CN103183736A - Anti-CLCN7 protein monoclonal antibody and application for same - Google Patents

Abstract

The invention provides a monoclonal antibody capable of specifically identifying CLCN7 protein and the function thereof in treatment for bone diseases and tooth diseases. The monoclonal antibody is good in stability and strong in specificity. The invention provides a variable region sequence of the antibody. The invention further provides a medicine composition.

Description

A kind of monoclonal antibody of anti-CLCN7 albumen and application thereof

Technical field

The invention belongs to the biological medicine technology field, particularly the invention provides a kind of monoclonal antibody, this antibody capable specific recognition ClCN7 albumen; And described antibody is for the preparation of the application in the medicine for the treatment of various bones and odontopathy, and the present invention also provides pharmaceutical composition, and it comprises described monoclonal antibody as activeconstituents.

Background technology

Antibody is as the existing developing history that goes up a century of the preparation of disease prevention, diagnosis and treatment.Along with the development of genetic engineering antibody technology and perfect, antibody drug develops into human mouse chimeric antibody, humanized antibody and human antibody from mouse monoclonal antibody.Surpass 40 antibody drugs by U.S. FDA approved in 2010.Also have 120 kinds of antibody drugs of surpassing to be in clinical study in addition, surpass 500 kinds of antibody drugs and be in preclinical study.These antibody are mainly used in autoimmune disorder, infectious diseases and tumor treatment.

Chloride channel is a kind of important anion channel in the organism, and they play specific biological function in the transmembrane transport of cytolemma and born of the same parents' inner cell organ plasma membrane.ClC type chloride channel is the widely distributed anion channel of a class, participates in the acidifying of cell intracellular vesicle, the various physiological processes such as voltage stability of muscle cell, and its transgenation or dysfunction all can cause the generation of multiple disease.ClC-7 is the member of ClC type chloride channel, and it mainly is positioned at the film of holding up of later stage endosome, lysosome and osteoclast.In Mammals, ClC type chlorion family has nine hypotypes, and can be divided into two big classes according to its cellular localization: the first kind is to be present in cytolemma, belongs to the plasmalemma chloride channel.It comprises ClC-1, two isomer of the ClC-ka of ClC-2 and high homology and ClC-kb.Another kind of is to be present in organoid, belongs to proton coupling Cl-transporter.It comprises ClC-3, ClC-4, ClC-5 and ClC-6, ClC-7.These ClC protein expressions only are positioned on the intracellular compartment, from the endosome to the lysosome in the different cell types of Different Organs.Mouse and people, loss ClC-7 or H+-ATP enzyme A3 subunit can cause sclerotin, it be a kind of be the disease of feature with defective bone resorption, the ClC-7 knock out mice demonstrates neurological dysfunction and serious lysosomal storage disease in addition.In people and mouse, CLCN7 transgenation meeting causes sclerotin and lysosomal storage disease.

People CLCN7 gene is positioned karyomit(e) 16p13 by the genomic dna coding of about 26kb.To the analysis revealed of genomic dna sequence, people CLCN7 gene comprises 25 exons, and coding contains 805 amino acid whose protein H+/Cl-exchange transporter 7.As if the CLCN7 gene of people and mouse has identical genomic organization, and the ClC-7 albumen of mouse homology contains 803 amino acid, have 96.1% identical with people ClC-7 albumen.ClC-7 albumen has tissue specificity, can detect transcribing of it in the fabric analysis of brain, muscle, kidney and testis, is a kind of member of ClC chloride channel family of wide expression, mainly is positioned at the later stage endosome, lysosome, and osteoclast.

At osteoclast, ClC-7 can cooperate H+-ATP enzyme acidifying bone resorption lacuna.The acidifying of cell inner chamber is to be driven by the H+-ATPase of vesica, and it is essential that it absorbs neurotransmitter for proenzyme activation, albumen go-no-go, receptor-mediated endocytosis and synaptic vesicle.The gauffer edge of osteoclast is rich in V-ATPase, and osteoclast carries out the absorption of bone by the degrade mineral substance of bone of its secretion hydrogen ion, is to be responsible for the multinuclear bone resorption cell constantly reinventing osseous tissue and keep calcium balance.The activation of osteoclast is created airtight space-a lacuna then from the adhesion of ground substance of bone between the boundary film of holding up and mineralising bone.ClC-7 can by late period endosome-lysosome membrane exocytosis be inserted into this special plasma membrane territory, by among the ClC-7 and electric current be that the acidifying of bone resorption lacuna is necessary efficiently.The acidifying pH value of bone resorption lacuna is close to 4.5, and it is by the H+-ATP enzyme mediation of holding up the border that produces electricity, is that the chemical dissolution of the inorganic materials of bone and organic ground substance of bone lytic enzyme is necessary.The mineralising composition that the bone that the acidifying environment is transferred is formed mainly is calcium phosphate, to expose it subsequently by the organic substrate of enzyme liberating.

Undertaken in the macromolecular degradation process by endocytic pathway, lysosome is equivalent to the stomach of cell terminal organoid.Lysosome contains lytic enzyme widely, and its high efficiency function depends on keeping of acid enteric cavity pH value.Though it is the acidifying by V-type proton ATP enzyme mediation, allow proton transportation in bulk by way of in parallel negatively charged ion approach be absolutely necessary.ClC-7 is an important CL-/H+ antiport body in the lysosome acidifying, and it has constituted lysosomal main Cl-permeability.Although the lysosomal pH value of neurone of cultivating is constant, the CLCN7 knock out mice demonstrates serious lysosomal storage disease.The bone phenotype of saving them by the genetically modified expression of ClC-7 in osteoclast can appropriateness increase its life-span, and has disclosed the pathology of the central nervous system of further progress.

Ostm1 is one and contains the amino terminal region of a large amount of saccharification and the I type membranin of short kytoplasm tail.ClC-7 needs Ostm1 to support its bone resorption and lysosome function as the B-subunit.Owing to be cut in lysosome height saccharification Ostm1 tube chamber territory, Ostm1 needs ClC-7 to reach lysosome, and ClC-7 can be issued to its point of destination in the situation that does not have Ostm1, but the stability of ClC-7 depends on the combination of it and Ostm1.Because glycosylation can protect lysosome membrane albumen not to be degraded, people guess that the Ostm1 protein of height saccharification can shield the mammalian proteins matter ClC-7 that unique shortage N-connects glycosylation site and not be degraded.The pathological observation the possibility of result of Ostm1 loss is because ClC-7 chlorion transhipment subsequently is unstable fully.In fact, the fatal mouse of grey not only have as Clcn7-/-sclerotin that mouse shows, also demonstrate similar lysosomal storage disease.

In the time of 2002 Kornak etc. utilize gene knockout set up Clcn7-/-mouse model, Clcn7-/-mouse gives birth to and 4 weeks of back serious osteosclerosis occurs, anaemia, hepatosplenomegaly, retina and optic nerve are degenerated.The loss of CLC-7 has caused serious sclerotin in mouse, it be a kind of be the disease of feature with defective bone resorption.Though the mouse amount of osteoclast of CLCN7 gene knockout is normal, bone resorption lacuna that can not the acidifying cell owing to them makes them can't absorb bone.CLCN7 gene knockout and the mouse that suffers from sclerotin can be rescued by the specific transgene expression ClC-7 of osteoclast.

Also determined the sudden change of ClC-7 in the autosomal dominant inheritance bone disease II type (ADOII) of the Osteopetrosis (ARO) of human malignant's autosomal recessive inheritance and " optimum ", these phenotypes are feature with the osteoclast of non-bone resorption, demonstrate the simple and crude border of holding up, this shows that the bone resorption lacuna is disturbed in acidization.Its different phenotypes can be interpreted as ClC-7 chloride channel function and reduce in various degree, because the negative sudden change of ClC-7 heterozygote dominance can only reduce, but can not cancel the function of ClC-7.The main iconography of the ADOII that CLCN7 causes is characterized as the spinal vertebrae increase in density, is typical " Layer cake " and levies, and the frontal bone density unevenness is even to be increased, and deep or light identical concentric annular ripple " os in os " phenomenon occurs.The result of study of ANNALISA in 2003 etc. shows: the ARO that recessive ClCN7 relies on may get involved relevant with central nervous system, and be difficult to predict, and the phenotype that heterozygote ClC-7 sudden change causes, even the phenotype scope is also very wide in same family, comprise from early stage severe to asymptomatic form almost.

Except the function of its bone resorption, CLC-7 also has other important effects in organism.People such as Su Ying in 2008 studies show that the expression that Clcn7mRNA is arranged in the mouse dental germ growth course, enameloblast is that LS8 also has the early stage inner enamel epithelium confluent monolayer cells of Clcn7mRNA. mouse dental germ mitriform strong positive to express, mitriform enameloblast in late period, odontoblast's strong positive are expressed, and result of study prompting CLC-7 may participate in enamel, Dentinal formation and mineralisation process.In addition, the CLC-7 deficient mice shows serious retinal degeneration.Visual disorder is often to occur serious infant's bone disease patient, and is optic nerve compression in the marble bones by attribution usually.Nearest studies show that, it is the central nervous system degeneration widely of characteristic feature that CLC-7 interrupts causing with neurone wax sample lipofuscin (NCL), and it is a hypotype of the mankind's lysosomal storage disease.

Characteristic of the present invention is to use recombinant expressed CLCN7 protein film outskirt directly to present in the expression library at non-immunized mice source ScFv phage surface to screen, and this antibody can be applied to the treatment field with the closely-related bone of CLCN7 albumen or tooth relative disease.

Monoclonal antibody called after HX2 provided by the invention, through detection monoclonal antibody to specific recognition in conjunction with CLCN7 albumen.

Summary of the invention

The purpose of this invention is to provide a kind of monoclonal antibody, described monoclonal antibody can be combined with the CLCN7 protein-specific, thereby can be for the preparation of the medicine for the treatment of bone and odontopathy.

Particularly, the invention provides following:

1. a monoclonal antibody is characterized in that, its variable region of heavy chain is made up of 124 amino-acid residues, sequence following (from the N-end):

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

Leu Ser Cys Lys Gly Ser Gly Phe Asn Ile Lys Asp Tyr Tyr Met His Trp Val Lys

Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Trp Ile Asp Pro Glu Asn Gly

Asn Thr Ile Tyr Asp Pro Lys Phe Gln Gly Lys Ala Ser Ile Thr Ala Asp Thr Ser

Ser Asn Thr Ala Tyr Leu His Leu Ile Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr

Tyr Cys Ala Arg Asp Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser

Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu

The cDNA sequence of encoding heavy chain variable region is (since 5 ' end):

gaggtgaagcttgtggagtctggggctgagtttgtgaggccaggggccttagtcaagttgtcctgcaaaggttct

ggcttcaacattaaagactactatatgcactgggtgaaacagaggcctgaacagggcctggagtggattggatg

gattgatcctgagaatggtaatactatatatgacccgaagttccagggcaaggccagtataacagcagacacatc

ctccaacacagcctacctgcacctcatcagcctgacatctgaggacactgccgtctattactgtgctagagactttg

actactggggccaaggcaccactctcacagtctcctcagccaaaacgacacccccatctgtctatccactg

Its variable region of light chain is made up of 113 amino-acid residues, sequence following (from the N-end):

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Gln Ala

Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu

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

Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp

Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Phe Cys Ser

Gln Asn Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

The cDNA sequence of encoded light chain variable region is (since 5 ' end):

GACATTGTCATGACCCAGACTCCACTCTCCCTGCCTGTCAGTCTTGG

AGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACA

GTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAG

TCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGT

CCCAGACAGGTTCAGTGGCAGTAGATCAGGGACAGATTTCACACTC

AAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAATTTATTTCTGCTC

TCAAAATACACATGTTCCGTGGACGTTCGGTGGAGGCACCAAACTG

GAAATCAAACGG

2. a monoclonal antibody is characterized in that, the aminoacid sequence of its sequence and claims 1 described monoclonal antibody has the sequence homology more than 90% or 90%.

3. a genetic engineering antibody is characterized in that, the heavy chain that it contains and light chain variable region sequence be with above 1 and the sequence of 2 described variable regions be consistent; This genetic engineering antibody can be: the people--mouse chimeric antibody (chimeric antibodies); Or humanized antibody (humanized antibody, HAb) or reshaping antibody (Reshaped antibody, RAb); Or the functional fragment Fab of antibody; Or single-chain antibody (single chain FV fragment, SCFv); Or the antibody function fragment VH-L of the fusion of variable region of heavy chain and complete light chain; Or the arrangement of one or more CDR of heavy chain and light chain, the antibody function fragment of connecting or combining; Or functional fusion rotein of above-mentioned antibody or antibody functional fragment and other various albumen or the polypeptide antibody-like that is connected, splices, merges and obtain.

4. as the described monoclonal antibody of claims 1-2 or claims 3 described genetic engineering antibodies, it is characterized in that it and CLCN7 albumen have special binding ability.

5. as the described monoclonal antibody of claims 1-4 or genetic engineering antibody, it is characterized in that it and CLCN7 albumen have special binding ability, and be used for the treatment of bone and odontopathy

6. a pharmaceutical composition is characterized in that, it contains just like the described monoclonal antibody of claims 1-4 or genetic engineering antibody; And pharmaceutically receivable carrier or damping fluid or additive or vehicle.

7. as claims 6 described pharmaceutical compositions, it is characterized in that it can be used for treating various bones and odontopathy.

The explanation of accompanying drawing table:

Fig. 1. the SDS-PAGE that is connected the total length HX2 antibody of expressing the back with constant region detects.Swimming lane

1 is HX2 heavy chain of antibody and the light chain after the DTT reduction; Swimming lane 2 is the reference of protein standard molecular weight; Swimming lane 3 is unreduced total length HX2 antibody.

Embodiment

In order more fully to understand and apply the invention, hereinafter describe reference example and accompanying drawing in detail the present invention, described embodiment only is intended as illustrative explanation the present invention, rather than intention limits the scope of the invention.Scope of the present invention is specifically limited by accompanying Claim.

Embodiment one, people sourceCLCN7 albumen Recombinant expressed and purifying

Design primer (Primer 1:5 ' GCATGCCATGGCCAACGTCTCTAAG

And Primer2:5 ' GTTCCTCGAGTTAGCGCTTGATCTCCACC

) from people's Skeletal Muscle Cell cDNA by pcr amplification (PCR principal reaction condition: 94 ℃ of sex change 30 seconds, 60 ℃ of annealing 30 seconds, 72 ℃ were extended 35 seconds; Totally 30 circulations), between the NcoI/XhoI site of the purpose fragment that obtains through being connected to expression vector pHAT2 behind the NcoI/XhoI double digestion, behind the screening positive clone, find that through order-checking the sequence and the theoretical sequence that obtain are in full accord:

atggccaacgtctctaagaaggtgtcctggtccggccgggaccgggacgacgaggaggcggcgccg

ctgctgcggaggacggcgcggcccggcggggggacgccgctgctgaacggggctgggcctggggctgcg

cgccagtcaccacgttctgcgcttttccgagtcggacatatgagcagcgtggagctggatgatgaacttttggacc

cggaTatggaccctccacatcccttccccaaggagatcccacacaacgagaagctcctgtccctcaagtatgag

agcttggactatgacaacagtgagaaccagctgttcctggaggaggagcggcggatcaatcacacggccttcc

ggacggtggagatcaagcgc；

126 amino acid residue sequences of its coding are:

MANVSKKVSWSGRDRDDEEAAPLLRRTARPGGGTPLLNGAGPGAARQSPRSALFRVGHMSSVELDDELLDPDMDPPHPFPKEIPHNEKLLSLKYESLDYDNSENQLFLEEERRINHTAFRTVEIKR

The recombinant plasmid pHAT2-clcn7 that obtains is transformed in the e. coli bl21 (DE3), chooses mono-clonal 37 ℃ of overnight incubation in 10ml fresh culture (penbritin that contains 100ug/ml) from this conversion flat board; The 10ml bacterium liquid that will spend the night change over to 1L fresh culture (penbritin that contains 100ug/ml) in 37 ℃ cultivate 4 hours after, the IPTG that adds final concentration and be 0.5mM induces the expression of target protein, induces 6 hours; In 4 ℃ of centrifugal collection bacterial sediments of 6000rpm.

With the bacterial sediment that obtains with the 60ml lysis buffer (25mM Tris, pH8,500mMNaCl) resuspended after, the ultrasonic degradation bacterium; Centrifugal 30 minutes of 15000rpm (4 ℃) collects supernatant, joins in the Ni-NTA filler of the good 1ml of lysis buffer balance last sample speed 30ml/ hour; Clean the adhesion protein of non-affine combination again with lysis buffer; Use 10ml elution buffer (25mM Tris, pH8,500mM NaCl, 200mM imidazole) target protein to be eluted 15%SDS-PAGE electrophoretic analysis purification result then.

The albumen desalination of purifying is changed in the PBS buffer system, and-80 ℃ frozen standby.

Embodiment two, Panning screening

It is the non-immunized mice source ScFv expression library that patent application person makes up before that phage surface presents the library.This patent is exactly the antibody of screening energy specific recognition CLCN7 albumen from this library.With CLCN7 proteantigen bag by plastic culture dish after, the recombinant phage supernatant is inclined on it, cultivate 2h, successively respectively wash plate 20 times with PBS and PBST for 37 ℃.The TG1 that will be cultured to logarithmic phase then inclines on the culture dish to immunosorption, cultivates 1 hour for 37 ℃.So the process of " adsorbing a wash-out one breeding " is the Panning screening, and again with the M13K07 superingection, the phage surface that just can generate the enrichment clone presents the library.Carry out 7 later on as stated above and take turns screening.

The evaluation of embodiment three, single recombinant phage clone's antigen-binding activity

1. prepare the mono-clonal recombinant phage

After the Panning screening, TGI mattress liquid is pressed stoste; 1: 10; 1: 100; 1: 10004 dilution is laid on the SOBAG agar plate, is inverted overnight incubation for 30 ℃.Select 90 single bacterium colonies, be inoculated in respectively among 2 * YTAG of 100ul, 30 ℃ of overnight incubation, get the 20ul culture, add 200ul and contain among 2 * YTAG of 5 * 10pfu/ml M13K07,37 ℃ of shaking culture 2h are centrifugal, with the resuspended precipitation of 2 * YTAK 200ul, 30 ℃ of overnight incubation. the supernatant after centrifugal is single recombinant phage.

2.ELISA detect antigen-binding activity (Douillard, et al., Enzyme-linked Immunosorbent Assay for Screening monoclonal antibody production using enzyme-labeled second antibody, Meth.Enzymol, .92:168-74,1983)

If 1 negative control hole, gaging hole is treated for 90 in 1 positive control hole.100u 10.5%BSA bag is by negative control hole, and 100ul goat-anti M13 phage antibody bag is by the positive control hole, and 100ul/ hole (10ug/ml) CLCN7 proteantigen bag is treated gaging hole.37 ℃ of sealings of 1%BSA 1 hour.Control wells adds the M13 phage, treats that gaging hole adds the mixture of 50ul supernatant to be measured and 50ul confining liquid, hatches 1h for 37 ℃.Wash plate 3 times with PBST, the HRP/ goat-anti M13 that each hole adds the 100ul working concentration bites mattress body antibody, and 37 ℃ were reacted 1 hour.PBST washes plate 4 times, adds freshly prepared H202-ABTS, surveys every hole OD value at 410nm.

ScFv gene sequencing, eukaryotic expression and the purifying of embodiment four, positive colony

The positive colony of selecting screening to obtain send company to carry out dna sequencing respectively.Be inserted into eukaryotic expression after the corresponding weight chain constant region connection of design primer with the weight chain variable region of positive colony and mouse IgG; Transfection CHO cell screening stably express strain respectively then.

Get the ProteinG-sepharose FF medium of 1ml in pillar, after the PBS balance, with the cells and supernatant of collecting through ProteinG-sepharose FF pillar purifying, after cleaning 50 column volumes with PBS then, with 100mM Glycine (pH3) target protein is eluted to 1M Tris, in the pH8.5 balance liquid.In carrying out dialysis the PBS damping fluid respectively.The pure antibody product that the 10%SDS-PAGE purification Identification obtains.

The activity identification of embodiment five, antibody

If 1 negative control hole, gaging hole is treated for 4 in 1 positive control hole.100ul 10.5%BSA bag is by negative control hole, and 100ul sheep anti-mouse antibody bag is by the positive control hole, and 100ul/ hole (10ug/ml) CLCN7 proteantigen bag is treated gaging hole.37 ℃ of sealings of 1%BSA 1 hour.Control wells adds IgG, treats that gaging hole adds the mixture of 50ul antibody to be measured and 50ul confining liquid, hatches 1h for 37 ℃.Wash plate 3 times with PBST, each hole adds the HRP/ sheep anti-mouse antibody of 100ul working concentration, and 37 ℃ were reacted 1 hour.PBST washes plate 4 times, adds freshly prepared H202-ABTS, surveys every hole OD value at 410nm.Found that and have only a strain antibody and antigen that very strong reaction is arranged.Called after HX1.

Reference

Douillard，et al.，Enzyme-linked Immunosorbent Assay for Screening monoclonal antibody production using enzyme-labeled second antibody，Meth.Enzymol，.92：168-74，1983

Leisle L et al.ClC-7 is a slowly voltage-gated 2Cl/1H-exchanger and requires Ostm1 for transport activity.EMBO J，2011，30：2140-2152

Zhao Q et al.ClC-7：A potential therapeutic target for the treatment of osteoporosis and neurodegeneration.BBRC，2009，384：277-279

Lange PF et al.ClC-7requires Ostm1 as a b-subunit to support bone resorption and lysosomal function.Nature，2006，440：220-223

Kasper D et al.Loss of the chloride channel ClC-7 leads to lysosomal storage disease and neurodegeneration.EMBO J，2005，24：1079-1091

Annalisa F et al.Chloride Channel ClCN7 Mutations Are Responsible for Severe Recessive，Dominant，and Intermediate Osteopetrosis.J.Bone，2003，10：1740-1747

Clinical and the genetic analysis of a family of .II type autosomal dominant inheritance osteopetrosises such as Su Nan.

China's osteoporosis and bone mineral content disease magazine, 2011,4:18-22

Chen Lie, the research of Xie Hao .ClC type chloride channel. the chemistry of life, 2010,30:545-549

Zhang Xinwei. the progress of Osteopetrosis. foreign medical science (blood transfusion and hematology fascicle), 2002,25:453-455

1

<211> 124

<212> PRT

＜213〉HX2 antibody gene

<220>

＜221〉HX2 antibody heavy chain variable region aminoacid sequence

<222> (1)..(124)

<223>

<400> 1

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

1 5 10 15

Ala Leu Val Lys Leu Ser Cys Lys Gly Ser Gly Phe Asn Ile Lys

20 25 30

Asp Tyr Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu

35 40 45

Glu Trp Ile Gly Trp Ile Asp Pro Glu Asn Gly Asn Thr Ile Tyr

50 55 60

Asp Pro Lys Phe Gln Gly Lys Ala Ser Ile Thr Ala Asp Thr Ser

65 70 75

Ser Asn Thr Ala Tyr Leu His Leu Ile Ser Leu Thr Ser Glu Asp

80 85 90

Thr Ala Val Tyr Tyr Cys Ala Arg Asp Phe Asp Tyr Trp Gly Gln

95 100 105

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

110 115 120

Val Tyr Pro Leu

<210> 2

<211> 113

<212> PRT

＜213〉HX2 antibody gene

<220>

＜221〉HX2 antibody chain variable region aminoacid sequence

<222> (1)..(113)

<223>

<400> 2

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu

1 5 10 15

Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val

20 25 30

His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro

35 40 45

Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe

50 55 60

Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp

65 70 75

Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Ile

80 85 90

Tyr Phe Cys Ser Gln Asn Thr His Val Pro Trp Thr Phe Gly Gly

95 100 105

Gly Thr Lys Leu Glu Ile Lys Arg

110

Claims (7)

1. a monoclonal antibody is characterized in that, its variable region of heavy chain is made up of 124 amino-acid residues, and sequence is as shown in the SEQ ID NO:1; Its variable region of light chain is made up of 113 amino-acid residues, and sequence is as shown in the SEQ ID NO:2.

2. a monoclonal antibody is characterized in that, the aminoacid sequence of its sequence and claims 1 described monoclonal antibody has the sequence homology more than 90% or 90%.

3. a genetic engineering antibody is characterized in that, the heavy chain that it contains and light chain variable region sequence are that the sequence with claims 1 and 2 described variable regions is consistent; This genetic engineering antibody can be: the people--mouse chimeric antibody (chimeric antibodies); Or humanized antibody (humanized antibody, HAb) or reshaping antibody (Reshaped antibody, RAb); Or the functional fragment Fab of antibody; Or single-chain antibody (single chain FV fragment, SCFv); Or the antibody function fragment VH-L of the fusion of variable region of heavy chain and complete light chain; Or the arrangement of one or more CDR of heavy chain and light chain, the antibody function fragment of connecting or combining; Or functional fusion rotein of above-mentioned antibody or antibody functional fragment and other various albumen or the polypeptide antibody-like that is connected, splices, merges and obtain.

4. as the described monoclonal antibody of claims 1-2 or claims 3 described genetic engineering antibodies, it is characterized in that it and ClCN7 albumen have special binding ability.

5. as the described monoclonal antibody of claims 1-4 or genetic engineering antibody, it is characterized in that it and ClCN7 albumen have special binding ability, and be used for the treatment of bone and odontopathy.

6. a pharmaceutical composition is characterized in that, it contains just like the described monoclonal antibody of claims 1-4 or genetic engineering antibody; And pharmaceutically receivable carrier or damping fluid or additive or vehicle.

7. as claims 6 described pharmaceutical compositions, it is characterized in that it can be used for treating various bones and odontopathy.

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