOPD (o-phenylenediamine, O-Phenylene Diamine) of new preparation joins in 4ml0.05M citrate buffer solution (containing 0.03g/ml Sodium peroxoborate), 50 μ l/ holes, room temperature 5min.
5) 2MH
2sO
4termination reaction, 50 μ l/ holes.
6) spectrophotometer 490nm reads plate.
7) twice independent experiment is at least done.
II. embodiment
Embodiment 1, HA specific memory B-cell
Use FITC-CD19/APC-IgG/Cy3-HA is Specific marker, obtains several HA specific b cells.
Embodiment 2, antibody gene
RT-PCR and Nested-PCR method obtains that antibody is heavy, chain variable region gene, molecular weight be about 400bp, β-actin as internal reference (343bp), electrophoretogram is shown in Fig. 2, Fig. 3 and Fig. 4.Same B cell antibody heavy and light chain gene variable region will be derived from and connect carrier T, and check order and carry out expression vector establishment.
1E1 heavy chain variable region gene sequence following (SEQIDNO:1):
GAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAAC
TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCA
CGGTTCACCATCTCCAGAGACAATTCCAAGAAGACGTTGTATCTTCAAATGACCAGCCTGAGAGCCGAGGACACGGCCGTTTATTACTGTGCGAAA
TTTGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTAATC
Remarks: what wherein use double underline mark is followed successively by heavy chain gene CDR1 (SEQIDNO:5), CDR2 (SEQIDNO:6), CDR3 (SEQIDNO:7) sequence.
1E1 heavy chain variable amino acid sequence following (SEQIDNO:2):
EVQLLESGGGLVQPGGSLRLSCAASGFTFN
WVRQAPGKGLEWVS
RFTISRDNSKKTLYLQMTSLRAEDTAVYYCAK
FDSWGQGTLVTVSS
Remarks: what wherein use double underline mark is followed successively by heavy chain amino CDR1 (SEQIDNO:8), CDR2 (SEQIDNO:9), CDR3 (SEQIDNO:10) sequence.
1E1 chain variable region gene sequence following (SEQIDNO:3):
GAAATTGTGTTGATTCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGC
TGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT
GGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCGCCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGT
ATCACCTTCGGCCAAGGGACACGACTGGAGATTAGACGAACTGTGGCTGCACCATCTGTCAATC
Remarks: what wherein use double underline mark is followed successively by light chain gene CDR1 (SEQIDNO:11), CDR2 (SEQIDNO:12), CDR3 (SEQIDNO:13) sequence.
1E1 chain variable region amino acid sequence following (SEQIDNO:4):
EIVLTQSPATLSLSPGERATLSC
WYQQKPGQAPRLLIY
GIPARFSGSGSGTDFTLAISSLEPEDFAVYYC
ITFGQGTRLEIK
Remarks: what wherein use double underline mark is followed successively by light chain amino acid CDR1 (SEQIDNO:14), CDR2 (SEQIDNO:15), CDR3 (SEQIDNO:16) sequence.
Embodiment 3, antibody expression
ELISA result shows successful expression human antibody, and because of the constant region containing heavy and light chain in expression vector, the 293T cell of therefore empty carrier transfection also can express the heavy constant region of light chain of antibody, sees Fig. 5.
Embodiment 4, antibody epitope analysis
ELISA shows, 1E1 can identify HA albumen, also can identify thermally denature HA albumen, and OD450nm value there is not change substantially, shows that the epi-position that 1E1 identifies is complete linear epitopes.But 1E1 nonrecognition HA1 albumen, and HA albumen is only made up 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 points out, and this two strain antibody has the broad spectrum identifying influenza virus.S-95-7 is mouse source specific recognition H1N1virus HA1 conformational epitope monoclonal antibody in 2009, and as positive control, Vector (supernatant of the 293T emiocytosis of empty carrier transfection) is negative control.
The present inventor obtains the fragment of HA2 albumen further, carries out the fragment that this antibody of ELISA experiment detection identifies.ELISA shows, one section of peptide section (G345-P504) between this human antibody identification HA2 albumen (calculating of sequence and figure place is based on the HA2 albumen of GenBank accession number ACP41105.1) the 345 to the 504 (A/California/07/2009 (H1N1) sequence), sees Fig. 7; In figure, non-peptide (nopeptide) is not wrapped in finger-hole by any peptide section, and vaccine refers to H1N1virus lysate.
Embodiment 5, antibody recognition influenza virus broad spectrum
ELISA result shows, 1E1 to 2009 influenza A virus (A/California/07/2009 (H1N1)) there is very high identity, to 2008-2009 annual seasons influenza virus (A/Brisbane/59/2007 (H1N1)), also there is very strong binding characteristic simultaneously, see Fig. 8.Show that this strain human monoclonal antibodies has identification broad spectrum.S-95-7 is mouse source specific recognition H1N1virus HA1 conformational epitope monoclonal antibody in 2009, and as positive control, Vector (supernatant of the 293T emiocytosis of empty carrier transfection) is negative control.
Embodiment 6, antibody have wide spectrum neutrality
Human monoclonal antibodies (1E1) uses 293T cell to express, and its concentration is adjusted to 1000 μ g/ml, carries out contrast dilution.Micro-neutralization test is used to measure it to A/Sichuan/1/2009 (H1N1) (being called for short SC09), the Neutralization effect of A/Jiangxi-Donghu/312/2006 (H3N2) and A/Guangzhou/333/99 (H9N2) (available from CDC) influenza virus.Experiment shows, 1E1 can not only in and H1 subtype influenza virus, and all there is Neutralization effect to H3, H9 subtype influenza virus, describe the wide spectrum Neutralization effect of this strain antibody, see Fig. 9.The Neutralization effect of 1E1 to these viruses presents concentration gradient dependency.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.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 (11)
1. the human monoclonal antibodies be separated, it is characterized in that, described human monoclonal antibodies comprises variable region of heavy chain and variable region of light chain, and the aminoacid sequence of its variable region of heavy chain and variable region of light chain is respectively as shown in SEQIDNO:2 and SEQIDNO:4.
2. human monoclonal antibodies as claimed in claim 1, it is characterized in that, its CH selects the constant region of one of heavy chain type in lower group: IgGl, IgG2a, IgG2b and IgG3, and its constant region of light chain selects one of constant region of lower group light chain type: κ chain and λ chain.
3. human monoclonal antibodies as claimed in claim 1, it is characterized in that, the aminoacid sequence of its CH and constant region of light chain is respectively as shown in Genbank ACK87036 and ACK87038.
4. the nucleic acid molecule of the human monoclonal antibodies described in the claim 1-3 that encodes is arbitrary.
5. the arbitrary described human monoclonal antibodies of claim 1-3 is for the preparation of diagnosis, the purposes treated and/or prevented in the medicine of the influenza infection caused by influenza virus.
6. purposes as claimed in claim 5, it 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.
7. an expression vector, is characterized in that, the DNA containing the arbitrary described human monoclonal antibodies of coding claim 1-3 in described expression vector.
8. a host cell, is characterized in that, containing expression vector according to claim 7 in described host cell.
9. a composition, is characterized in that, it contains the arbitrary described monoclonal antibody of claim 1-3 of significant quantity, and pharmaceutically acceptable carrier.
10. detect a test kit for influenza virus, it comprises the arbitrary described human monoclonal antibodies of claim 1-3.
11. 1 kinds of nondiagnostic ground detect the method for influenza virus, it is characterized in that, the arbitrary described human monoclonal antibodies of claim 1-3 is utilized to contact with testing sample, by detect described human monoclonal antibodies and given the test agent in conjunction with situation, there is situation and amount in what obtain influenza virus.
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