CN106084051B - A kind of ZnT8 specific single-chain antibodies scFv C27 and its application - Google Patents
A kind of ZnT8 specific single-chain antibodies scFv C27 and its application Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- 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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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- Genetics & Genomics (AREA)
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Abstract
The invention discloses a kind of anti-type 1 diabetes ZnT8 specific single-chain antibody scFv C27 and its application.The amino acid sequence of described anti-type 1 diabetes ZnT8 specific single-chain antibodies 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 libraries, screens and identifies ZnT8 specificity scFv, and 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, is related to a kind of anti-type 1 diabetes ZnT8 specific single-chain antibodies scFv-
C27 and its application.
Background technology
Zinc transporter (ZnT) is one group and zinc ion is gone out into extracellular transmembrane protein by intracellular transport, is total to ZIP family proteins
With the balance for safeguarding intraor extracellular zinc ion.2004, Cheimienti etc. had found ZnT8 albumen in ZnT families first, it
By SLC30A8 gene codes, the expression of high special is rear further study show that expression special ZnT8 is in pancreas islet in pancreas
On β cells, ZnT8 is overexpressed in INS-1 cells can promote the aggregation of zinc ion and strengthen the post-stimulatory insulin releasing of sugar.
These researchs show that ZnT8 plays a significant role in the synthesis and secretion of insulin.
Type 1 diabetes (T1D) are by T cell mediateds, and the organ characterized by selective destruction beta Cell of islet is special
Property autoimmune disease.There is a variety of autoantigens, such as insulin, glutamate decarboxylase (GAD), junket on beta Cell of islet
Propylhomoserin phosphatase and ZnT8.Wherein, the important autoantigen, Wenzlau etc. that ZnT8 is considered as T1DM finds part
T1D high-risk patients premorbid can detect ZnT8 autoantibodies in 2 years and maintain higher level for a long time.We send out early-stage Study
There is multiple epitopes identify by type 1 diabetes patient autoreactive T cell by existing ZnT8, and ZnT8 antibody levels and
There is significant correlation for T1D development.Early detection ZnT8 antibody and ZnT8 specific C D8+T cells are prediction and diagnosis
T1D important means.The potential method being in progress for the specific treatment of ZnT8 target spots as prevention T1D.
Antibody research has more than 100 years history so far, and Kohler in 1975 and Milstein found B lymphocyte hybridoma
Technology, mouse resource monoclonal antibody are widely used in the diagnosis and treatment of disease, but because mouse resource monoclonal antibody has to human body
Immunogenicity, be also easy to produce HAMA reaction (human anti-mouse antibody, HAMA and hypersensitivity, and due to
Its molecular weight is larger, it is difficult to the shortcomings of wearing film, limits its application in disease treatment field.From mid-1980s in 20th century
Phase, with the technical progress of molecular biology, foreign scholar establishes phage antibody library technique.Due to phage expression
ScFv antibody molecule amounts are smaller, only 1/6th of complete antibody molecule, and are penetrated with preferable blood vessel or tissue barrier
Property, half-life short, Stability Analysis of Structures, without Fc sections, reduce with internal Fc acceptors with reference to and the advantage such as adverse effect for bringing,
The medical domain such as molecular biology and immunology, pharmacology is widely used in, is current study hotspot.But have not yet to see
The relevant report of anti-type 1 diabetes ZnT8 specific single-chain antibodies.
The content of the invention
The purpose of the present invention is the above-mentioned deficiency for prior art, there is provided a kind of anti-type 1 diabetes ZnT8 specificity is single
Chain antibody.
It is a further object of the present invention to provide the application of the 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 amino acid sequence of the variable region of described light chain is as shown in SEQ ID NO.1；
The amino acid sequence of the variable region of described heavy chain is as shown in SEQ ID NO.2.
The amino acid sequence of described anti-type 1 diabetes ZnT8 specific single-chain antibodies is preferably as shown in SEQ ID NO.5.
A kind of gene for encoding anti-type 1 diabetes ZnT8 specific single-chain antibodies scFv-C27 of the present invention, wherein,
The gene order of the variable region of light chain is encoded 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 anti-type 1 diabetes ZnT8 specific single-chain antibodies scFv-C27 of the present invention gene
Row are preferably as shown in SEQ ID NO.6.
Anti- type 1 diabetes ZnT8 specific single-chain antibodies scFv-C27 of the present invention prepare type 1 diabetes diagnosis,
Application in treatment and/or Index for diagnosis reagent.
Anti- type 1 diabetes ZnT8 specific single-chain antibodies scFv- described in fgs encoder claim 1 of the present invention
Application of the C27 gene in type 1 diabetes diagnosis, treatment and/or Index for diagnosis reagent is prepared.
Beneficial effect：
The present invention constructs a storage capacity up to 1 × 10 from patient's T1D peripheral blood gene first8ScFv phage antibodies
Storehouse.We have recruited 50 initial T1D patients to ensure the specificity in storehouse, diversity and representativeness.From the storehouse, Wo Menke
To screen T1D various autoantibodies.
ZnT8 antigens are T1D important autoantigens, have six transmembrane structures, and it is by SCL30A8 gene codes, positioning
In on No. 8 chromosome q24.11 of people, comprising 8 extrons, 369 amino acid are encoded.Generation of the ZnT8 autoantibodies in T1D
Important function has been played in development.At present, the amino terminal (1-74aa) of ZnT8 albumen and carboxyl terminal (268-369aa) are recognized
For be autoreactive T cell identification main region.Because ZnT8 albumen affects the synthesis and secretion of insulin, so right
The deep understanding of its antibody mechanism of action is significant to treatment T1D.The present invention has successfully screened ZnT8 specificity scFv.For
Ensure the diversity of the selection result, we have selected the amino carboxyl fusogenic peptide progress scFv of ZnT8 albumen screening.Have
A large amount of reports find that there is point mutation, junket ammonia is sported by arginine (Arg) for the amino acids of ZnT8 antigens 325 in T1D patient
Sour (Trp), the identification of the site mutation and ZnT8 antibody is closely related, the mutation dimer of ZnT8 carboxyl terminals
(Arg325Trp) there is the specificity and sensitiveness of height to detection ZnT8 antibody.So we further use ZnT8 carboxyls
The dimer (Arg325Trp) of end identifies scFv-C27, the results showed that scFv-C27 can be combined with the dimer.Immune group
Change result and further show scFv energy specific recognition islet cells, there is good biological activity.
The present invention successfully constructs T1D scFv phage libraries, screens and identifies ZnT8 specificity scFv, and the ZnT8 is special
Different in nature scFv can be used for type 1 diabetes diagnosis, treatment and/or the preparation of Index for diagnosis reagent.
Brief description of the drawings
The gene constructed .M of Fig. 1 restructuring ZnT8:Nucleic acid standards；Swimming lane 1:ZnT8 amino terminals gene (1-74aa) is about
250bp；Swimming lane 2:ZnT8 carboxyl terminals gene (268-369aa) about 350bp；Swimming lane 3:ZnT8 amino carboxyl fusions (1-
74aa, 268-369aa) about 550bp；Swimming lane 4:ZnT8 c-terminus dimerization mutant gene (Arg325Trp) about 650bp.
The expression and purification and identification of Fig. 2 restructuring ZnT8 albumen
A:The Western blotting detection (swimming lanes 1 of ZnT8 amino carboxyl fusion proteins:Before protein purification；Swimming lane 2:
Albumen after purification；Swimming lane 3:Flow through)；B:SDS-PAGE detections (the M of ZnT8 amino carboxyl fusion proteins: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 proteins
Western blotting detect (swimming lane 1:Before protein purification；Swimming lane 2:Albumen after purification；Swimming lane 3:Flow through)；D:ZnT8 carboxylics
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 proteins and c-terminus dimerization mutant protein molecular weight about 20kD and 25kD respectively.
Fig. 3 .ELISA detect the binding ability of amino carboxyl fusion protein and commercialization ZnT8 antibody.
Fig. 4 .T1D scFv gene magnifications .M:Nucleic acid standards；Swimming lane 1:V λ PCR primers (~350bp)；Swimming lane 2:Vκ
PCR primer (~350bp)；Swimming lane 3:VH PCR primers (~450bp)；Swimming lane 4:ScFv PCR primers (~800bp).
Fig. 5 .scFv-C27 purifying and identification
A.SDS-PAGE detection 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 is flowed through)；B.Western blotting analyze detect scFv (swimming lanes 1:scFv-
C27 is before purification；Swimming lane 2:ScFv-C27 after purification；Swimming lane 3:ScFv-C27 is flowed through).
Fig. 6 .Western blotting detect scFv and ZnT8 amino carboxyl fusion proteins binding ability
Swimming lane 1:ScFv-C27 detects ZnT8 amino carboxyl fusion proteins；Swimming lane 2:Negative control.
Fig. 7 .scFv-C27 and ZnT8 amino carboxyl fusion proteins affinity detect.
Fig. 8 .ELISA detect binding abilities of the scFv-C27 to ZnT8 c-terminus dimerization mutant proteins.
Fig. 9 SABCs detect the recognition capability (A of scFv antibody on human pancreatic tissues:Islet cells HE coloration results；
B:C27 detects islet cells result.Row 1:40 times of amplification；Row 2:200 times of amplification)
Embodiment
Embodiment 1 recombinates the construction and expression of ZnT8 prokaryotic expression plasmids
Design primer ZF and LR (table 1), with ZnT8 plasmids (be purchased from Sino Biological Inc.,
HG11621-M it is) template, expands people ZnT8 amino terminals (1-74aa) gene, design primer LF and CR (table 1), with ZnT8 matter
Grain is template, expands people ZnT8 carboxyl terminals (269-368aa) gene, passed through after PCR primer glue reclaim with primer ZF and CR
Overlap builds amino carboxyl fusion.
ZnT8 carboxyl terminal dimerization mutant bases are designed with reference to the SEQ ID No.55 in US9023984 (B2) sequence table
Cause, as shown in SEQ ID No.7, and biotech firm is entrusted to synthesize, the dimer contains 2 people's ZnT8 carboxyl terminal genes, and first
Individual 325 is that amino acid is arginine (Arg), and second is tyrosine (Trp) at 325,2 people's ZnT8 carboxylics in the dimer
Base terminal gene passes through one section of flexible peptide linkage.Design primer CF and CR are used to expand ZnT8 c-terminus dimerization mutant genes.
The primer sequence of table 1.
Primer ZF and LR are used to expand ZnT8 amino terminals gene (1-74aa), and primer LF and CR are used to expand ZnT8 carboxyls
Terminal gene (268-369aa), primer ZF and CR are used for overlap amplification ZnT8 amino carboxyl fusions, primer CF and CR
For expanding ZnT8 c-terminus dimerization mutant genes.
Above-mentioned two recombination is inserted in the plasmids of pcold II after SacI and XbaI double digestions respectively.Connection product turns
Select positive colony after dissolving into e. coli bl21 (DE3) culture and be sequenced.Sequencing is correctly cloned in LB culture mediums
37 DEG C are expanded to D (600) values up to adding IPTG derivants after 1.0 to final concentration of 1mM/L, 15 DEG C of induced expression 24h.4 DEG C, 10
- 80 DEG C of preservations of 000 × g collected after centrifugation thalline.
People's ZnT8 amino carboxyls fusion protein of purifying is coated with elisa plate, from 1.6ug/ holes doubling dilution to 0.1ug/
The dose-effect change of the ZnT8 antibody bindings of hole, detection fusion albumen and commercialization.
ZnT8 amino carboxyl fusion about 550bp, carboxyl dimerization mutant gene about 650bp, PCR primer electrophoresis result
See Fig. 1, be successively inserted into the carriers of pcold II, 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 detections show that ZnT8 amino carboxyls fusion protein can be combined with the ZnT8 antibody specificities of commercialization, can use
In ZnT8 specificity scFv screening (Fig. 3).
The structure of the T1D scFv phage antibody libraries of embodiment 2
Experimental specimen is the peripheral blood of 50 initial T1D volunteers.Wherein 31 volunteers sera's ZnT8 antibody positives.
Serum Zn T8 antibody levels detected by radio-immunity part method (method detailed referring to 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 type1diabetes.Diabetes/metabolism
research and reviews 2011；27:899-905).All patients sign informed consent form.Sort T1D volunteer
Peripheral blood PBMC.T1D volunteer peripheral blood PBMC RNA are extracted, then reverse transcription synthesis cDNA, concrete operation step is illustratively
Book is carried out.By RT-PCR method, heavy chain and light chain are expanded with human single chain variable fragments antibody (scFv) universal primer (table 2), is passed through
Overlap PCR are linked into scFv, and pcomb3XSS plasmids are inserted after Sfi I digestions and (are purchased from BioVector NTCC preservations
The heart) in.Connection product is transformed into Escherichia coli XL1-Blue (being purchased from Stratagene, Cat.#200228) by multiple electricity, uses
SB culture mediums add helper phage VCSM13 superinfection after expanding culture.Next day collects supernatant after PEG-8000/NaCl is precipitated
Obtain T1D scFv phage antibody libraries.
The people source scFv universal primer sequences of table 2.
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
It is as follows to expand the combination of VH strand primers:
Expand VκPrimer combination is as follows:
Expand 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 exist
750bp or so (Fig. 4), insert in pcomb3XSS carriers after glue reclaim, electricity is transformed into Escherichia coli XL1-Blue, through VCSM13
Phage antibody library is obtained after superinfection, storage capacity is 1 × 10 after testing8。
1.2.4 ZnT8 specificity scFv screening
By the ZnT8 amino carboxyls fusion protein of purifying by 1 μ g/ holes coating elisa plate, by " absorption " of 4 wheels, " wash
It is de- ", " amplification " (Zhang X, Qi X, Zhang Q, Zeng X, Shi Z, Jin Q, et al.Human 4f5 single- 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 is detected.Select the high positive colony extraction plasmid of OD values and send survey
Sequence, sequencing result and human immunoglobulin sequence (IMGT databases) (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
Compare and carry out the division of CDR region.
By the screening of 4 wheels, ZnT8 specificity scFv is enriched with.Each round phage titre is shown in Table 3.Last is taken turns
After the phage-infect Escherichia coli XL1-Blue of elution, 160 clones are randomly choosed.Phage ELISA results show 23
Individual positive colony, wherein there are 11 clone's OD values higher, 7 plants of scFv are obtained through sequencing.We have carried out CDR to this 7 plants of scFv
The division in area, meet people source scFv antibody characteristics.Wherein, scFv-C27 and C22OD values highest, therefore we select scFv-
C27 makes further research.
The enrichment of table 3.ZnT8 specific bacteriophage antibody
The phagocytosis volume density (pfu) before phagocytosis volume density (pfu) × 100/ absorption after yield (%)=elution
Correct positive colony will be sequenced and be transformed into Escherichia coli Top10F ', choose monoclonal colony inoculation in containing 20mM
In MgCl2 SB culture mediums, IPTG to final concentration 1mM, 37 DEG C of induced expression 24h are added after 37 DEG C of shaking 8h.4 DEG C, 10 000*
- 80 DEG C of preservations of g collected after centrifugation thalline.
Bacterial precipitation is resuspended with the phosphate buffer of the urea containing 8M, is placed on ice, is split with sonicated cells instrument
Solution.4 DEG C of centrifugation 30min of 10000*g, supernatant use HisTrap affinity column purifying proteins after 0.22 μm of membrane filtration.Wash
The destination protein taken off is less than 0.125mM by concentration gradient dialysis renaturation to urea, and high concentration albumen is obtained after ultrafiltration concentration.
Western and SDS-PAGE results show our successful expressions and purified scFv-C27, its molecular weight about 30kD (Fig. 5)
The ZnT8 specificity scFv of embodiment 3 specificity analysis
Western blot are detected：By the restructuring ZnT8 amino carboxyl fusion proteins of purifying after 12% protein adhesive electrophoresis
Go on pvdf membrane, after the closing of 5% milk, by the use of the scFv of purifying as primary antibody, the HA antibody of HRP marks is as secondary antibody, detection
ScFv-C27 and ZnT8 binding activity.As a result show that scFv-C27 can detect amino carboxyl fusion protein, in 20kD or so
It can be seen that obvious band, is consistent with expection, and negative control has no band, illustrates that scFv-C27 and ZnT8 albumen have good knot
Conjunction ability (Fig. 6).
Affinity detects:Bioisystech Co., Ltd is opened up by Suzhou hundred to complete.Affinity result show scFv-C27 with
ZnT8 amino carboxyls fusion protein has higher affinity, and affinity is 5.397 × 10-8KD (M) (Fig. 7).
ELISA is detected：The ZnT8 carboxyl terminal dimerization mutant protein of purifying is coated with elisa plate by 0.5 μ g/ holes,
ScFv-C27 is from 1:10 doubling dilutions are to 1:The dose-effect change that 20480, detection scFv-C27 are combined with ZnT8.As shown in Figure 8, with
The doubling dilution of scFv-C27 concentration, the binding ability of itself and ZnT8 c-terminus dimerization mutant proteins gradually reduces, explanation
ScFv-C27 is combined with concentrationdependent manner with ZnT8 Antigenic Peptides.
SABC：We carry out immunohistochemical experiment by the method for indirect labelling, resisted using C27 antibody as primary antibody
The islet cells of Human Pancreas section after body incubation is in strong positive, illustrates that C27 antibody has good bioactivity, can
The ZnT8 albumen of special identification people beta Cell of islet expression.
Result above shows that scFv-C27 can be specifically bound with people ZnT8.
Claims (2)
1.ZnT8 specific single-chain antibodies scFv-C27, it is characterised in that：
The amino acid sequence of the variable region of light chain is as shown in SEQ ID NO.1；
The amino acid sequence of the variable region of heavy chain is as shown in SEQ ID NO.2;
Described ZnT8 specific single-chain antibodies scFv-C27 amino acid sequence is as shown in SEQ ID NO.5.
A kind of 2. gene for encoding the ZnT8 specific single-chain antibodies scFv-C27 described in claim 1, it is characterised in that：
The nucleotide sequence of the variable region of light chain is encoded 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;
Its nucleotide sequence is as shown in SEQ ID NO.6.
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