CN109071638A - Antibody or its antigen-binding fragment in conjunction with from the antigentic specificity of mycobacteria - Google Patents
Antibody or its antigen-binding fragment in conjunction with from the antigentic specificity of mycobacteria Download PDFInfo
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- C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
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Abstract
The present invention relates to the antigen-binding fragments specifically bound with the CFP-10 or Ag85B as the antigen from mycobacteria, the polynucleotides, the recombinant vector comprising the polynucleotides and the host cell comprising the recombinant vector that encode it.When using antigen-binding fragment of the invention, the kit that can quickly and accurately detect mycobacteria can be developed using highly sensitive detection as the CFP-10 or Ag85B of the antigen from mycobacteria.
Description
[technical field]
This disclosure relates to culturing filtrate protein 10 (CFP-10) as the antigen from mycobacteria or anti-
The antibody or its antigen-binding fragment of former 85B (Ag85B) specific binding, include the multicore glycosides at the polynucleotides for encoding it
Sour recombinant vector and the host cell comprising the recombinant vector.
[background technique]
In general, tuberculosis is disease caused by being infected by mycobacterium tuberculosis (Mycobacterium tuberculosis)
Disease, and be considered as the highest disease of disease incidence in legal infectious diseases in the world.It is most important that being used for
Obtain the early diagnosis and therapy of authentic communication infected with the patient of tuberculosis from suspection in a short time.
Tuberculin skin test (tuberculin skin test, TST) is common for being diagnosed to tuberculosis
Method, but there is low specificity and be not therefore highly reliable in terms of the standard of tuberculosis infection.Nevertheless, the test
It is still widely used due to highly sensitive and cost allowance.In addition, the method cultivated mycobacterium tuberculosis is each
Most reliable test method in kind of Diagnosis of Tuberculosis method, and be the side of tuberculosis of being clarified a diagnosis by M. tuberculosis multiplication
Method.However, the risk of experimenter's infection is very important, and cultivates since this method needs to separate and cultivate bacterium
Bacterium at least spends about 4 weeks to about 6 weeks, therefore early diagnosis is difficult.
Recently, it is used for using fluid nutrient medium or Mycobacter Growth Indicator Tube (Mycobacteria growth
Indicator tube, MGIT) method BACTEC system.By this method, when can shorten culture using fluid nutrient medium
Between, and therefore can determine mycobacterium tuberculosis proliferation in about two weeks.However, this method needs other test of clarifying a diagnosis
To distinguish non tuberculous Mycobacterium, and presence such as following problems: as using waste caused by radioactive isotope to handle
Problem, the risk of mycobacterium tuberculosis infection, and the demand to expensive device.
Therefore, be continuously needed exploitation for quickly and accurately detect mycobacterium tuberculosis technology, and for that purpose it is necessary to
Develop the method for specifically detecting the antigen secreted in M. tuberculosis multiplication.
[detailed description of the invention]
[technical problem]
It provides and the antibody specifically bound of the CFP-10 as the antigen from mycobacteria or its antigen binding
Segment, the polynucleotides for encoding it, the recombinant vector comprising the polynucleotides and the host comprising the recombinant vector are thin
Born of the same parents.
Provide the antibody or its antigen binding fragment specifically bound with the Ag85B as the antigen from mycobacteria
Section, polynucleotides, the recombinant vector comprising the polynucleotides and the host comprising the recombinant vector for encoding it are thin
Born of the same parents.
[solution to the problem]
One embodiment of present disclosure provides in conjunction with from the CFP-10 antigentic specificity of mycobacteria
Antibody or its antigen-binding fragment, the antibody or its antigen-binding fragment include: the amino acid sequence containing SEQ ID NO:5
The heavy chain variable region of column；And the light chain variable region of the amino acid sequence comprising SEQ ID NO:6.
One of present disclosure embodiments further provide with the CFP-10 antigentic specificity knot from mycobacteria
The antibody of conjunction or its antigen-binding fragment, the antibody or its antigen-binding fragment include: the amino acid containing SEQ ID NO:7
The heavy chain variable region of sequence；And the light variable domains of the amino acid sequence containing SEQ ID NO:8.
Present disclosure another embodiment provides in conjunction with from the Ag85B antigentic specificity of mycobacteria
Antibody or its antigen-binding fragment, the antibody or its antigen-binding fragment include: the amino acid sequence containing SEQ ID NO:9
The heavy chain variable region of column；And the light chain variable region of the amino acid sequence containing SEQ ID NO:10.
Present disclosure another embodiments further provide with the Ag85B antigentic specificity knot from mycobacteria
The antibody of conjunction or its antigen-binding fragment, the antibody or its antigen-binding fragment include: the amino containing SEQ ID NO:11
The heavy chain variable region of acid sequence；And the light chain variable region of the amino acid sequence containing SEQ ID NO:12.
Present disclosure another embodiments further provide with the Ag85B antigentic specificity knot from mycobacteria
The antibody of conjunction or its antigen-binding fragment, the antibody or its antigen-binding fragment include: the amino containing SEQ ID NO:13
The heavy chain variable region of acid sequence；And the light chain variable region of the amino acid sequence containing SEQ ID NO:14.
Term " mycobacteria " used herein refers to the thick cell wall comprising waxy hydrophobic mycolic acid
Aerobic antiacid gram-positive bacteria.Mycobacteria can be classified as the nonpathogenic mycobacteria mushroomed out in the training period and
The pathogenic mycobacterium slowly grown during culture.CFP-10 or Ag85B antigen may be from pathogenic mycobacterium, such as
Mycobacterium tuberculosis, Mycobacterium bovis (Mycobacterium bovis), Mycobacterium leprae (Mycobacterium
Leprae), mycobacterium kansasii (Mycobacterium kansasii), Mycobacterium intracellulare (Mycobacterium
Intracellulare), mycobacterium abscessus (Mycobacterium abscessus) or mycobacterium avium
(Mycobacterium avium)。
Term " antibody " used herein, which refers to, has specifically Ag85B the or CFP-10 antigen from Mycobacterium
The antibody of property, the antigen knot in conjunction with Ag85B or CFP-10 antigentic specificity, and including complete antibody and antibody molecule
Close segment.
Complete antibody includes two full-length light chains and two total length heavy chains, wherein every light chain is connected by disulfide bond and heavy chain
It connects.Heavy chain constant region is γ, μ, α, δ or ε type, can be categorized further, as γ 1, γ 2, γ 3, γ 4, α 1 or α 2.Light chain is permanent
Determining area is κ or λ type.
Term " heavy chain " used herein includes total length heavy chain and its segment, and the total length heavy chain includes to contain to be enough to determine
Surely for the variable region (V of the amino acid sequence of the specificity of antigenH) and tool there are three constant domain (CH1、CH2And CH3)
Constant region.
In addition, term " light chain " used herein is intended to include full-length light chains and its segment, the full-length light chains include
Variable region (V containing the amino acid sequence for being enough to determine the specificity for antigenL) and constant region (CL)。
Term " antigen-binding fragment " used herein refers to the piece of the intact immunoglobulins comprising antigen binding domain
Section, and any polypeptide portion comprising antigen binding domain.
For example, antigen-binding fragment can be 2 segment of F (ab '), Fab ' segment, Fab segment, Fv segment or scFv segment,
But present disclosure is without being limited thereto.In antigen-binding fragment, Fab segment has the variable region of light chain and heavy chain, light chain
First constant region C of constant region and heavy chainH1, and there is an antigen binding site.Fab ' segment it is different from Fab's it
It is in Fab ' in addition with the hinge area of heavy chain, in heavy chain CH1The C-terminal of structural domain includes at least one cysteine residues.When
When the cysteine residues of the hinge area of Fab ' segment are connected by disulfide bond, F (ab ') 2 segment is generated.Fv segment is that only have
The minimum antibody fragment of heavy chain variable region and light chain variable region, and the recombinant technique for generating Fv segment is public in this field
Know.Double chain Fv fragments can have the structure that wherein heavy chain variable region is connect by non-covalent bond with light chain variable region, and single
Chain Fv segment can usually have dimeric structure as in double chain Fv fragments, and wherein heavy chain variable region passes through peptide linker and light chain
Variable region covalent bond or heavy chain variable region and light chain variable region are connected to each other directly in its C-terminal.Antigen-binding fragment can make
It is obtained with protease and (for example, by complete antibody papain digestion to obtain Fab segment, and uses pepsin digestion
To obtain 2 segment of F (ab ')), and can be prepared by gene recombination technology.
Term " specific binding " used herein or " specific recognition " are to those skilled in the art
It is well known, and indicates that antigen and antibody (or its antigen-binding fragment) are specifically immunoreacted each other.
Antigen-binding fragment in conjunction with from CFP-10 the or Ag85B antigentic specificity of mycobacteria may include in energy
The change of amino acid sequence described in appended sequence number in the range of enough specific recognition CFP-10 antigen or Ag85B antigen
Body.For example, in order to enhance the binding affinity of antibody and/or other biological characteristics, the amino acid sequence of antibody can be mutated.
For example, such mutation includes missing, insertion and/or the replacement of the amino acid sequence residue of antibody.Amino acid mutation is based on ammonia
The relative similarity of base acid side chain substituents and occur, such as hydrophobicity, hydrophily, charge or size.For example, arginine, rely
Propylhomoserin and histidine are individually positively charged residue, and alanine, glycine and serine have similar size, and phenylpropyl alcohol
Propylhomoserin, tryptophan and tyrosine have similar shape.Therefore, it is based on above-mentioned consideration, arginine, lysine and histidine can be with
It is functional biological equivalent, alanine, glycine and serine can be functional biological equivalent, and phenylalanine, color ammonia
Acid and tyrosine can be functional biological equivalent.
Meanwhile wherein the amino acid substitution in protein that molecular activity does not change completely is as known in the art.Most
Common amino acid substitution can be between Ala and Ser, between Val and IIe, between Asp and Glu, between Thr and Ser, Ala
Between Gly, between Ala and Thr, between Ser and Asn, between Ala and Val, between Ser and Gly, between Thy and Phe,
Between Ala and Pro, between Lys and Arg, between Asp and Asn, between Leu and Ile, between Leu and Val, Ala and Glu it
Between or the replacement between Asp and Gly.The mutation with biology equivalent activity is considered, with CFP-10 antigen or Ag85B antigen
The antigen-binding fragment of specific binding can also be interpreted to include the sequence substantially the same with the sequence disclosed in sequence number
Column.Substantially the same sequence can be following sequence: when the amino acid sequence and other any amino acid sequence ratios of sequence number
When to corresponding to each other as much as possible and analyze institute's aligned amino acid sequence using algorithms most in use as known in the art,
There is at least 60% homology, at least 70% homology, at least 80% homology or at least 90% with sequence described in sequence number
Homology.The comparison method compared for sequence is as known in the art.Pass through NCBI Local Alignment Search for example, can be used
Obtainable sequence analyzes journey to basic tool (Basic Local Alignment Search Tool, BLAST) on the internet
One of sequence, such as blastp, blastx, tblasm and tblastx.
Present disclosure another embodiment provides: coding with from mycobacteria CFP-10 antigen-specific
Property combine antibody heavy chain variable region polynucleotides, the heavy chain variable region include SEQ ID NO:5 or SEQ ID NO:7
Amino acid sequence；And encode the light chain variable region of the antibody in conjunction with from the CFP-10 antigentic specificity of mycobacteria
Polynucleotides, the light chain variable region include SEQ ID NO:6 or SEQ ID NO:8 amino acid sequence.
Present disclosure another embodiments further provide: coding and the Ag85B antigen from mycobacteria are special
The polynucleotides of the heavy chain variable region for the antibody that the opposite sex combines, the heavy chain variable region include SEQ ID NO:9, SEQ ID NO:
The amino acid sequence of 11 or SEQ ID NO:13；And coding is in conjunction with from the Ag85B antigentic specificity of mycobacteria
The polynucleotides of the light chain variable region of antibody, the light chain variable region include SEQ ID NO:10, SEQ ID NO:12 or SEQ
The amino acid sequence of ID NO:14.
Term " polynucleotides " used herein refers to as deoxyribonucleotide existing for single-stranded or double-stranded form
Or the polymer of ribonucleotide.Unless in addition particularly illustrating herein, otherwise polynucleotides include rna gene group sequence
Column, DNA (gDNA and cDNA) and the RNA sequence transcribed by it, and the analog including native polynucleotide.
Polynucleotides further include the ammonia of antigen-binding fragment of the coding in conjunction with CFP-10 antigen or Ag85B antigentic specificity
The nucleotide sequence and its complementary series of base acid sequence.Complementary series not only includes the sequence of complete complementary, but also including base
Complementary sequence in sheet.This means for example can be with antigen knot of the coding in conjunction with CFP-10 antigen or Ag85B antigentic specificity
Close the sequence of the nucleotide sequence hybridization of the amino acid sequence of segment.
In addition, the amino acid sequence of antigen-binding fragment of the coding in conjunction with CFP-10 antigen or Ag85B antigentic specificity
Nucleotide sequence can be mutated.Such mutation includes insertion, missing or the non-conservative or conservative replacement of nucleotide.It compiles
The polynucleotides of the amino acid sequence of antigen-binding fragment of the code in conjunction with CFP-10 antigen or Ag85B antigentic specificity are explained
To further include the nucleotide sequence substantially the same with the nucleotide sequence.Substantially the same sequence can be following sequence:
When nucleotide sequence and other any nucleotide sequence comparisons to correspond to each other and using commonly used in the art as much as possible
Algorithm when analyzing compared nucleotide sequence, with nucleotide sequence at least 80% homology, at least 90% homology
Or at least 95% homology.
According to an embodiment, the multicore glycosides of the heavy chain variable region of antibody of the coding in conjunction with CFP-10 antigentic specificity
Acid can have the nucleotide sequence of SEQ ID NO:15 or SEQ ID NO:16.
According to an embodiment, the multicore glycosides of the light chain variable region of antibody of the coding in conjunction with CFP-10 antigentic specificity
Acid can have the nucleotide sequence of SEQ ID NO:17 or SEQ ID NO:18.
According to an embodiment, the multicore glycosides of the heavy chain variable region of antibody of the coding in conjunction with Ag85B antigentic specificity
Acid can be any one of the polynucleotides of nucleotide sequence with SEQ ID NO:19 to 21.
According to an embodiment, the multicore glycosides of the light chain variable region of antibody of the coding in conjunction with Ag85B antigentic specificity
Acid can be any one of the polynucleotides of nucleotide sequence with SEQ ID NO:22 to 24.
Present disclosure another embodiment provides the recombinant vectors comprising above-mentioned polynucleotides.
Term " carrier " used herein refers to the tool for expressing target gene in host cell.For example, carrier
Can be plasmid vector, cosmid vector and viral vectors, for example, phage vector, adenovirus vector, retroviral vector and
Gland relevant viral vector, but present disclosure is not limited to examples detailed above.Recombinant vector can be by being commonly used in performance domain
The plasmid of recombinant vector is (for example, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/
9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, pUC19 etc.), bacteriophage is (for example, λ
Gt4 λ B, λ-Charon, λ Δ z1, M13 etc.) or viral (for example, CMV, SV40 etc.) prepare.
Recombinant vector may include the nucleotide sequence of polynucleotides and the promoter that effectively connect with the nucleotide sequence.
Term " effectively connecting " used herein mean nucleotide Expression modulation sequence (for example, promoter sequence) with
Functional connection between other nucleotide sequences, and therefore nucleotide expression withers and saves other adjustable described nucleotide of sequence
The transcription and/or translation of sequence.
It usually can be the carrier for being used to clone or express by construction of recombinant vector.Carrier for expression can be for
The universal support of foreign protein is expressed in plant, animal or microorganism.A variety of sides as known in the art can be used in recombinant vector
Method constructs.
In addition, protokaryon or eukaryotic can be used as host cell to construct recombinant vector.For example, when using protokaryon thin
When born of the same parents are as host cell, used expression vector generally comprises the strong promoter that can originate transcription (for example, pL starts
Son, trp promoter, lac promoter, tac promoter, T7 promoter etc.), for initiation of translation ribosome bind site, with
And transcription/translation termination sequence.When using eukaryocyte as host cell, used carrier is included in eukaryocyte
The replication orgin to play a role, such as f1 replication orgin, SV40 replication orgin, pMB1 replication orgin, adenovirus origin of replicationAAV replication orgin, CMV replication orgin, BBV replication orgin etc., but present disclosure is not
It is limited to examples detailed above.In addition, also can be used the promoter of the genome from mammalian cell (for example, metallothionein
Promoter) or from mammalian virus promoter (for example, adenovirus late promoter, vaccinia virus 7.5K promoter,
The tk promoter of SV40 promoter, cytomegalovirus (CMV) promoter and HSV), and transcription terminator usually can be it is more
Polyadenylation sequence.
At the same time, can be used for present disclosure recombinant vector can by plasmid common in performance domain (for example,
PSC101, ColE1, pBR322, pUC8/9, pHC79, pUC19, pET etc.), bacteriophage is (for example, λ gt4 λ B, λ-Charon, λ Δ
Z1, M13 etc.) or viral (for example, SV40 etc.) prepare.
The recombinant vector of present disclosure can be merged with other sequences to be conducive to purify the antigen binding fragment expressed from it
Section.The sequence of fusion include for example glutathione S-transferase (Pharmacia, USA), maltose-binding protein (NEB, USA),
FLAG (IBI, USA), 6xHis (six histidines；Qiagen, USA) etc., and most preferably 6x His.Due to for such pure
The appended sequence of change, the protein expressed in host can quickly and easily be purified by affinity chromatography.
At the same time, the expression vector of present disclosure may include antibiotics resistance gene commonly used in the art as choosing
Select label.For example, antibiotics resistance gene may include for ampicillin, gentamicin, Carbenicillin, chloramphenicol, streptomysin,
Kanamycins, Geneticin, neomycin, hygromycin and tetracycline resistant gene.
Present disclosure another embodiment provides the host cells comprising above-mentioned recombinant vector, that is, with recombination
The cell of carrier conversion.
In order to prepare the transformed cells of present disclosure, genomic dna sequence and its transcript can be used.Transcript can make
With method as known in the art prepare, and when using recombinant vector generate transcript when, first can by vector linearization with
Prepare transcript.
Can stablize and continuously clone or express recombinant vector host cell can be it is as known in the art any
Host cell.Prokaryotic cell can be such as Escherichia coli (E.coli) JM109, e. coli bl21, Escherichia coli RR1, big
Enterobacteria LE392, Escherichia coli B, Escherichia coli X1776, Escherichia coli W3110；Bacillus (Bacillus genus)
Bacterium, such as bacillus subtilis (Bacillus subtilis) and bacillus thuringiensis (Bacillus
thuringiensis)；Enterobacteria, such as salmonella typhimurium (Salmonella typhimurium), Serratia marcescans
(Serratia marcescens) and a variety of pseudomonas (Pseudomonas) species；Deng.The eukaryotic host cell of conversion
Yeast be can be (for example, saccharomyces cerevisiae (Saccharomyces cerevisiae), insect cell, plant cell or animal are thin
Born of the same parents, for example, Sp2/0, CHO (Chinese hamster ovary) K1, CHO DG44, PER.C6, W138, BHK, COS-7,293, HepG2,
Huh7,3T3, RIN, mdck cell system etc..
Polynucleotides transfer method as known in the art can be used to be transferred to host cell comprising its recombinant vector
In.When using prokaryotic cell as host cell, CaCl can be used2Method, electroporation etc. are shifted, and true when using
When nucleus is as host cell, it can be banged by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, gene
It hits etc. and to be shifted, but present disclosure is not limited to examples detailed above.
When using microorganism (such as Escherichia coli) as host cell, the generation of antibody is higher than anti-in zooblast
Body generates.However, microorganism is unsuitable for generating complete Ig type antibody, but microorganism can be used for producing due to glycosylating problem
Raw antigen-binding fragment (such as Fab and Fv).
The host cell of conversion can use the phenotype expressed by selective key by any method as known in the art
To select.For example, culture turns in wrapping antibiotic culture medium when selective key is specific antibiotics resistance gene
Change body, and is therefore readily able to select transformant.
[advantageous effect of the invention]
When using antigen-binding fragment of the invention, it can be detected using high sensitivity as anti-from mycobacteria
Former CFP-10 or Ag85B, can develop the kit that can quickly and accurately detect mycobacteria.
[brief description]
By the description below in conjunction with attached drawing to embodiment, these and or other aspects will be apparent and be easier to
Understand, in the accompanying drawings:
Fig. 1 shows the Ag85B human antibodies specific-phage library qualification result recycled according to biopanning；
Fig. 2 shows the single bacteriophages of Ag85B specificity separated from human antibody-phage libraryWith
The definitive result combined between Ag85B antigen；
Fig. 3 shows the qualification result of the single bacteriophage of Ag85B specificity and the binding site of Ag85B；
Fig. 4 shows the CFP-10 human antibodies specific-phage library qualification result recycled according to biopanning；
Fig. 5 shows the definitive result of the bond strength between the single bacteriophage of CFP-10 specificity and CFP-10 antigen；
Fig. 6 is shown for the human IgG heavy chain expression vector of Ag85B or the figure of light chain expression vector；
Fig. 7 is to show the surface plasma body resonant vibration result figure of the antigen-binding activity of C12 and 8B3 antibody；
Fig. 8 is to show the surface plasma body resonant vibration result figure of the antigen-binding activity of 50B14 and 8B3 antibody；
Fig. 9 is shown for the human IgG heavy chain expression vector of CFP-10 or the figure of light chain expression vector；And
Figure 10 shows the surface plasma body resonant vibration result of the antigen-binding activity of the 2nd and 3 group of antibody of display.
[embodiment of the present invention]
Hereinafter, one or more embodiments will be described in further detail referring to following embodiment.However, providing
These embodiments are only for exemplary purposes, it is not intended that are limited the scope of the disclosure.
Embodiment 1: the generation and purifying of the recombinant protein of antigen of mycobacterium tuberculosis CFP-10 and Ag85B
In order to generate CFP-10 antibody and Ag85B (p85) antibody, recombinant antigen CFP-10 and Ag85B are produced.Every kind right
It answers gene to use the genomic DNA of mycobacterium tuberculosis (M.tuberculosis) as template and uses CFP-10-F primer
(SEQ ID NO:1), CFP-10-R primer (SEQ ID NO:2), Ag85B-F primer (SEQ ID NO:3) and Ag85B-R primer
(SEQ ID NO:4) expanded by polymerase chain reaction (PCR) (Shin etc., Clin.Vaccine Immunol., 15:
1788,2008) (referring to table 1).
The PCR product of amplification is digested with restriction enzyme BamHI and EcoRI, and is inserted into pET28a (Novagen, USA)
The site BamHI/EcoRI in.By recombinant plasmid transformed to Escherichia coli (Escherichia coli, E.coli) BL21
(DE3) it in, and then cultivates at 37 DEG C until OD600Value reaches 0.4 to 0.5.After culture, pass through the Escherichia coli obtained by
Middle addition IPTG induces overexpression to final concentration of 1mM, and culture solution is centrifuged to recycle the Escherichia coli of culture.It will return
The Escherichia coli of receipts are suspended in 20mM Tris-HCl (pH 8.0), 0.5M NaCl, 20mM imidazoles and 1mM phenylmethylsulfonyl fluoride
(PMSF) in.After this, it by ultrasonic disruption cell, and is purified by Ni-NTA agarose column (Invitrogen, USA)
Recombinant protein.
<table 1>
(indicating restriction enzyme site added with the part of underscore)
Embodiment 2: selection and purifying with the antigen-binding fragment of antigen of mycobacterium tuberculosis Ag85B specific binding
The antigen-binding fragment in conjunction with Ag85B antigentic specificity is identified using human antibody-phage library.
2-1: the selection with the antigen-binding fragment of Ag85B (p85) specific binding
By the immune pipe of Ag85B antigen (10 μ g/ml) coating generated with 2ml according to embodiment 1 and make coated antigen
It is reacted with human antibody-phage library to carry out the biopanning for selecting initially to combine bacteriophage.At this point, phagocytosis in library
The sum of body is about 2 × 1013CFU.Then, by washing 10 removal Non-specific phages, and then with remaining phagocytosis
Body ehec infection XL-1Blue is to expand bacteriophage.Primary sublibrary is recycled from the bacteriophage of amplification, and uses the son
Library carries out in total antigen-phagocytosis precursor reactant and selection course three times.The number of each panning process pnagus medius such as the following table 2 institute
Show.
<table 2>
In order to determine whether the protein shown on bacteriophage identifies Ag85B antigen, make with the plate of Ag85B antigen coat
With selected phagocytosis precursor reactant, and make combine bacteriophage and secondary antibody (anti-M13-HRP) combine.Then, addition is adjacent thereto
Phenylenediamine dihydrochloride/H2O2Substrate (OPD/H2O2Substrate), and with post analysis 490nm wavelength signal.At this point, for phase
Compare the sum with the number of the bacteriophage of Ag85B specific binding and bacteriophage over the ground, is all expressed using all bacteriophages
Recombinant C-myc label carries out compared with myc-ELISA analyzes result.
It is bitten from as a result, it was confirmed that the number of the bacteriophage obtained in each elutriation from library is similar to each other, and at these
In thallus, as the progress of panning rounds and the number of the bacteriophage of Ag85B specific binding increase (referring to Fig. 1).
2-2: the purifying with the antigen-binding fragment of Ag85B specific binding
Since the bond strength of phage library and Ag85B do not significantly improve after level Four elutriation, from four
The phage library separation monoclonal phage-antibodies obtained in grade elutriation.Particularly, about 40 are collected from level Four phage library
A colony (XL-1blue), and individually express bacteriophage and harvested in culture solution.Then, in the phagocytosis individually generated
Single Phage-ELISA analysis is carried out to Ag85B and c-myc in body.
As a result, it was confirmed that 60% or more bacteriophage shows in total and the combination of Ag85B is active (referring to fig. 2), and
As make obtain single bacteriophage with two the subunits p32 and p33 of Ag85B ining conjunction with determine gained bacteriophage and Ag85B where
It is that a site combines as a result, determine that list bacteriophage is selectively combined with p32, without in conjunction with p33 (referring to Fig. 3).
Then, in order to analyze the feature of single bacteriophage, the phasmid of corresponding monoclonal is extracted, and pass through BstI restriction enzyme
Processing carries out DNA fingerprint analysis to it.As a result, obtaining the scFv antibody (8B3,50B14 and C12) of three types, and analyze
Every kind of antibody amino acid sequence (heavy chain variable region of 8B3: the light chain variable region of SEQ ID NO:9,8B3: SEQ ID NO:
10,50B14 heavy chain variable region: the light chain variable region of SEQ ID NO:11,50B14: the heavy chain of SEQ ID NO:12, C12 can
Become area: the light chain variable region of SEQ ID NO:13 and C12: SEQ ID NO:14).
Embodiment 3: selection and purifying with the antigen-binding fragment of antigen of mycobacterium tuberculosis CFP-10 specific binding
3-1: the selection with the antigen-binding fragment of CFP-10 specific binding
Bacteriophage is selected in mode in the same manner as in Example 2, the difference is that the process of elutriation is in triplicate, and really
It is fixed to show whether the protein on bacteriophage identifies CFP-10 antigen (10kDa culture filtrate antigen).
As a result, it was confirmed that c-myc is similar in each elutriation, and with the progress of panning rounds, it is special with CFP-10
Property the number of bacteriophage that combines increase (referring to fig. 4).
3-2: the purifying with the antigen-binding fragment of CFP-10 specific binding
Since the bond strength of phage library and CFP-10 significantly improve in three-level elutriation, with embodiment 2
In the phage library that is obtained from three-level elutriation of identical mode separate single bacteriophage.
96 clones are selected from single bacteriophage of separation, and as passing through clone and CFP- selected by elisa assay
10 bond strength as a result, identifying the antibody with high proportion in conjunction with CFP-10 (referring to Fig. 5).In this 96 clones,
Analyze the sequences of 62 with strong signal clones, and the sequence as the antigen recognition site that is compared to each other as a result, mirror
Make the antibody of 6 seed types (referring to table 3).
<table 3>
| The sum of clone: 62 | Number | |
| 1st group | IGHV1-69*01/IGKV2D-40*01 | 32 |
| 2nd group | IGHV3-9*01/IGLV1-47*02 | 20 |
| 3rd group | IGHV3-33*01/IGLV1-47*01 | 5 |
| 4th group | IGHV1-69*06 | 1 |
| 5th group | IGHV3-30*18 | 1 |
| 6th group | IGHV3-72*01/IGKV1-17*02 | 2 |
| No signal | 1 |
In order to determine each group monoclonal antibody bond strength, by the concentration of CFP-10 antigen from 1 μ g/ml according to 1/5
It dilutes in a step-wise fashion, and is coated with plate with it, then carry out elisa assay.
As a result, it was confirmed that even if in the case that antigen is diluted 1/3125 times (about 350pg/ml) wherein, quantitative detection
It is possible, and the 4th and 6 group shows strongest bond strength.In this six groups, two kinds of scFv antibody is obtained
(the 2nd group and the 3rd group), and analyze every kind of antibody amino acid sequence (the 2nd group of heavy chain variable region: SEQ ID NO:5, the 2nd
The light chain variable region of group: SEQ ID NO:6, the 3rd group of heavy chain variable region: SEQ ID NO:7 and the 3rd group of light chain variable
Area: SEQ ID NO:8).
Embodiment 4: the generation with the IgG antibody of Ag85B specific binding
Full IgG antibodies are generated using the sequence of the scFV antibody obtained according to embodiment 2.
4-1: for the human IgG heavy chain of Ag85B or the building of light chain expression vector
In the sequence for every kind of scFV antibody of Ag85B, corresponding primer amplification heavy chain variable region and light chain are used
Variable region, and the heavy chain variable region and light chain variable region expanded with restriction enzyme SfiI/NheI or SfiI/BgIII digestion.With
Afterwards, segment is inserted into and is connected in pNATABH and pNATABL, be the human IgG heavy chain expression with identical restriction enzyme digestion
Carrier or light chain expression vector (referring to Fig. 6).Carrier is transformed into XL-1blue and is cultivated, and bacterium colony then is carried out to it
PCR is to determine whether antibody sequence is correctly inserted into carrier (referring to Fig. 6).
The expression and purifying of 4-2:Ag85B monoclonal antibody
By HEK293 cell and heavy chain expression vector and light chain expression vector cotransfection, and cell culture is recycled every three days
Solution is to obtain the antibody expressed and secreted by HEK293 cell.By 10 times of the cell culture solution concentration of about 500ml recycling, and
Then use albumin A pearl (agarose: Pharmacia) antibody purification.
It is obtained using the measurement of surface plasma body resonant vibration (surface plasmon resonance, SPR) method
The antigen-binding affinity of Ag85B monoclonal antibody.Make to be combined with the golden C12 in conjunction with polypeptide (GBP) respectively with 25 μ g/ml, 50 μ
The concentration of g/ml, 100 μ g/ml and 200 μ g/ml are reacted on the coated chip of gold, and are then gone using the BSA of 500 μ g/ml
Except nonspecific reaction.Then, the Ag85B antigen for preparing 10 μ g/ml according to embodiment 1 is in connection, and reuses 100 μ
The BSA of g/ml removes nonspecific reaction, then reacts with the 8B3 that concentration is 50 μ g/ml.
As a result, as shown in Figure 7, determining C12 and 8B3 in conjunction with Ag85B antigen high degree of specificity (referring to Fig. 7).In addition,
Make the 50B14 for being combined with GBP respectively with the concentration of 25 μ g/ml, 50 μ g/ml, 100 μ g/ml and 200 μ g/ml coated with gold
Reacted on chip, remove nonspecific reaction using the BSA of 500 μ g/ml, make the Ag85B antigen of the embodiment 1 of 10 μ g/ml with
It is combined, and then reuses the BSA removal nonspecific reaction of 100 μ g/ml, then anti-for the 8B3 of 50 μ g/ml with concentration
It answers.As a result, as shown in figure 8, combining (referring to Fig. 8) with determining 50B14 and 8B3 and Ag85B antigen high degree of specificity.
Embodiment 5: the generation with the IgG antibody of CFP-10 specific binding
5-1: for the human IgG heavy chain expression vector of CFP-10 or the building of light chain expression vector
The sequence of CFP-10 antibody is inserted into carrier and is transformed into a manner of identical with above-described embodiment 4-1
(referring to Fig. 9) in XL-1blue.
The expression and purifying of 5-2:CFP-10 monoclonal antibody
By HEK293 cell and the light chain expression vector and heavy chain expression vector cotransfection for the 2nd group or the 3rd group, and
Recycling cell culture solution is every three days to obtain the antibody expressed and secreted from the HEK293 cell of transfection.About 500ml is recycled
Cell culture solution be concentrated into about 50ml, and then using albumin A pearl (agarose: Pharmacia) binding antibody and using
Glycine purifying.At purified CFP-10 antibody (about 10ml) is stored in -20 DEG C after dialysis, and pass through dodecyl
Sodium sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining identify obtained antibody.
The antigen binding for measuring obtained CFP-10 monoclonal antibody by surface plasma body resonant vibration (SPR) method is affine
Power.Make to be combined with the 3rd group of GBP respectively with the concentration of 100 μ g/ml, 50 μ g/ml, 10 μ g/ml and 1 μ g/ml coated with gold
It is reacted on chip, removes nonspecific reaction using the BSA of 100 μ g/ml, make the CFP-10 antigen of the embodiment 1 of 100 μ g/ml
It is in connection, and then reuse 100 μ g/ml BSA removal nonspecific reaction, then and concentration be 100 μ g/ml combination
There is the 2nd group of reaction of silica binding protein (silica binding protein, SBP).As a result, as shown in Figure 10,
Determine that the 2nd group and the 3rd group with CFP-10 antigen high degree of specificity combines (referring to Figure 10).
The specificity analysis of embodiment 6:Ag85B antibody and CFP-10 antibody
In order to measure the specificity of the antibody generated according to embodiment 4 and 5, other branches are present in by ELISA detection
Antigen in bacillus culture solution.
As a result, it was confirmed that only specifically detecting that CFP-10 antigen and Ag85B are anti-in mycobacterium tuberculosis culture solution
Former (referring to table 4).
<table 4>
Although it have been described that the exemplary implementation scheme of present disclosure, but present disclosure fields is common
The skilled person will understand that the present invention can by it is a variety of it is modified in the form of embody be made without departing from the scope of the present invention or its
Substantive characteristics.Therefore, embodiment described herein should be to be considered merely as illustrative rather than for limitation mesh
's.Scope of the present disclosure being defined by the appended claims, without being limited by the description of front, and of equal value with it
All differences in range should be interpreted scope of the present disclosure interior.
<110>Univ Chung Ang Ind
<120>with the antibody or antigen-binding fragment of the antigen binding from mycobacteria
<130> PX160035PCT
<160> 24
<170> KopatentIn 2.0
<210> 1
<211> 31
<212> DNA
<213>artificial sequence
<220>
<223>CFP-10-F primer
<400> 1
ggccggggat ccatggcaga gatgaagacc g 31
<210> 2
<211> 31
<212> DNA
<213>artificial sequence
<220>
<223>CFP-10-R primer
<400> 2
ggccgggaat tcgaagccca tttgcgagga c 31
<210> 3
<211> 31
<212> DNA
<213>artificial sequence
<220>
<223>Ag85-F primer
<400> 3
ggccggggat ccagcgaaga gccggacgat g 31
<210> 4
<211> 31
<212> DNA
<213>artificial sequence
<220>
<223>Ag85-R primer
<400> 4
ggccgggaat tcgagcatga gactcgatca g 31
<210> 5
<211> 112
<212> PRT
<213>artificial sequence
<220>
<223>the 2nd groups of heavy chain
<400> 5
Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Arg Ser Leu Arg
1 5 10 15
Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Asp Asp Phe Ala Met His
20 25 30
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Gly Ile
35 40 45
Thr Trp Asn Ser Gly Thr Ile Ala Tyr Ala Asp Ser Val Lys Gly Arg
50 55 60
Phe Thr Leu Ser Arg Asp Asn Ala Lys Asn Ser Leu Ser Leu Glu Met
65 70 75 80
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly
85 90 95
His Tyr Gly Leu Asp Val Trp Gly His Gly Thr Lys Val Thr Val Ser
100 105 110
<210> 6
<211> 114
<212> PRT
<213>artificial sequence
<220>
<223>the 2nd groups of variable light
<400> 6
Asp Val His Ser Gln Phe Val Leu Thr Gln Pro Pro Ser Val Ser Gly
1 5 10 15
Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Tyr Ser Asn
20 25 30
Ile Gly Thr Asn Tyr Val Tyr Trp Tyr His Gln Leu Pro Gly Thr Ala
35 40 45
Pro Lys Leu Val Ile Gln Lys Asn Thr Gln Arg Pro Ser Gly Val Ser
50 55 60
Asp Arg Phe Ser Gly Ser Arg Ser Gly Thr Ser Ala Ser Leu Ala Ile
65 70 75 80
Ser Gly Leu Arg Ser Glu Asp Glu Gly Asn Tyr Phe Cys Ser Ala Trp
85 90 95
Asp Asp Ser Leu Ser Ala Val Leu Phe Gly Gly Gly Thr Lys Leu Thr
100 105 110
Val Leu
<210> 7
<211> 125
<212> PRT
<213>artificial sequence
<220>
<223>the 3rd groups of variable heavy chain
<400> 7
Asp Val His Ser Gln Val Gln Leu Val Lys Ser Gly Gly Gly Leu Val
1 5 10 15
Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
20 25 30
Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Glu Gly
35 40 45
Leu Glu Trp Val Ser Ala Ile Thr Gly Gly Gly Gly Ser Thr Tyr Tyr
50 55 60
Ala Asp Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
65 70 75 80
Asn Thr Leu Tyr Val Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
85 90 95
Val Tyr Tyr Cys Ala Lys Pro Lys Tyr Ser Ser Gly Trp Tyr Asp Ala
100 105 110
Phe Asp Ile Trp Asp Gln Gly Thr Met Val Thr Val Ser
115 120 125
<210> 8
<211> 100
<212> PRT
<213>artificial sequence
<220>
<223>the 3rd groups of variable light
<400> 8
Val Ser Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser
1 5 10 15
Tyr Ser Asn Ile Gly Thr Asn Tyr Val Tyr Trp Tyr His Gln Leu Pro
20 25 30
Gly Thr Ala Pro Lys Leu Val Ile Gln Lys Asn Thr Gln Arg Pro Ser
35 40 45
Gly Val Ser Asp Arg Phe Ser Gly Ser Arg Ser Gly Thr Ser Ala Ser
50 55 60
Leu Ala Ile Ser Gly Leu Arg Ser Glu Asp Glu Gly Asn Tyr Phe Cys
65 70 75 80
Ser Ala Trp Asp Asp Ser Leu Ser Ala Val Leu Phe Gly Gly Gly Thr
85 90 95
Lys Val Thr Val
100
<210> 9
<211> 134
<212> PRT
<213>artificial sequence
<220>
<223>variable heavy chain of 8B3
<400> 9
Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Gly Ser
20 25 30
Ala Met His Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr Ala Tyr Ala Ala
50 55 60
Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr
65 70 75 80
Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ala Tyr Tyr Asp Phe Trp Ser Gly Tyr Tyr Arg Leu
100 105 110
Val Lys Val Ala Arg Pro Pro Thr Tyr Phe Asp Tyr Trp Gly Gln Gly
115 120 125
Thr Leu Val Thr Val Ser
130
<210> 10
<211> 107
<212> PRT
<213>artificial sequence
<220>
<223>variable light of 8B3
<400> 10
Arg Leu Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ala Lys Thr Asn
20 25 30
Val Ala Trp Tyr Gln Gln Lys Phe Gly Gln Ala Pro Arg Val Leu Ile
35 40 45
Tyr Gly Ala Ser Thr Arg Ala Thr Asp Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Ser Arg Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro Trp
85 90 95
Thr Phe Gly Gln Gly Thr Gln Val Glu Ile Lys
100 105
<210> 11
<211> 123
<212> PRT
<213>artificial sequence
<220>
<223>variable heavy chain of 50B14
<400> 11
Gln Val Gln Leu Val Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn
20 25 30
Ser Ala Thr Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu
35 40 45
Trp Leu Gly Arg Thr Tyr Phe Arg Ser Lys Trp Tyr Asp Asp Tyr Ala
50 55 60
Val Ser Val Lys Ser Arg Leu Thr Ile Asn Pro Asp Thr Ser Lys Asn
65 70 75 80
Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95
Tyr Tyr Cys Ala Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala
100 105 110
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120
<210> 12
<211> 111
<212> PRT
<213>artificial sequence
<220>
<223>variable light of 50B14
<400> 12
Arg Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asn Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Ser Leu Thr Ile Thr Asn Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr His Cys Gln Gln Tyr Asn Ser Phe Pro Pro
85 90 95
Lys Thr Phe Gly Gln Gly Thr Gln Val Glu Ile Lys Arg Leu Glu
100 105 110
<210> 13
<211> 121
<212> PRT
<213>artificial sequence
<220>
<223>variable heavy chain of C12
<400> 13
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Arg Thr Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu
50 55 60
Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ala Phe Gly Glu Ala Ala Gly Arg Tyr Gly Met Asp Val Trp
100 105 110
Gly Gln Gly Thr Leu Val Thr Val Ser
115 120
<210> 14
<211> 110
<212> PRT
<213>artificial sequence
<220>
<223>variable light of C12
<400> 14
Asp Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Thr Gly
1 5 10 15
Asp Arg Val Asn Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Gly Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile
35 40 45
Tyr Lys Val Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asp Ser Val Pro Val
85 90 95
Thr Phe Gly Gln Gly Thr Arg Leu Asp Ile Lys Arg Leu Glu
100 105 110
<210> 15
<211> 336
<212> DNA
<213>artificial sequence
<220>
<223>polynucleotides of the 2nd group of variable heavy chain are encoded
<400> 15
ctggtggagt ccgggggaga cttggtacag cctggcaggt ccctgagact ctcctgtgta 60
gcctctggat tcacctttga tgattttgcc atgcactggg tccggcaagc tccggggaag 120
ggcctggagt gggtctcagg tattacttgg aatagtggta ccatcgccta tgcggactct 180
gtgaagggcc gcttcaccct ctccagagac aacgccaaga attccctgtc tctggaaatg 240
aacagtctga gagccgagga cacggccgtg tattactgtg cgagaggcca ctacggtttg 300
gacgtctggg gccatgggac caaggtcacc gtctcc 336
<210> 16
<211> 375
<212> DNA
<213>artificial sequence
<220>
<223>polynucleotides of the 3rd group of variable heavy chain are encoded
<400> 16
gatgtccact cgcaggtgca gctggtaaag tctgggggag gcttggtaca gcctgggggg 60
tccctgagac tctcctgtgc agcctctgga ttcaccttta gcagctatgc catgagctgg 120
gtccgccagg ctccagggga ggggctggag tgggtctctg ctattactgg tggtggcggt 180
agcacatact acgcagactc cgtgaggggc cggttcacca tctcccgaga caattccaag 240
aacacgctgt atgtgcaaat gaacagtctg agagccgagg acacggccgt gtattactgt 300
gcgaaaccga agtatagcag tggctggtat gatgcttttg atatctggga ccaagggaca 360
atggtcaccg tctcc 375
<210> 17
<211> 342
<212> DNA
<213>artificial sequence
<220>
<223>polynucleotides of the 2nd group of variable light are encoded
<400> 17
gatgtccact cgcagttcgt gctgactcag ccgccctcag tctctgggac ccccgggcag 60
agggtcacca tctcttgttc tggaagttac tccaacatcg gaactaatta tgtctactgg 120
taccaccaac tcccgggaac ggcccccaaa cttgtcatcc aaaagaatac tcagcggccc 180
tcaggtgtca gtgaccgatt ctcgggctct aggtctggca cctcagcctc cctggccatc 240
agtgggctcc ggtccgagga tgagggtaat tatttttgtt ccgcatggga tgacagcctg 300
agtgccgtac ttttcggcgg agggaccaag ctgaccgtcc ta 342
<210> 18
<211> 300
<212> DNA
<213>artificial sequence
<220>
<223>polynucleotides of the 3rd group of variable light are encoded
<400> 18
gtctctggga cccccgggca gagggtcacc atctcttgtt ctggaagtta ctccaacatc 60
ggaactaatt atgtctactg gtaccaccaa ctcccgggaa cggcacccaa acttgtcatc 120
caaaagaata ctcagcggcc ctcaggtgtc agtgaccgat tctcgggctc taggtctggc 180
acctcagcct ccctggccat cagtgggctc cggtccgagg atgagggtaa ttatttttgt 240
tccgcatggg atgacagcct gagtgccgta cttttcggcg gagggaccaa ggtcaccgtc 300
300
<210> 19
<211> 402
<212> DNA
<213>artificial sequence
<220>
<223>polynucleotides of the variable heavy chain of 8B3 are encoded
<400> 19
caggtgcagc tggtgcagtc tgggggaggc ttggtccagc ctggggggtc cctgaaactc 60
tcctgtgcag cctctgggtt caccctcagt ggctctgcta tgcactgggt ccgccaggct 120
tccgggaaag ggctggagtg ggttggccgt attagaagca aatctaacaa ttacgcgaca 180
gcatatgctg cgccggtgaa aggcaggttc accatctcca gagatgattc aaagaacacg 240
gcgtatcttc aaatgaacag cctgaaaacc gaggacacgg ccgtctatta ctgtgcaaga 300
gcctattacg atttttggag tggttattat cggttagtga aagtggctcg acctccgaca 360
tactttgact actggggcca aggaaccctg gtcaccgtct cc 402
<210> 20
<211> 369
<212> DNA
<213>artificial sequence
<220>
<223>polynucleotides of the variable heavy chain of 50B14 are encoded
<400> 20
caggtgcagc tggtgcagtc tggtccagga ctggtgaagc cctcacagac cctctcactc 60
acctgtgcca tctccgggga cagtgtctct agcaacagtg ctacttggaa ctggatcagg 120
cagtccccat cgagaggcct tgagtggctg ggaaggacat acttcaggtc caagtggtat 180
gatgattatg cagtatctgt gaaaagtcga ctaaccatca acccagacac atccaagaac 240
cagttctccc tgcagctgaa ctctgtgact cccgaggaca cggccgtcta ttactgtgct 300
ccgtttggtt actacgtgtc tgactatgct atggcctact ggggccaagg gaccctggtc 360
accgtctcc 369
<210> 21
<211> 363
<212> DNA
<213>artificial sequence
<220>
<223>polynucleotides of the variable heavy chain of C12 are encoded
<400> 21
caggtgcagc tggtgcagtc tggagctgag gtgaggacgc ctggggcctc agtgaaggtc 60
tcctgcaagg cttctggtta cacctttacc agctatggta tcagctgggt gcgacaggcc 120
cctggacaag ggcttgagtg gatgggatgg atcagcgctt acaatggtaa cacaaactat 180
gcacagaagc tccagggcag agtcaccatg accacagaca catccacgag cacagcctac 240
atggagctga ggagcctgag atctgacgac acggccgtct attactgtgc aagagcattc 300
ggggaagcag ctggtaggta cggtatggac gtctggggcc aaggaaccct ggtcaccgtc 360
tcc 363
<210> 22
<211> 321
<212> DNA
<213>artificial sequence
<220>
<223>polynucleotides of the variable light of 8B3 are encoded
<400> 22
cgactcgtgt tgacgcagtc tccaggcaca ctgtcagtgt ctccagggga aagagccacc 60
ctctcctgca gggccagtca gagtgctaaa accaacgtag cctggtacca acagaaattt 120
ggccaggctc ccagggtcct catctatggt gcatccacca gggccactga tatcccagcc 180
aggttcagtg gcagtgggtc tgggacagat ttcagtctca ccatcagcag actggagcct 240
gaagattttg cagtgtatta ctgtcagcag tatggtagct caccgtggac gttcggccaa 300
gggacacagg tggagatcaa a 321
<210> 23
<211> 333
<212> DNA
<213>artificial sequence
<220>
<223>polynucleotides of the variable light of 50B14 are encoded
<400> 23
cgaattgtga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaaa cagagtcacc 60
atcacttgcc gggcgagtca gggcattagc aattatttag cctggtatca gcagaaacca 120
gggaaagttc ctaagctcct gatctatgct gcatccactt tgcaatcagg ggtcccatct 180
cggttcagcg gcagtggatc tgggacagag ttcagtctca ccatcaccaa tctgcaacct 240
gaagattttg caacttacca ctgtcaacag tataacagtt tccctccgaa gacgttcggc 300
caagggacac aggtggaaat taaacgtctc gag 333
<210> 24
<211> 330
<212> DNA
<213>artificial sequence
<220>
<223>polynucleotides of the variable light of C12 are encoded
<400> 24
gatattgtga tgacccagtc tccttccacc ctgtctgcat ctactggaga cagggtcaac 60
atcacttgcc gggccagtca gagtattagt ggctggttgg cctggtatca gcagaagcca 120
gggaaagccc ctaacctcct aatctataag gtgtctagtt tacaaagtgg ggtcccatca 180
aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240
gaagattttg caacttacta ctgtcaacag agtgacagtg tcccggtcac cttcggccaa 300
gggaccagac tggatattaa acgtctcgag 330
Claims (15)
1. antibody or its antigen-binding fragment in conjunction with from the CFP-10 antigentic specificity of mycobacteria, the antibody or
Its antigen-binding fragment includes:
The heavy chain variable region of amino acid sequence containing SEQ ID NO:5；And
The light chain variable region of amino acid sequence containing SEQ ID NO:6.
2. antibody or its antigen-binding fragment in conjunction with from the CFP-10 antigentic specificity of mycobacteria, the antibody or
Its antigen-binding fragment includes:
The heavy chain variable region of amino acid sequence containing SEQ ID NO:7；And
The light chain variable region of amino acid sequence containing SEQ ID NO:8.
3. antibody described in claim 1 or claim 2 or its antigen-binding fragment, wherein the mycobacteria is selected from: knot
Core mycobacteria (Mycobacterium tuberculosis), Mycobacterium bovis (Mycobacterium bovis), leprosy point
Branch bacillus (Mycobacterium leprae), mycobacterium kansasii (Mycobacterium kansasii), branch intracellular
Bacillus (Mycobacterium intracellulare), mycobacterium abscessus (Mycobacterium abscessus) and bird
Mycobacteria (Mycobacterium avium).
4. the polynucleotides of the heavy chain variable region of the antibody in conjunction with from the CFP-10 antigentic specificity of mycobacteria are encoded,
Wherein the heavy chain variable region includes the amino acid sequence of one of SEQ ID NO:5 and 7.
5. the polynucleotides of the light chain variable region of the antibody in conjunction with from the CFP-10 antigentic specificity of mycobacteria are encoded,
Wherein the heavy chain variable region includes the amino acid sequence of one of SEQ ID NO:6 and 8.
6. recombinant vector, it includes polynucleotides described in claim 4 or claim 5.
7. host cell, it includes recombinant vectors as claimed in claim 6.
8. antibody or its antigen-binding fragment in conjunction with from the Ag85B antigentic specificity of mycobacteria, the antibody or
Its antigen-binding fragment includes:
The heavy chain variable region of amino acid sequence containing SEQ ID NO:9；And
The light chain variable region of amino acid sequence containing SEQ ID NO:10.
9. antibody or its antigen-binding fragment in conjunction with from the Ag85B antigentic specificity of mycobacteria, the antibody or
Its antigen-binding fragment includes:
The heavy chain variable region of amino acid sequence containing SEQ ID NO:11；And
The light chain variable region of amino acid sequence containing SEQ ID NO:12.
10. antibody or its antigen-binding fragment in conjunction with from the Ag85B antigentic specificity of mycobacteria, the antibody or
Its antigen-binding fragment includes:
The heavy chain variable region of amino acid sequence containing SEQ ID NO:13；And
The light chain variable region of amino acid sequence containing SEQ ID NO:14.
11. antibody described in any one of claim 8 to 10 or its antigen-binding fragment, wherein the mycobacteria is selected from:
Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium leprae, mycobacterium kansasii, Mycobacterium intracellulare, mycobacterium abscessus
And mycobacterium avium.
12. polynucleotides encode the weight chain variable of the antibody in conjunction with from the Ag85B antigentic specificity of mycobacteria
Area, wherein the heavy chain variable region includes the amino acid sequence of one of SEQ ID NO:9,11 and 13.
13. polynucleotides encode the light chain variable of the antibody in conjunction with from the Ag85B antigentic specificity of mycobacteria
Area, wherein the light chain variable region includes the amino acid sequence of one of SEQ ID NO:10,12 and 14.
14. recombinant vector, it includes polynucleotides described in claim 12 or claim 13.
15. host cell, it includes the recombinant vectors described in claim 14.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
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| KR20150191461 | 2015-12-31 | ||
| KR10-2015-0191461 | 2015-12-31 | ||
| KR10-2016-0055153 | 2016-05-04 | ||
| KR1020160055153A KR101631054B1 (en) | 2015-12-31 | 2016-05-04 | Antibody or Antigen Binding Fragment Binding to CFP-10 or Ag85B from Mycobacteria |
| PCT/KR2016/015587 WO2017116212A1 (en) | 2015-12-31 | 2016-12-30 | Antibody specifically binding to antigen derived from mycobacteria or antigen-binding fragment thereof |
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| CN109071638A true CN109071638A (en) | 2018-12-21 |
| CN109071638B CN109071638B (en) | 2022-01-25 |
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| KR101631054B1 (en) * | 2015-12-31 | 2016-06-16 | 중앙대학교 산학협력단 | Antibody or Antigen Binding Fragment Binding to CFP-10 or Ag85B from Mycobacteria |
| WO2019117585A2 (en) * | 2017-12-11 | 2019-06-20 | 주식회사 딕스젠 | Composition for diagnosing tuberculosis and method for diagnosing tuberculosis on basis of change in optical characteristics |
| CN110172097B (en) * | 2019-05-22 | 2020-11-13 | 东方海洋(北京)医学研究院有限公司 | Monoclonal antibody of mycobacterium tuberculosis CFP-10 protein and application thereof |
| CA3164027A1 (en) * | 2019-12-09 | 2021-06-17 | Versiti Blood Research Institute Foundation Inc. | Bcg car constructs and methods of their manufacture and use |
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- 2016-05-04 KR KR1020160055153A patent/KR101631054B1/en active IP Right Grant
- 2016-12-30 BR BR112018013381-7A patent/BR112018013381A2/en active Search and Examination
- 2016-12-30 WO PCT/KR2016/015587 patent/WO2017116212A1/en active Application Filing
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| BR112018013381A2 (en) | 2018-12-18 |
| WO2017116212A1 (en) | 2017-07-06 |
| CN109071638B (en) | 2022-01-25 |
| KR101631054B1 (en) | 2016-06-16 |
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