CN106084051A - A kind of anti-type 1 diabetes ZnT8 specific single-chain antibody scFv C27 and application thereof - Google Patents
A kind of anti-type 1 diabetes ZnT8 specific single-chain antibody scFv C27 and application thereof Download PDFInfo
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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Abstract
The invention discloses a kind of anti-type 1 diabetes ZnT8 specific single-chain antibody scFv C27 and application thereof.The aminoacid sequence of described anti-type 1 diabetes ZnT8 specific single-chain antibody is as shown in SEQ ID NO.5, and its coding gene sequence is as shown in SEQ ID NO.6.The present invention successfully constructs T1D scFv phage library, screens and identify ZnT8 specificity scFv, and this ZnT8 specificity scFv can be used for type 1 diabetes diagnosis, treatment and/or the preparation of Index for diagnosis reagent.
Description
Technical field
The invention belongs to biomedicine technical field, relate to a kind of anti-type 1 diabetes ZnT8 specific single-chain antibody scFv-
C27 and application thereof.
Background technology
Zinc transporter (ZnT) is one group of transmembrane protein being gone out outside born of the same parents by intracellular transport by zinc ion, with ZIP family protein altogether
With the balance safeguarding intraor extracellular zinc ion.2004, Cheimienti etc. found ZnT8 albumen first in ZnT family, it
By SLC30A8 gene code, the expression of high special in pancreas, after further study show that expression special for ZnT8 is at islets of langerhans
On β cell, in INS-1 cell, process LAN ZnT8 can promote the gathering of zinc ion and strengthen sugared post-stimulatory insulin releasing.
These researchs show that ZnT8 plays a significant role in the synthesis of insulin and secretion.
Type 1 diabetes (T1D) is by T cell mediated, special with the organ that selective destruction beta Cell of islet is characterized
Property autoimmune disease.Multiple autoantigen is there is, such as insulin, glutamate decarboxylase (GAD), cheese on beta Cell of islet
Propylhomoserin phosphatase and ZnT8.Wherein, ZnT8 is considered as an important autoantigen of T1DM, and Wenzlau etc. finds part
T1D high-risk patient premorbid i.e. can detect that ZnT8 autoantibody and long term maintenance are in higher level for 2 years.We send out early-stage Study
Existing ZnT8 also exist multiple epitope can by type 1 diabetes patient's autoreactive T cell identification, and ZnT8 antibody horizontal with
The development of T1D also exists significant correlation.Detection ZnT8 antibody and ZnT8 specific C D8+T cell are prediction and diagnosis in early days
The important means of T1D.Specific treatment for ZnT8 target spot will become the potential method stoping T1D progress.
Antibody research has more than 100 year history so far, and within 1975, Kohler and Milstein foundes B lymphocyte hybridoma
Technology, Mus resource monoclonal antibody is widely used in diagnosis and the treatment of disease, but owing to human body is had by Mus resource monoclonal antibody
Immunogenicity, be easily generated human antimouse antibody reaction (human anti-mouse antibody, HAMA and allergy, and due to
Its molecular weight is bigger, it is difficult to wear the shortcomings such as film, limits its application in disease treatment field.From mid-1980s in 20th century
Phase, along with the technical progress of molecular biology, foreign scholar establishes phage antibody library technique.Due to phage expression
ScFv antibody molecule amount is less, only 1/6th of complete antibody molecule, and has preferable blood vessel or tissue barrier penetrates
Property, half-life be short, Stability Analysis of Structures, without Fc section, decrease the advantages such as the adverse effect that is combined and bring with internal Fc receptor,
It is widely used in molecular biology and the medical domain such as immunology, pharmacology, is current study hotspot.But have not yet to see
The relevant report of anti-type 1 diabetes ZnT8 specific single-chain antibody.
Summary of the invention
It is an object of the invention to the above-mentioned deficiency for prior art, it is provided that a kind of anti-type 1 diabetes ZnT8 specificity list
Chain antibody.
It is a further object of the present invention to provide the application of this single-chain antibody.
The purpose of the present invention can be achieved through the following technical solutions:
Anti-type 1 diabetes ZnT8 specific single-chain antibody scFv-C27, including heavy chain and light chain,
The aminoacid sequence of the variable region of described light chain is as shown in SEQ ID NO.1;
The aminoacid sequence of the variable region of described heavy chain is as shown in SEQ ID NO.2.
The aminoacid sequence of described anti-type 1 diabetes ZnT8 specific single-chain antibody is preferably as shown in SEQ ID NO.5.
A kind of gene encoding anti-type 1 diabetes ZnT8 specific single-chain antibody scFv-C27 of the present invention, wherein,
The gene order of the variable region of coding light chain is as shown in SEQ ID NO.3;
The gene order of the variable region of encoding heavy chain is as shown in SEQ ID NO.4.
Encode the nucleotides sequence of the gene of anti-type 1 diabetes ZnT8 specific single-chain antibody scFv-C27 of the present invention
Row are preferably as shown in SEQ ID NO.6.
Anti-type 1 diabetes ZnT8 specific single-chain antibody scFv-C27 of the present invention preparation type 1 diabetes diagnosis,
Application in treatment and/or Index for diagnosis reagent.
Anti-type 1 diabetes ZnT8 specific single-chain antibody scFv-described in fgs encoder claim 1 of the present invention
The application in preparation type 1 diabetes diagnosis, treatment and/or Index for diagnosis reagent of the gene of C27.
Beneficial effect:
The present invention constructs a storage capacity first from patient's T1D peripheral blood gene and reaches 1 × 108ScFv phage antibody
Storehouse.We have recruited the T1D patient of 50 example onsets to ensure the specificity in storehouse, multiformity and representativeness.From this storehouse, Wo Menke
To screen the various autoantibodys of T1D.
ZnT8 antigen is the important autoantigen of T1D, has six transmembrane structure, and it is by SCL30A8 gene code, location
On No. 8 chromosome q24.11 of people, comprise 8 exons, encode 369 aminoacid.ZnT8 autoantibody is in the generation of T1D
Development has played important function.At present, amino terminal (1-74aa) and the carboxyl terminal (268-369aa) of ZnT8 albumen is recognized
For being the main region of autoreactive T cell identification.Owing to ZnT8 albumen affects synthesis and the secretion of insulin, thus right
Treatment T1D is significant by the deep understanding of its antibody mechanism of action.The present invention has successfully screened ZnT8 specificity scFv.For
Ensureing the multiformity of the selection result, we have selected the amino carboxyl fusogenic peptide of ZnT8 albumen and carry out the screening of scFv.Have
A large amount of reports find that in T1D patient, ZnT8 antigen 325 amino acids also exists point mutation, is sported cheese ammonia by arginine (Arg)
Acid (Trp), this site mutation is closely related with the identification of ZnT8 antibody, the sudden change dimer of ZnT8 carboxyl terminal
(Arg325Trp) detection ZnT8 antibody is had specificity and the sensitivity of height.So we use ZnT8 carboxyl further
The dimer (Arg325Trp) of end identifies scFv-C27, and result shows that scFv-C27 can be combined with this dimer.Immune group
Change result and show scFv energy specific recognition islet cells further, there is good biologic activity.
The present invention successfully constructs T1D scFv phage library, screens and identify ZnT8 specificity scFv, and this ZnT8 is special
Opposite sex scFv can be used for type 1 diabetes diagnosis, treatment and/or the preparation of Index for diagnosis reagent.
Accompanying drawing explanation
Fig. 1. the restructuring gene constructed .M of ZnT8: nucleic acid standards;Swimming lane 1:ZnT8 amino terminal gene (1-74aa) is about
250bp;Swimming lane 2:ZnT8 carboxyl terminal gene (268-369aa) about 350bp;Swimming lane 3:ZnT8 amino carboxyl fusion gene (1-
74aa, 268-369aa) about 550bp;Swimming lane 4:ZnT8 c-terminus dimerization mutant gene (Arg325Trp) about 650bp.Fig. 2.
The expression and purification of restructuring ZnT8 albumen and qualification.
The Western blotting of A:ZnT8 amino carboxyl fusion protein detects (swimming lane 1: before protein purification;Swimming lane 2:
Albumen after purification;Swimming lane 3: flow through);The SDS-PAGE of B:ZnT8 amino carboxyl fusion protein detects (M: protein standard substance;Swimming
Road 1: before protein purification;Swimming lane 2: albumen after purification;Swimming lane 3: flow through);C:ZnT8 c-terminus dimerization mutant protein
Western blotting detects (swimming lane 1: before protein purification;Swimming lane 2: albumen after purification;Swimming lane 3: flow through);D:ZnT8 carboxylic
Cardinal extremity dimerization mutant protein SDS-PAGE detects (M: protein standard substance;Swimming lane 1: before protein purification;Swimming lane 2: egg after purification
In vain;Swimming lane 3: flow through).
ZnT8 amino carboxyl fusion protein and c-terminus dimerization mutant protein molecular weight the most about 20kD and 25kD.
Fig. 3 .ELISA detection amino carboxyl fusion protein and the binding ability of commercialization ZnT8 antibody.
Fig. 4 .T1D scFv gene amplification .M: nucleic acid standards;Swimming lane 1:V λ PCR primer (~350bp);Swimming lane 2:V κ
PCR primer (~350bp);Swimming lane 3:VH PCR primer (~450bp);Swimming lane 4:scFv PCR primer (~800bp).
The purification of Fig. 5 .scFv-C27 and qualification.
A.SDS-PAGE detects scFv (M: protein standard substance;Swimming lane 1:scFv-C27 is before purification;Swimming lane 2: after purification
scFv-C27;Swimming lane 3:scFv-C27 flows through);B.Western blotting analyze detects scFv (swimming lane 1:scFv-
C27 is before purification;Swimming lane 2: scFv-C27 after purification;Swimming lane 3:scFv-C27 flows through).
The binding ability of Fig. 6 .Western blotting detection scFv Yu ZnT8 amino carboxyl fusion protein.
Swimming lane 1:scFv-C27 detects ZnT8 amino carboxyl fusion protein;Swimming lane 2: negative control.
Fig. 7 .scFv-C27 detects with the affinity of ZnT8 amino carboxyl fusion protein.
Fig. 8 .ELISA detects the scFv-C27 binding ability to ZnT8 c-terminus dimerization mutant protein.
Fig. 9. identification ability (A: the islet cells HE coloration result of SABC detection scFv antibody on human pancreatic tissue;
B:C27 detects islet cells result.Row 1: amplify 40 times;Row 2: amplify 200 times)
Detailed description of the invention
Embodiment 1 is recombinated the construction and expression of ZnT8 prokaryotic expression plasmid
Design primer ZF and LR (table 1), with ZnT8 plasmid (purchased from Sino Biological Inc.,
HG11621-M) being template, expand people's ZnT8 amino terminal (1-74aa) gene, design primer LF and CR (table 1), with ZnT8 matter
Grain is template, expands people's ZnT8 carboxyl terminal (269-368aa) gene, PCR primer glue passes through with primer ZF and CR after reclaiming
Overlap builds amino carboxyl fusion gene.
ZnT8 carboxyl terminal dimerization mutant base is designed with reference to the SEQ ID No.55 in US9023984 (B2) sequence table
Cause, as shown in SEQ ID No.7, and entrusts biotech firm to synthesize, this dimer contain 2 people's ZnT8 carboxyl terminal genes, first
Individual is arginine (Arg) 325 for aminoacid, and second is tyrosine (Trp) at 325,2 people's ZnT8 carboxylics in this dimer
Base terminal gene passes through one section of flexible peptide linkage.Design primer CF and CR is used for expanding ZnT8 c-terminus dimerization mutant gene.
Table 1. primer sequence
Primer ZF and LR is used for expanding ZnT8 amino terminal gene (1-74aa), and primer LF and CR is used for expanding ZnT8 carboxyl
Terminal gene (268-369aa), primer ZF and CR expands ZnT8 amino carboxyl fusion gene, primer CF and CR for overlap
For expanding ZnT8 c-terminus dimerization mutant gene.
Above-mentioned two recombination inserts in pcold II plasmid respectively after SacI and XbaI double digestion.Connect product to turn
Select positive colony after dissolving e. coli bl21 (DE3) cultivation to check order.By correct being cloned in LB culture medium of checking order
37 DEG C are expanded to after D (600) value reaches 1.0 add IPTG derivant extremely final concentration of 1mM/L, 15 DEG C of abduction delivering 24h.4 DEG C, 10
-80 DEG C of preservations of 000 × g collected after centrifugation thalline.
People's ZnT8 amino carboxyl fusion protein of purification is coated elisa plate, from 1.6ug/ hole doubling dilution to 0.1ug/
Hole, detection fusion albumen changes with the dose-effect of the ZnT8 antibodies of commercialization.
ZnT8 amino carboxyl fusion gene about 550bp, carboxyl dimerization mutant gene about 650bp, PCR primer electrophoresis result
Seeing Fig. 1, be all successively inserted into pcold II carrier, sequencing result is consistent with theory.
Antigen is shown in Fig. 2 through expression and purification renaturation rear electrophoresis result, it is seen that ZnT8 amino carboxyl fusogenic peptide molecular weight after purification
About 20kD, carboxyl dimerization mutant molecule amount about 25kD.
ELISA detection display ZnT8 amino carboxyl fusion protein can be combined with the ZnT8 antibody specificity of commercialization, available
Screening (Fig. 3) in ZnT8 specificity scFv.
The structure of embodiment 2T1D scFv phage antibody library
Experimental specimen is the peripheral blood of the T1D volunteer of 50 onsets.Wherein 31 volunteers sera's ZnT8 antibody positives.
Serum Zn T8 antibody horizontal by radioimmunity part method carry out detecting (method detailed sees Gu Y, Zhang M, Chen H,
Wang Z,Xing C,Yang H,et al.Discordant association of islet autoantibodies
with high-risk hla genes in chinese type 1diabetes.Diabetes/metabolism
research and reviews 2011;27:899-905).All patients all sign Informed Consent Form.Sorting T1D volunteer
Peripheral blood PBMC.Extracting T1D volunteer peripheral blood PBMC RNA, then reverse transcription synthesis cDNA, concrete operation step is illustratively
Book is carried out.By RT-PCR method, with human single chain variable fragments antibody (scFv) universal primer (table 2) amplification heavy chain and light chain, pass through
Overlap PCR is linked into scFv, inserts pcomb3XSS plasmid (purchased from BioVector NTCC preservation after Sfi I enzyme action
The heart) in.Connect product and be transformed into escherichia coli XL1-Blue (purchased from Stratagene, Cat.#200228) through repeatedly electricity, use
Helper phage VCSM13 superinfection is added after SB culture medium amplification culture.Collect supernatant next day after PEG-8000/NaCl precipitates
Obtain T1D scFv phage antibody library.
Table 2. people source scFv universal primer sequence
Note: R=A or G Y=C or C M=A or C K=G or T S=C or G W=A or T H=A
Or C or T B=C or G or T V=A or C or G D=A or G or T N=A or C or G or T
Amplification VH strand primer combination is as follows:
Amplification VκPrimer combination is as follows:
Amplification VλPrimer combination is as follows:
Above-mentioned Successful amplification heavy chain of antibody and light chain after RT-PCR, size about 400bp, single-chain antibody gene band exists
About 750bp (Fig. 4), glue inserts in pcomb3XSS carrier after reclaiming, and electricity is transformed into escherichia coli XL1-Blue, through VCSM13
Obtaining phage antibody library after superinfection, storage capacity is 1 × 10 after testing8。
1.2.4ZnT8 the screening of specificity scFv
The ZnT8 amino carboxyl fusion protein of purification is coated elisa plate by 1 μ g/ hole, through 4 take turns " absorption ", " wash
De-", " amplification " (Zhang X, Qi X, Zhang Q, Zeng X, Shi Z, Jin Q, et al.Human 4f5single-afterwards
chain fv antibody recognizing a conserved ha1epitope has broad neutralizing
potency against h5n1influenza a viruses of different clades.Antiviral
research 2013;99:91-9), phage-ELISA detects.Select the high positive colony of OD value extract plasmid and send survey
Sequence, sequencing result and human immunoglobulin sequence (IMGT data base) (Ehrenmann F, Kaas Q, Lefranc
MP.Imgt/3dstructure-db and imgt/domaingapalign:A database and a tool for
immunoglobulins or antibodies,T cell receptors,mhc,igsf and mhcsf.Nucleic
acids research 2010;38:D301-7.Lefranc MP,Giudicelli V,Duroux P,Jabado-
Michaloud J,Folch G,Aouinti S,et al.Imgt(r),the international immunogenetics
information system(r)25years on.Nucleic acids research 2015;43:D413-22.) carry out
Comparison also carries out the division of CDR region.
Through 4 screenings taken turns, ZnT8 specificity scFv is enriched with.Each phage titre of taking turns is shown in Table 3.Last is taken turns
After the phage-infect escherichia coli XL1-Blue of eluting, randomly choose 160 clones.Phage ELISA result shows 23
Individual positive colony, wherein has 11 clone's OD values higher, obtains 7 strain scFv through order-checking.We have carried out CDR to this 7 strain scFv
The division in district, all meets people source scFv antibody characteristic.Wherein, scFv-C27 and C22OD value is the highest, and therefore we select scFv-
C27 makes further research.
The enrichment of table 3.ZnT8 specific bacteriophage antibody
The phage density (pfu) before phage density (pfu) × 100/ absorption after yield (%)=eluting
By checking order, correct positive colony is transformed into escherichia coli Top10F ', chooses monoclonal colony inoculation in containing 20mM
In the SB culture medium of MgCl2, after 37 DEG C of shaking 8h, add IPTG to final concentration 1mM, 37 DEG C of abduction delivering 24h.4 DEG C, 10 000*
-80 DEG C of preservations of g collected after centrifugation thalline.
Bacterial precipitation is resuspended with the phosphate buffer containing 8M carbamide, is placed on ice, splits with sonicated cells instrument
Solve.4 DEG C of centrifugal 30min of 10000*g, supernatant uses HisTrap affinity column purifying protein after 0.22 μm membrane filtration.Wash
The destination protein taken off less than 0.125mM, obtains high concentration albumen by Concentraton gradient dialysis renaturation to carbamide after ultrafiltration concentration.
Western Yu SDS-PAGE result shows our successful expression purification scFv-C27, its molecular weight about 30kD (Fig. 5)
The specificity analysis of embodiment 3ZnT8 specificity scFv
Western blot detects: by the restructuring ZnT8 amino carboxyl fusion protein of purification after 12% protein adhesive electrophoresis
Going on pvdf membrane, after 5% milk is closed, resist as one with the scFv of purification, the HA antibody of HRP labelling resists as two, detection
The combination activity of scFv-C27 Yu ZnT8.Result display scFv-C27 can detect that amino carboxyl fusion protein, at about 20kD
Visible obvious band, is consistent with expection, and negative control has no band, illustrates that scFv-C27 Yu ZnT8 albumen has good knot
Conjunction ability (Fig. 6).
Affinity detects: is opened up Bioisystech Co., Ltd by Suzhou hundred and completes.Affinity result display scFv-C27 with
ZnT8 amino carboxyl fusion protein has higher affinity, and affinity is 5.397 × 10-8KD (M) (Fig. 7).
ELISA detects: by 0.5 μ g/ hole, the ZnT8 carboxyl terminal dimerization mutant protein of purification is coated elisa plate,
ScFv-C27 is from 1:10 doubling dilution to 1:20480, the dose-effect change that detection scFv-C27 with ZnT8 is combined.As shown in Figure 8, with
The doubling dilution of scFv-C27 concentration, it is gradually lowered with the binding ability of ZnT8 c-terminus dimerization mutant protein, explanation
ScFv-C27 is combined with ZnT8 antigenic peptides with concentrationdependent manner.
SABC: C27 antibody is resisted by we as one, carries out immunohistochemical experiment by the method for indirect labelling, anti-
Body hatch after Human Pancreas section islet cells be strong positive, illustrate that C27 antibody has good biological activity, it is possible to
Special identifies the ZnT8 albumen that people's beta Cell of islet is expressed.
Result above all shows that scFv-C27 can be specific binding with people ZnT8.
Claims (6)
- The most anti-type 1 diabetes ZnT8 specific single-chain antibody scFv-C27, including light chain and heavy chain, it is characterised in that:The aminoacid sequence of the variable region of described light chain is as shown in SEQ ID NO.1;The aminoacid sequence of the variable region of described heavy chain is as shown in SEQ ID NO.2.
- Anti-type 1 diabetes ZnT8 specific single-chain antibody scFv-C27 the most according to claim 1, it is characterised in that described The aminoacid sequence of anti-type 1 diabetes ZnT8 specific single-chain antibody as shown in SEQ ID NO.5.
- 3. encoding a gene of anti-type 1 diabetes ZnT8 specific single-chain antibody scFv-C27 described in claim 1, it is special Levy and be:The nucleotide sequence of the variable region of coding light chain is as shown in SEQ ID NO.3;The nucleotide sequence of the variable region of encoding heavy chain is as shown in SEQ ID NO.4.
- Gene the most according to claim 3, it is characterised in that its nucleotide sequence is as shown in SEQ ID NO.6.
- 5. the anti-type 1 diabetes ZnT8 specific single-chain antibody scFv-C27 described in claim 1 or 2 is in preparation type 1 diabetes Application in diagnosis, treatment and/or Index for diagnosis reagent.
- 6. the anti-type 1 diabetes ZnT8 specific single-chain antibody scFv-described in the coding claim 1 described in claim 3 or 4 The application in preparation type 1 diabetes diagnosis, treatment and/or Index for diagnosis reagent of the gene of C27.
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CN106771233A (en) * | 2016-12-05 | 2017-05-31 | 上海良润生物医药科技有限公司 | ZnT8A autoantibody detection kits |
WO2020037174A1 (en) | 2018-08-16 | 2020-02-20 | The Johns Hopkins University | Antibodies to human znt8 |
CN114184793A (en) * | 2021-12-09 | 2022-03-15 | 深圳市亚辉龙生物科技股份有限公司 | Zinc transport protein 8 antibody chemiluminescence immunoassay kit and preparation method thereof |
US11841363B2 (en) | 2016-04-25 | 2023-12-12 | The Johns Hopkins University | ZnT8 assays for drug development and pharmaceutical compositions |
US11892457B2 (en) | 2017-07-12 | 2024-02-06 | The Johns Hopkins University | Proteoliposome-based ZnT8 self-antigen for type 1 diabetes diagnosis |
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Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
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US11841363B2 (en) | 2016-04-25 | 2023-12-12 | The Johns Hopkins University | ZnT8 assays for drug development and pharmaceutical compositions |
CN106771233A (en) * | 2016-12-05 | 2017-05-31 | 上海良润生物医药科技有限公司 | ZnT8A autoantibody detection kits |
US11892457B2 (en) | 2017-07-12 | 2024-02-06 | The Johns Hopkins University | Proteoliposome-based ZnT8 self-antigen for type 1 diabetes diagnosis |
WO2020037174A1 (en) | 2018-08-16 | 2020-02-20 | The Johns Hopkins University | Antibodies to human znt8 |
CN113166241A (en) * | 2018-08-16 | 2021-07-23 | 约翰霍普金斯大学 | Human ZNT8 antibodies |
CN114184793A (en) * | 2021-12-09 | 2022-03-15 | 深圳市亚辉龙生物科技股份有限公司 | Zinc transport protein 8 antibody chemiluminescence immunoassay kit and preparation method thereof |
CN114184793B (en) * | 2021-12-09 | 2023-08-11 | 深圳市亚辉龙生物科技股份有限公司 | Zinc transport protein 8 antibody chemiluminescence immunoassay kit and preparation method thereof |
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