CN103374070B - A kind of binding molecule 2F5 of rabies poison - Google Patents
A kind of binding molecule 2F5 of rabies poison Download PDFInfo
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- CN103374070B CN103374070B CN201210122898.0A CN201210122898A CN103374070B CN 103374070 B CN103374070 B CN 103374070B CN 201210122898 A CN201210122898 A CN 201210122898A CN 103374070 B CN103374070 B CN 103374070B
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Abstract
The present invention relates to a kind of binding molecule 2F5 of rabies poison.Binding molecule of the present invention can in conjunction with the rabies virus with native conformation, and compared with the binding molecule that some are animal derived, binding molecule immunogenicity of the present invention greatly reduces, and affinity is good.Not only result for the treatment of is good, and side effect is low.
Description
Technical field
The invention belongs to biotechnology and field of immunology; More specifically, the present invention relates to a kind of binding molecule of rabies poison.
Background technology
Rabies are the common transmissible diseases of a kind of viral people and animals, can propagate between human and animal.The rabies virus infection of most of mankind starts from by the animal bite of RV virus infection or scratches, and virus enters human body by animal saliva.The mad clinical manifestation of the mankind can be divided into four-stage, (average 1-3 month latent period, without any symptom), prodromal stage (the infected starts to occur general malaise, fever, the symptom such as tired, uneasy), excitation period (general spasticity, illusion, the water funk wind that keeps in dark place), coma stage (degree of depth is gone into a coma, and finally suffocates dead because of bottleneck throat spasm or dies of exhaustion).In not vaccinated animals or humans, running mad is almost 100% mortality ratio (with reference to AHumanMonoclonalAntibodyCocktailasaNovelComponentofRabie sPostexposureProphylaxis, 2007).
The whole world, nearly 100,000,000 people need the exposure aftertreatment of rabies virus every year, and about have 4-7 ten thousand people to die from rabies every year, 95% mainly concentrates on Africa, the nations of China and India.Therefore, at present in the urgent need to for rabic safe, effective, cheap prophylactic treatment method.
Rabies virus (Rabiesvirus, RABV) belongs to Rhabdoviridae Rhabdovirus, is to cause rabic pathogenic agent.Its profile is in playing shape, and nucleocapsid is helically symmetrical, and surface has coating, interior containing single stranded RNA.There is cyst membrane outside virion, inside have nucleocapsid.The outermost layer of cyst membrane has the many fibres be made up of glycoprotein to dash forward, and arrangement is relatively more neat, and this projection has antigenicity, and body can be stimulated to produce neutralizing antibody.Virus is containing 5 kinds of major protein (L, N, G, M1 and M2) and 2 kinds of small albumen (P40 and P43).L albumen presents Transcription; N protein is the main nucleoprotein of composition virus particle, is the main component of induction rabies cellular immunization, is usually used in the diagnosis of rabies virus, classification and epidemiological study; G-protein forms the prominent glycoprotein of virus surface fibre, and having the erythrocytic characteristic of aggegation, is the structure that rabies virus is combined with cell receptor, causes a disease and play keying action in immunity rabies virus; M1 albumen is specific antigens, and forms cell-surface antigens with M2.
Be used for preventing those people be exposed under rabies virus to occur the main method of similar rabic symptom at present, use the rabies virus of attenuation to combine the immunoglobulin (Ig) obtained in the people or horse body of immunity again as vaccine exactly.But the safety problem of these blood productss and batch between unstable etc. greatly limit the applicability of the method, force people to seek the rabic method of new prevention and therapy.
The treatment that therapeutic antibodies is used for disease of viral infection 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 human monoclonal antibodies with the rabies poison of 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.1986, first strain treating organs transplants mouse monoclonal antibody---the Orthoclone OKT 3 (murmonabCD3 of the rejection occurred, hocloneOKT3) be approved listing by U.S. FDA, but because it can human anti-murine antibodies (HAMA) react in human body, limit application.Along with immunology and molecular biological development, genetic engineering antibody develops rapidly, chimeric antibody, humanized antibody and human antibody production technology development, is down to minimum and even eliminates HAMA reaction.2009,4 are had for human antibody in 14 medicines of U.S. FDA approval, this indicates the arriving in total man's therapeutic monoclonal antibodies epoch, human antibody becomes the developing direction (with reference to AHumanMonoclonalAntibodyCocktailasaNovelComponentofRabie sPostexposureProphylaxis, 2007) in antibody drug future.
G-protein in rabies virus major protein, can be combined with acetylcholine receptor thus make virus have neurotoxicity, and is the albumen uniquely making to produce in body protectiveness neutralizing antibody known at present, thus becomes the object of scientists study Dispersal risk medicine.Fig. 8 is the structural representation of G-protein.Rabies virus G protein is made up of 524 amino acid usually, wherein front 19 Amino acid profile hydrophobic signal peptides, and mediation G-protein enters endoplasmic reticulum, the ripe G-protein that the cut rear formation of signal peptide is made up of 505 amino acid.Ripe G-protein is structurally divided into 3 regions: 1 ~ 439 amino acids forms film outskirt, is positioned at the surface of viral particle envelope membrane, carries the epitope point that rabies virus is main, and participates in the process of Receptor recognition and film fusion; 440 ~ 461 is cross-film district, with fixing relevant on viral lipid duplicature of G-protein; 462 ~ 505 is film inner region, is positioned at peplos internal surface, provides the action site (with reference to AHumanMonoclonalAntibodyCocktailasaNovelComponentofRabie sPostexposureProphylaxis, 2007) of M and N.
To 2011, the neutralizing antigenic site of clear and definite G-protein had three: GI only containing an epi-position, is minor antigen site; GII is positioned at 34 ~ 200 amino acids sections, and wherein two Primary epitope (34 ~ 42 with 198 ~ 200 sections) are connected by disulfide linkage, are typical space conformation antigen sites; GIII is roughly positioned at 330 ~ 357 sections, is topmost antigenic region, is also spatial dependence antigen site.In addition, G also exist some linear neutralizing epitopes: 244 ~ 281 amino acids epi-positions, 218 ~ 240 amino acids epi-positions, 251 tryptophane (Trp251) epi-positions.Rabies virus neutralizing monoclonal antibodies SO57 most widely used in the market acts in the epi-position of the antigenic region I shown in Fig. 8.
As can be seen here, the binding molecule developed for rabies virus G protein, future will produce important effect in for rabies virus post-exposure prophylaxis and follow-up treatment.
Summary of the invention
The object of the present invention is to provide a kind of binding molecule of rabies poison.
In a first aspect of the present invention, a kind of binding molecule of separation is provided, it can identify and in conjunction with rabies virus envelope protein G-protein, 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; With
In a 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.
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 is human monoclonal antibodies.
In another aspect of this invention, the nucleic acid molecule of the binding molecule described in coding is provided.
In another aspect of this invention, provide described binding molecule for the preparation of diagnosis, the purposes treated and/or prevented in the medicine of rabies virus infection.
In another aspect of this invention, provide a kind of expression vector, the DNA containing the binding molecule described in coding in described expression vector.
In another aspect of this invention, provide a kind of host cell, containing described expression vector in described host cell.
In another aspect of this invention, provide a kind of composition, it contains the described monoclonal antibody of significant quantity, and pharmaceutically acceptable carrier.
In another aspect of this invention, provide a kind of test kit detecting rabies virus, it comprises described binding molecule.
In another aspect of this invention, there is provided a kind of method suppressing rabies virus (preferably, method for non-therapeutic), described method comprises and gives object (as experimenter, patient, or be separated from the place of doubtful rabies virus (as public domain, vehicle, furniture etc.)) the described binding molecule of significant quantity.
In another aspect of this invention, there is provided a kind of method detecting rabies virus (preferably, method for nondiagnostic), 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 rabies virus.
Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.
Accompanying drawing explanation
Fig. 1, FACS selected by flow cytometry apoptosis specific b cells.
The electrophoretogram of Fig. 2, β-actin internal reference.
The electrophoretogram of Fig. 3, heavy chain gene.
The electrophoretogram of Fig. 4, light chain gene.
The concentration determination result of Fig. 5, human monoclonal antibodies (2F5 etc.).
Fig. 6, human monoclonal antibodies (2F5) antigen and antibody specific detect.
Fig. 7, human monoclonal antibodies (2F5) pseudovirus Neutralization effect detect.
The structural representation of Fig. 8, rabies virus G protein.
Embodiment
The present inventor is through extensive and deep research, and obtain a kind of binding molecule of the rabies poison wide spectrum neutrality containing unique CDR district, preferred human monoclonal antibodies, this binding molecule has good neutralizing effect for rabies virus.Complete the present invention on this basis.
Binding molecule
The invention provides the binding molecule of energy specific binding rabies virus.Preferably, described binding molecule is human binding molecules, and it can identify and in conjunction with rabies virus envelope protein G-protein.Binding molecule of the present invention presents the Neutralization effect for rabies virus.
Binding molecule of the present invention can be complete immunoglobulin molecules such as polyclone or monoclonal antibody or 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 containing being enough to (many) skins or its fragment of giving the fragment of immunoglobulin (Ig) be combined with the specific antigens of rabies virus strain.In preferred embodiments, binding molecule of the present invention is human monoclonal antibodies.
According to Kabatetal. (1991), 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 rabies 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 rabies virus or its fragment.Preferably, described functional variant can competitive specific binding by least two (or more) the different rabies 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 rabies virus, preferably at least two (or multiple) rabies 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 be imported by standard technique known in the art, 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 limits 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 rabies virus envelope protein G-protein 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 rabies virus envelope protein G-protein or its fragment.Preferably, variable region includes but not limited to framework region, the aminoacid sequence of hypervariable region, particularly CDR3 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 common molecular biology method known in the art 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.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 anti-rabies monoclonal antibodies, 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 anti-rabies monoclonal antibodies of the present invention is brand-new, and technical conceive is different from existing anti-rabies virus antibody.
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, its have especially excellent identification and in and rabies virus effect while, also there is the feature that immunogenicity is low, security is high.
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.Whether infect rabies virus strain for such as evaluating object or monitored the generation of rabies virus infection as a part for clinical experiment program or be in progress with the effect such as determining TA scheme with comprising the immunoconjugates diagnosticability of detectable substance.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 rabies virus envelope protein G-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 rabies virus and the antigen being attached to binding molecule the strong T cell caused for described conjugate is attacked, finally cause the destruction of rabies 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 anti-rabies monoclonal antibodies variable region of heavy chain (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 rabies virus.
Based on new discovery of the present invention, additionally provide a kind of composition suppressing rabies virus or rabies virus 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 rabies virus 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) rabies 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, be characterised in that described binding molecule in and rabies virus in play synergy.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 rabies virus.Calculating synergistic mode is calculated by combinatorial index.The concept of combinatorial index (CI) is described by ChouandTalalay (1984).Described composition also can comprise a kind of binding molecule and a kind of non-neutral rabies virus specific binding molecules with Neutralization effect.
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 rabies virus.
Dosage regimen can be adjusted to provide response (such as treating response) needed for the best.Suitable dosage range can be such as 0.1-100mg/kg body weight, preferred 0.5-15mg/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 rabies 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 comprise cultured cells, cell conditioned medium and cell lysates " testing sample " also to comprise and being separated from doubtful place of carrying rabies virus (as public domain, vehicle, furniture etc.).
After obtaining binding molecule provided by the invention, the detection kit for specific detection rabies 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 rabies 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 whether the donor of testing sample infects rabies virus, and 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 rabies 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 rabies virus in testing sample is combined with binding molecule, forms the solid phase carrier with " rabies 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 " rabies 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 rabies 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 rabies virus content in testing sample according to concentration standard curve.
Major advantage of the present invention is:
Provide and a kind of there is brand-new binding molecule, can in conjunction with the envelope protein (G-protein) of rabies virus (RABV) with native conformation, thus stop rabies virus be combined with cell receptor and infect permissive cell.Binding molecule of the present invention is total man source, and compared with the molecule of animal derived (as mouse) rabies poison, immunogenicity greatly reduces, and affinity is good.Not only result for the treatment of is good, and side effect is low.
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, Science Press, the condition described in 2002, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number calculate by weight.
Unless otherwise defined, all specialties used in literary composition and scientific words and one skilled in the art the same meaning be familiar with.In addition, any method similar or impartial to described content and material all can be applicable in the present invention.The use that better implementation method described in literary composition and material only present a demonstration.
The acquisition of embodiment 1, peripheral blood lymphocytes (PBMC)
In the volunteer's body injecting hydrophobia, extract peripheral blood, (producer is to adopt conventional Ficoll-Paque
-H (CEDARLANE) company) density gradient centrifugation, obtain 10
7with last peripheral blood lymphocytes (PBMC).
Ficoll separation method:
(1) in 50ml centrifuge tube (in advance containing 4% (w/v) Trisodium Citrate 1ml), collect whole blood 10ml, put upside down mixing 8-10 time (wherein Trisodium Citrate final concentration is 0.4%);
(2) equal-volume RPMI1640 (containing Trisodium Citrate) is added, mixing;
(3) with the transparent centrifuge tube of 15ml, paving 3ml lymphocyte separation medium, carefully adds 6ml blood sample thereon.Form separating interface (or 4ml parting liquid adds 8ml blood sample) B to manage: 500 μ lOpti-MEM+20 μ lLipofectamineReagent;
(4) room temperature centrifugal 800g, 20min (2000rpm, 20min);
(5) careful absorption interfacial layer cell, is transferred to new pipe;
(6) add RPMI1640 (containing Trisodium Citrate), dilution reduces fluid density.Centrifugal, 800g/2000rpm, 10min, remove supernatant.
(7) RPMI1640 washes cell 2-3 time, for subsequent use.
Embodiment 2, the sorting of G-protein specific memory B-cell
Use FITC-CD19/APC-IgG/Cy3-RV-G is mark, obtains specific b cells to 96 hole RT-PCR plate, one, every hole cell through flow cytometer, obtains G-protein specific memory B-cell.
1) rabies virus envelope protein G-protein (the RV-G) (G-protein of RabiesvirusstrainSRV9, GenBank:AF499686.2,3317-4894 in completegenome sequence) expressed (purchased from Invitrogen company, according to the preparation of description of test book) by mammalian cell CHO expression system;
2) G-protein carries out vitamin H (Biotin) mark: No-WeighSulfo-NHS-LC-Biotin (purchased from PIERCE) 10mM reagent; Another two mark FITC-CD19A and PC-IgG are all purchased from BDBioscience company;
3) mark of sorting cells: PBMC cell divides into groups, and experimental group+control group, adds mark by cell count, lucifuge dyes, and marks, after resuspended with PBS, uses 40 μm of BDfalcon membrane filtrations;
4) sorting of specific b cells: use BDFACSARIAII screening, from PBMC, lymphocyte is screened according to forward angle and lateral angle, then compensated by the adjustment of different control group, obtain the specific memory B-cell of mad dog G-protein, be sorted in 96 orifice plates and carried out RT-PCT (reverse transcription PCR), one, every hole cell, plate is placed on dry ice.
As a result, use FITC-CD19/APC-IgG/Cy3-RV-G is Specific marker, obtains the specific b cells of several RV-G albumen, sees the figure that hives off of Fig. 1 Flow Cytometry sorting cells.The position of 96 orifice plates is positioned at, for B cell called after 1A1,1A2,1A6,2F5,3G5 etc. of secretory antibody according to the individual cells be sorted into.
Embodiment 3, antibody gene
For the antibody of B cell 2F5 secretion, experiment is carried out according to the method that (JournalofImmunologicalMethods329 (2008) 112-124) in document reports, namely antibody heavy and light chain variable region gene is obtained by RT and Nested-PCR method, molecular weight is about 400bp, β-actin is as internal reference (343bp), and the electrophoretogram of β-actin, heavy chain gene and light chain gene is shown in Fig. 2, Fig. 3 and Fig. 4 respectively.Same B cell antibody heavy and light chain gene variable region will be derived from and connect pGEMT carrier (purchased from Invitrogen company), sequence verification antibody gene.The 2F5 human antibody gene order obtained is as follows:
2F5 heavy chain variable region gene sequence (SEQIDNO:1) (5 ' → 3 '):
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCCTCGGAGGCCCTGTCCCTCACCTGCACTGTCTCAGGTGTCCCGCTCGGT
TGGATCCGGCAGTCCCCAGGGAAGCGACTGGAATGGATTGGC
CGAGTCACGATATCAGCAGACACGTCCAAGAACCAGATCTCCCTGAGCCTGAGTTCTGTGACCGCTGCGGACACGGCCGTGTATTACTGT
TGGGGCCAGGGAATCCCGGTCACCGTCTCCTCAG
Remarks: what wherein use double underline mark is followed successively by hypervariable region sequence in heavy chain gene variable region and CDR district encoding sequence, hold by 5 ' and are initially followed successively by CDR1 (SEQIDNO:5), CDR2 (SEQIDNO:6), CDR3 (SEQIDNO:7).
2F5 heavy chain variable amino acid sequence (SEQIDNO:2) (N ' → C '):
QVQLQESGPGLVKPSEALSLTCTVSGVPLG
WIRQSPGKRLEWIG
RVTISADTSKNQISLSLSSVTAADTAVYYC
WGQGIPVTVSS
Remarks: wherein use
be followed successively by hypervariable region in heavy chain gene variable region and CDR sequence, held by N and be initially followed successively by CDR1 (SEQIDNO:8), CDR2 (SEQIDNO:9), CDR3 (SEQIDNO:10).
2F5 chain variable region gene sequence (SEQIDNO:3) (5 ' → 3 '):
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGC
TGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCTTGATCTCT
GGGGTCCCTTCAAGGTTCAGTGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTATGCAACTTACTACTGT
GCTCACTTTCGGCGGAGGGACCA
Remarks: wherein double underline mark be followed successively by hypervariable region sequence in light chain gene variable region and CDR district encoding sequence, hold by 5 ' and be initially followed successively by CDR1 (SEQIDNO:11), CDR2 (SEQIDNO:12), CDR3 (SEQIDNO:13).
2F5 chain variable region amino acid sequence (SEQIDNO:4) (N ' → C '):
QMTQSPSSLSASVGDRVTITC
WYQQKPGKAPKLLIS
GVPSRFSGSGSGTEFTLTISSLQPEDYATYYC
LTFGGGT
Remarks: wherein use double underline and overstriking mark be followed successively by hypervariable region in light chain gene variable region and CDR sequence, held by N and be initially followed successively by CDR1 (SEQIDNO:14), CDR2 (SEQIDNO:15), CDR3 (SEQIDNO:16).
Heavy chain, the light chain expression vector of antibody are respectively ABVec-hIgG1, ABVec-hIgKappa.(CloningvectorAbVec-hIgG1，completesequenceGenBank：FJ475055.1；CloningvectorAbVec-hIgKappa，completesequenceGenBank：FJ475056.1)
Two carriers, all containing ammonia benzyl resistant gene, can carry out clone's resistance screening in intestinal bacteria.Carrier adopts CMV as promoter sequence, SV40 and polyA ending signal is as the transcription termination signal of antibody gene.All there is a segment signal peptide sequence before the multiple clone site of expression vector, guide the secretion membrane of antibody; Constant-region sequences containing antibody after multiple clone site.
Utilize molecule clone technology, need to insert weight chain variabl area sequence between the restriction enzyme site AgeI/SalI of AbVec-hIgG1; Between the restriction enzyme site AgeI/BsiWI of AbVec-hIgKappa, insert light-chain variable sequence, can expression vector establishment be completed.
Embodiment 4, antibody expression and antibody concentration measure
Liposome method transient transfection 293T cell, carries out human antibody expression:
(1) at day before transfection by 1.0 × 10
6cell is inoculated in 6 porocyte culture plates;
(2) A pipe: 500 μ lOpti-MEM+4 μ gIgG+4 μ gIgL;
(3) B pipe: 500 μ lOpti-MEM+20 μ lLipofectamineReagent;
(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 LipofectamineReagentDNA, every hole adds 2mlFreeStyle
tM293ExpressionMedium;
(7) after 72h, collecting cell supernatant, 4 DEG C, 3000rpm, 5min.
ELISA method measures human antibody concentration:
(1) wrap by Goatanti-HumanIgG (FabSpecific) antibody 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: 1% (w/v) BSA, every hole 200 μ l, 37 DEG C, 2h;
(4) PBST washes plate, 3 times;
(5) human IgG of doubling dilution is used to do typical curve, concentration 400ng/ml, 200ng/ml, 100ng/ml, 50ng/ml, 25ng/ml, 12.5ng/ml, 6.25ng/ml, 0ng/ml, 100 μ l/ holes; Human antibody cell (1A6,1A2,2F5,1A1,3G5) supernatant dilutes 5000 times, every hole 100 μ l, 37 DEG C, 2h;
(6) PBST washes plate, 3 times;
(7) GoatAnti-HumanIgG (Fcspecific)-Peroxidaseantibody, 1: 10000 dilution, every hole 100 μ l, 37 DEG C, 1h;
(8) PBST washes plate, 3 times;
(9) substrate A liquid: B liquid=1: 1, 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.
ELISA result shows successful expression human antibody, and its concentration is 50 μ about g/ml.Because of the constant region containing heavy and light chain in expression vector, therefore empty carrier also can express incomplete antibody, sees Fig. 5.
Embodiment 5, antigen-specific detect
Detect the human antibody of expressing and whether identify inactivated rabies virus vaccine (purchased from Novartis Co., Ltd), and with the binding ability of antigen.
(1) bag is inactivated hydrophobia (SRV9 vaccine strain) in elisa plate (purchased from NUNC company), 10 μ g/ml, the multiple hole of two, each sample, every hole 100 μ l, and 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, human antibody cell conditioned medium is adjusted to same concentrations, every hole 100 μ l, people many anti-(H.Ab, preparation method is: the blood extracting the people after rabies vaccine immunity, whole blood is placed 37 degree make it coagulate after, place 4 and spend night, get supernatant after centrifugal and be human serum; Human serum ammonium sulfate precipitation or ProteinG purifying all can be obtained at once many anti-) and mouse serum (M.Ab, preparation method is with human serum preparation method) immune mouse BalB/C obtain) be positive control, 37 DEG C, 2h;
(6) PBST washes plate, 3 times;
(7) GoatAnti-HumanIgG (Fc specificity)-Peroxidaseantibody, 1: 10000 dilution, every hole 100 μ l, is added in the elisa plate of sample (2F5,1A6,1A2, H.Ab, M.Ab) and human antibody SO57; GoatAnti-MouseIgG (Fab specificity)-Peroxidaseantibody, 1: 10000 dilution, every hole 100 μ l, is added to the elisa plate of mouse serum, 37 DEG C, 1h;
(8) PBST washes plate, 3 times;
(9) substrate A liquid: B liquid=1: 1, substrate is TMB, TMB (purchased from Sigma, using method presses the description of product).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.
ELISA result shows, 2F5 human antibody has specific binding capacity for inactivated rabies virus vaccine SRV9 strain, and the ability of other human antibody of same batch of this energy force rate preparation is significantly eager to excel in whatever one does.As shown in Figure 6, this experiment, with the polyclonal antibody of the people of inoculated rabies vaccine and the mouse serum positive control of rabies vaccine immunity, the heavy constant region of light chain Vector of the antibody of expressing with empty expression vector is as negative control.
Embodiment 6, antibody neutralization measure---pseudovirus neutralization test
(1) pcDNA3.1 (-)-RV-G plasmid construction: the G-protein total length (G-protein of RabiesvirusstrainSRV9 choosing the above-mentioned SRV9 rabies virus strain mentioned, GenBank:AF499686.2,3317-4894 in completegenome sequence), molecule clone technology is utilized to be inserted into by gene between the EcoRI/XhoI multiple clone site of pcDNA3.1 (-).
Packaging plasmid pNL4-3Luc
+env
-vpr
-build:
This plasmid is on the basis of HIV virion, lack its encoded packets membranin gene Env-and rdrp gene Vpr-, make it cannot translate HIV envelope protein thus can be combined with external source envelope protein, and the pseudovirus of preparation cannot copy the biological safety that can ensure experimental implementation.In addition, also add the luciferase plain gene Luc+ for late detection.This packaging plasmid is purchased from Shanghai Immune Biotech Co., Ltd..
(2) RV-G pseudovirus is packed: by pcDNA3.1 (-)-RV-G plasmid and packaging plasmid pNL4-3Luc
+env
-vpr
-cotransfection 293T cell together, after 48-72h cultivated by 37 DEG C of incubators, collects cells and supernatant, after measuring pseudovirus concentration, is adjusted to proper concn;
(3) infect the day before yesterday, the BHK-21 cell (purchased from ATCC) good with growth conditions spreads 24 orifice plates, and density is 5 × 10
4individual/hole;
(4) same day is infected, the human antibody (2F5) of the RV-G pseudovirus and aforementioned preparation that are adjusted to proper concn or SO57 antibody (available from North China Pharmaceutical Group Company Ltd) are hatched, 1h postoperative infection cell, 1ml/ hole, is placed in 37 DEG C of incubators; Arrange simultaneously and do not hatch infected group, as negative control;
(5), after infecting 24h, the fluid infusion of 1ml/ hole, also adds the human antibody of equal proportion dilution simultaneously.
(6) after infecting 48h, lysing cell, detects fluorescein enzyme values, determines that rabies virus and human antibody hatch the infectivity of front and back for BHK-21 cell, calculates the inhibiting rate of human antibody for virus.
Human monoclonal antibodies (2F5) uses 293T cell to express, and its concentration is adjusted to 0.5ug/ml, 1ug/ml and 2ug/ml and carries out pseudovirus neutralization test and measure its Neutralization effect, namely to the inhibiting rate of mad dog pseudovirus.As shown in Figure 7,2F5 has the inhibiting rate more than 98.5% for virus, and the concentration of antibody becomes positive correlation with to the inhibiting rate of virus.
The improvements that the 2E1 that the present invention obtains contrasts existing SO57 antibody are as follows:
1,2E1 adopts prepared by brand-new single-cell RT-PCR human antibody technology of preparing.With SO57 antibody prepared by the viral immortalizing technology of traditional EBV, not only efficiency is lower to have the method, and the shortcoming of prepared most of antibody, namely affinity of antibody is lower, and antigen and antibody specific is easy to lose.And human antibody prepared by single-cell RT-PCR, not only easy to operate, utilize conventional laboratory conditions to complete, and fast, whole process only uses 30 days, be applicable to the research and development in early stage of the antibody of anti-virus infection.
2,2E1 still keeps when lower concentration preferably acting on.As shown in Figure 7, pseudovirus neutralization test shows, and in lower concentration situation (0.5ug/ml), 2E1 is better than contrast SO57 for the inhibiting rate of rabies virus.This represents, 2E1 is still effective in low dosage situation, can reduce the toxic side effect of medicine to greatest extent.
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 (10)
1. a monoclonal antibody, it can identify and in conjunction with rabies virus envelope protein G-protein, it is characterized in that, described monoclonal antibody comprises variable region of heavy chain, and the aminoacid sequence of described variable region of heavy chain is as shown in SEQIDNO:2; With
Described monoclonal antibody comprises variable region of light chain, and the aminoacid sequence of described variable region of light chain is as shown in SEQIDNO:4.
2. monoclonal antibody as claimed in claim 1, it is characterized in that, described monoclonal antibody is human monoclonal antibodies.
3. the nucleic acid molecule of the arbitrary described monoclonal antibody of claim 1-2 of encoding.
4. the arbitrary described monoclonal antibody of claim 1-2 is for the preparation of diagnosis, the purposes treated and/or prevented in the medicine of rabies virus infection.
5. an expression vector, is characterized in that, the DNA containing the arbitrary described monoclonal antibody of coding claim 1-2 in described expression vector.
6. a host cell, is characterized in that, containing expression vector according to claim 5 in described host cell.
7. a composition, is characterized in that, it contains the arbitrary described monoclonal antibody of claim 1-2 of significant quantity, and pharmaceutically acceptable carrier.
8. detect a test kit for rabies virus, it comprises the arbitrary described monoclonal antibody of claim 1-2.
9. suppress a method for the non-therapeutic of rabies virus, it is characterized in that, described method comprises the arbitrary described monoclonal antibody of the claim 1-2 giving object significant quantity.
10. one kind is detected the method for the nondiagnostic of rabies virus, it is characterized in that, the arbitrary described monoclonal antibody of claim 1-2 is utilized to contact with testing sample, by detect described monoclonal antibody and given the test agent in conjunction with situation, there is situation and amount in what obtain rabies virus.
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| WO2006084006A1 (en) * | 2005-02-02 | 2006-08-10 | University Of Massachusetts | Human antibodies against rabies and uses thereof |
| CN101235086A (en) * | 2007-02-02 | 2008-08-06 | 吉林圣元科技有限责任公司 | Recombination human rabies viruses resisting antibody |
| CN101708331A (en) * | 2009-09-29 | 2010-05-19 | 福尔生物制药股份有限公司 | Novel human rabies vaccine and method for preparing same |
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| WO2006084006A1 (en) * | 2005-02-02 | 2006-08-10 | University Of Massachusetts | Human antibodies against rabies and uses thereof |
| CN101235086A (en) * | 2007-02-02 | 2008-08-06 | 吉林圣元科技有限责任公司 | Recombination human rabies viruses resisting antibody |
| CN101708331A (en) * | 2009-09-29 | 2010-05-19 | 福尔生物制药股份有限公司 | Novel human rabies vaccine and method for preparing same |
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