CN110128539A - The monoclonal antibody and its preparation method and application of anti-dipeptidyl peptidase 4 - Google Patents
The monoclonal antibody and its preparation method and application of anti-dipeptidyl peptidase 4 Download PDFInfo
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- CN110128539A CN110128539A CN201810125462.4A CN201810125462A CN110128539A CN 110128539 A CN110128539 A CN 110128539A CN 201810125462 A CN201810125462 A CN 201810125462A CN 110128539 A CN110128539 A CN 110128539A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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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
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising 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
Abstract
The present invention relates to immunologys, pharmacy and medicine related fields, disclose method and purposes that the B cell epitope based on dipeptidyl peptidase 4 (DPP-4) prepares hybridoma cell strain and monoclonal antibody, in particular to the B epitope of DPP-4 is coupled with intramolecular carrier peptides to prepare small molecule antigens peptide and be quickly obtained by synthetic method, its immune mouse generates strong immune response, it can be used for preparing immune spleen lymphocyte, prepare hybridoma and antibody etc., monoclonal antibody of the present invention is by hybridoma cell strain (deposit number is CCTCC No:C2017227) secretion, for Th2 type antibody, it is IgG1 in mouse, it inhibits DPP-4 enzymatic activity, specific bond DPP-4, DPP-4 detection reagent and DPP-4 inhibitor can be developed, it is significant hypoglycemic With uric acid and creatinine four items of blood lipid tests, raising GLP-1, adjusting oxidative stress and immunologic balance from Th1 toward Th2, mitigate pancreas inflammatory, drug can be prepared for preventing and treating diabetes and its complication, high lithemia, gout, kidney hepatopathy, hyperlipidemia, AS, cardiovascular disease and the higher related disease of Th1.
Description
Technical field
The present invention relates to immunology, pharmacy and medicine related fieldss.The present invention relates to anti-dipeptidyl peptidases 4
The monoclonal antibody and its preparation method and application of (Dipeptidyl peptidase 4, DPP-4), in particular to employment dipeptides
The B cell epitope of base peptase 4 is related particularly to as haptens by being coupled people with the intramolecular carrier peptides of a small molecule
The B cell epitope peptide of dipeptidyl peptidase 4 (DPP-4) is to prepare multi-epitope peptide or Antigenic Peptide or peptide based immunogens and quick by synthesizing
It obtains, mouse, which is immunized, in peptide based immunogens can generate strong immune response, can be used for preparing immune spleen lymphocyte, this immune spleen leaching
Bar cell, which can be used for preparing hybridoma preparation monoclonal antibody or extract its gene by prior art, passes through antibody library skill
Art or genetic engineering prepare Multiple Antibodies.The monoclonal antibody specific bond and inhibition DPP-4 activity of preparation, can develop
DPP-4 detection reagent and DPP-4 inhibitor, the monoclonal antibody belongs to Th2 subclass antibodies, and (Th2 subclass antibodies are in mouse
IgG1 subclass antibodies, in artificial IgG2a subclass antibodies), this monoclonal antibody can inhibit DPP-4 active, special and DPP-4
And the peptide or protein containing this B cell epitope combines, and can develop DPP-4 detection reagent and DPP-4 inhibitor, the monoclonal
Antibody is Th2 type antibody, and energy specific recognition combines and inhibits DPP-4, increases GLP-1 and insulin, reduces glucagon,
Internal blood glucose uric acid and creatinine and four items of blood lipid tests are dropped, oxidative stress is adjusted, immunologic balance is adjusted from Th1 toward Th2, reduces
Th17, increase Treg, mitigate pancreas inflammatory, protect pancreas kidney, can be used for preparing drug for prevent and treat due to blood glucose and uric acid
And gout caused by creatinine and dyslipidemia, oxidative stress disorder, kidney hepatopathy, diabetes and complication, hyperlipidemia, AS, painstaking effort
Pipe disease and the higher related disease of Th1 immunological regulation have significant application value.
Background technique
In the diseases such as type 1 diabetes, rheumatoid arthritis, multiple sclerosis and chronic thyroiditis, Th1 or Th1
The cell factor of secretion is dominant, and Th1 induction morbidity aggravates the state of an illness；Illness can then be mitigated to Th2 drift, or even prevent morbidity,
This is related with the inflammatory of Th1/Th2 and anti-inflammatory response.Relationship based on Th1/Th2 drifting state Yu various diseases, increasingly
Discovery is just tended in more research, exploitation can reverse or stablize the drug and method of Th1/Th2 state.The immune biology of antigen,
Th1 can generate IgG2a subclass antibodies in Mice Body, and Th2 type can then generate IgG1 subclass antibodies, and Th1 can be generated in mankind's body
IgG1 subclass antibodies, Th2 type can then generate IgG2a subclass antibodies, and Th2 subclass antibodies can increase the production of Th2 type cytokines
The higher disease of Th1 is given birth to and controlled, disease symptoms are alleviated.
Dipeptidyl peptidase 4 is otherwise known as t cell surface antigen CD26, is a kind of serine protease of cell surface.People
Earliest discovery DPP4 can be used as one treatment diabetes B important target spot.Exactly because it is to other intestines such as GLP-1, GIP
The degradation of pancreotropic hormone.GLP-1 has the insulin secretion accelerating of glucose dependency, and glucagon suppression secretion promotes β thin
Born of the same parents' regeneration and reparation, and delay postprandial gastric emptying, increasing functions, the GIP such as satiety equally has the function of insulin secretion accelerating.So
And the half-life period in vivo such as GLP-1, GIP only has 1-2min, is degraded rapidly inactivate by DPP4 later.DPP4 inhibitor passes through competing
The active site of striving property combination DPP4, reduces the catalytic activity of enzyme, so that the level for increasing internal GLP-1 and GIP reaches promotion
The effect of insulin secretion, stability contorting blood glucose improve β cell function, and patient's weight will not be caused to increase, and can avoid
Risk of hypoglycemia.The chemical synthetic drugs such as current a variety of DPP4 inhibitor such as Xi Gelieting, saxagliptin, are widely used to face
Bed.
Although at present also have been reported that DPP4 inhibitor for type 1 diabetes (1 diabetes mellitus of Type,
T1DM treatment), but since it is chemicals, is frequently accompanied by that take side effect for a long time strong, especially with secretion metabolism
For disease with the middle-aged and the old of hypertension, hyperlipidemia and hyperglycemia, control bad leads to compromised kidneys for a long time.And T1DM is one
The autoimmune disease of kind organ specificity.The insulin secretory cell of patient's body --- beta Cell of islet is by the immune of itself
System attack leads to persistent high blood sugar and long-term metabolic disorder, makes body tissue to cause the absolute shortage of internal insulin
Organ, especially eye, kidney, the damage of angiocarpy and nervous system and its dysfunction and failure.Therefore it needs to develop newly
DPP4 inhibitor, especially antibody or immunosuppressor control the higher disease of Th1 such as type 1 diabetes etc., alleviate disease disease
Shape.B cell epitope based on DPP-4 and the small-molecular peptides carrier for being coupled the preparation of this laboratory obtain Antigenic Peptide or peptide based immunogens come
Preparation Th2 hypotype monoclonal antibody is not reported both at home and abroad.
Small peptide-carrier protein couplet is chiefly used in preparing anti-polypeptide antibody, and individual polypeptide is typically too small to be not enough to evoke
Adequately immune response, therefore by becoming with small molecule B cell epitope or peptide haptens and carrier protein couplet or after merging
The immune biological adaptive immune spleen lymphocyte of Antigenic Peptide, is used to prepare hybridoma to prepare monoclonal antibody or by mentioning
Immune spleen lymphocyte gene phage antibody library or genetic engineering etc. is taken to prepare Multiple Antibodies.The load most generally used
Body is that keyhole limpet hemocyanin (KLH, keyhole limpet hemocyanin) than bovine serum albumin(BSA) (BSA) has higher exempt from
Epidemic focus, thus be the carrier protein being most often selected.Immune system is that this peptide-albumen is immune to evoke as a whole
Reaction, thus have in the antibody generated for polypeptide, have for linking agent, also have for carrier protein, because hereafter
The antibody that phase generates excessive antibody species and needs screening becomes more difficult, in addition, the small-molecular peptides such as B epitope and KLH or BLH
The antibody generated after coupling is immune is on the high side with Th1 antibody, and Th2 antibody is less than Th1 antibody, for connecting B cell epitope or peptide
Immunogene that haptens is formed and its for the unsuitable treatment Th1 of the antibody for preparing higher disease (such as type 1 diabetes and simultaneously
Send out disease and related disease, atherosclerosis, cardiovascular disease, senile dementia, kidney trouble, ephritis, rheumatoid arthritis, reaction
Property arthritis, multiple sclerosis, autoimmune thyroiditis, contact dermatitis, erythema nodosum, habitual abortion etc.)
It is ineffective.
The preparation of monoclonal antibody of the invention is using the B cell epitope of DPP-4 as haptens, with the preparation of this laboratory
The coupling of Inner adjuvant peptide, design synthesis multi-epitope peptide are directly exempted from as Antigenic Peptide or peptide based immunogens by such peptide based immunogens
Epidemic disease biology prepares immune spleen lymphocyte, this Inner adjuvant by HSP60 437~460 one section of 24 amino
The main Th2 epitope P2 peptide fragment (448~460) of sour specific polypeptide P277 peptide and islet cells specific antigen-insulinoma phase
Close 626~630 linear sequences of the membrane-proximal region JM2 of albumen (Insulinoma associated protein-2, IA-2)
It is a B cell epitope (626FEYQD630), it is named to be made up of link peptide for IA2 (5), currently without with P2 and IA2
(5) carrier peptides formed are coupled the report of small peptide preparation monoclonal antibody antibody and application thereof.
The present invention relates to immune, pharmacy and medicine related fieldss, are related to the B cell table of people's dipeptidyl peptidase 4 (DPP-4)
Serum after position and the peptide based immunogens containing such epitope and coupling protein or the immune animal of the fusion protein immunization original of expression contains
There is the high-caliber Th2 antibody that can and specifically bind, more anti-, monoclonal antibodies and genetic engineering antibody etc. can be used to prepare,
Further relate to the B cell epitope based on naive DPP-4 changes structure peptide and peptide based immunogens and coupling egg containing such epitope peptide
Serum after white or expression the immune animal of fusion protein immunization original contains high-caliber energy and dipeptidyl peptidase 4 is specifically bound
Th2 antibody, can be used for preparation more anti-, monoclonal antibodies and genetic engineering antibody etc., further relate to the B cell epitope containing DPP4
Prevention and treatment diabetes are used to prepare with the fusion protein for changing the immunogene and coupling protein of structure epitope peptide, composition or expression to be especially
The antibody of the related diseases such as type 1 diabetes and its complication especially monoclonal antibody and genetic engineering antibody, the antibody of preparation
It can be used for preparing the related diseases such as medical treatment diabetes and its complication drug and exploitation detection be anti-containing such epitope peptide
Former purposes.
Summary of the invention
Goal of the invention:
In view of this, one of the objects of the present invention is to provide with a kind of 4 (DPP- of specific binding dipeptidyl peptidase
4) monoclonal antibody mAb and preparation method thereof and its purposes on medicine and immunology.
The second object of the present invention provides the hybridoma cell strain of DPP-4 mouse monoclonal antibody mAb a kind of, classification naming
Are as follows: hybridoma cell strain CPULTM-4E9；Be preserved in No. 299 Wuhan Universitys of Wuhan, China city Wuchang District Bayi Road in the school in
State's Type Tissue Collection, the deposit date is on October 24th, 2017, deposit number was CCTCC NO:C2017227.
The third object of the present invention additionally provides the monoclonal antibody mAb (being named as CPU-KD4) of anti-DPP-4 a kind of, this
Monoclonal antibody is generated by above-mentioned hybridoma cell strain.
The monoclonal antibody CPU-KD4 of hybridoma cell strain CPULTM-4E9 and secretion based on above-mentioned acquisition can use survey
Sequence technology obtains heavy chain variable region VHSequence and light chain variable region VLSequence and its VH3 CDR regions sequence and VL3
The sequence of CDR region, above-mentioned sequence include amino acid sequence and nucleotide sequence and its application based on above-mentioned sequence.
The above-mentioned amino acid sequence and nucleotide sequence obtained can be used for recombinant microorganism and transgenic animals or plant
Object and its cell make them as production plant and produce antibody or for being transformed.
Based on above-mentioned amino acid sequence or nucleotide sequence and based on its obtain recombinant microorganism and transgenic animals or
Plant and its cell, preparation immunogene and prepare immune spleen lymphocyte to prepare antibody, various pharmaceutically may be used for developing
With the drug of receiving or composition or conjugate or polymer.
The present invention provides the preparation methods of the monoclonal antibody CPU-KD4 to realize in accordance with the following steps:
(1) preparation of immunogene and immunological lymphocyte: by IA2 (5) peptide fragment FEYQD, (amino acid sequence is shown in SEQ ID
NO.1 the N-terminal of C-terminal and Th2 epitope P2 peptide fragment (amino acid sequence is shown in SEQ ID NO.2)) pass through flexible peptide or link peptide (by
The amino acid of identical or different amino acid and different number composition) connection form carrier peptides, be named as IA2 (5)-P2-1 and (write a Chinese character in simplified form
Pass through flexible peptide with B cell epitope or the C-terminal of synthetic peptide haptens (indicating with B) again for IP) or link peptide is covalently attached, is formed
Multi-epitope peptide is Antigenic Peptide or is peptide based immunogens B-IA2 (5)-P2-1 (being abbreviated as B-IP), and the B of this patent is dipeptidyl peptidase
4 B cell epitope O (amino acid sequence is shown in SEQ ID NO.3), peptide based immunogens D41-IA2 (5)-P2-1 (be shown in by amino acid sequence
SEQ ID NO.4) it can be obtained by chemical synthesis, this peptide is not required to coupling BSA or KLH, can be small directly as immunogen immune
Mouse can generate strong immune response, be B cell epitope or synthetic peptide half that intramolecular carrier peptides in this structure significantly increase connection
The immunogenicity of antigen is immunized the immune spleen lymphocyte that organism preparation is used to prepare hybridoma with this peptide based immunogens, exempts from
Epidemic disease spleen lymphocyte can be used for preparing hybridoma to prepare monoclonal antibody or by extracting immune spleen lymphocyte
Gene prepares Multiple Antibodies by antibody library or genetic engineering.Antigenic Peptide can be used for screening hybridoma cell clone.It is special at this
B cell epitope comes from people DPP-4 in benefit.The immune spleen lymphocyte obtained can produce the antibody with DPP-4 specific bond.
Further, D41-KLH is obtained with B cell epitope and the KLH coupling of this person DPP-4, is immunized with same method small come Balb/c
Mouse generates the immune serum (antibody containing Th1 and Th2 type, Th1 are slightly more) for being greater than 10000 potency after D41-KLH is immune,
And D41-IA2 (5)-P2-1 of the invention it is immune after can also generate that (Th2 antibody has comparative advantage and is higher than greater than 10000 potency
The equally immune Th2 antibody generated of D41-KLH), therefore, under same immune condition, B-IA2 is used on preparation Th2 antibody
(5)-P2-1 is immunized Balb/c mouse and prepares immune spleen lymphocyte to prepare antibody.
(2) hybridoma technology prepares monoclonal antibody CPU-KD4: further, by above-mentioned peptide based immunogens D41-IA2 (5)-
The immune spleen lymphocyte that Balb/c mouse obtains is immunized in P2-1 and Sp2/0 myeloma cell's fusion prepares hybridoma,
Limiting dilution assay obtains monoclonal, and Antigenic Peptide ELISA method screens positive hybridoma cell, and acquisition can secrete anti-DPP-4 specificity
The hybridoma cell strain of antibody, is named as hybridoma cell strain CPULTM-4E9, and ELISA subtype identification is that IgG1 (is in mouse
Th2 antibody), antibody is produced using the method for producing monoclonal antibody in animal body, octanoic acid-ammonium sulfate precipitation method can recycle most
Monoclonal antibody, ProteinG column chromatography, which is further purified, obtains monoclonal antibody CPU-KD4.Pass through Western Blot, hypotype respectively
And titration experiment, inhibit DPP-4 activity experiment, the experiment for the treatment of diabetic mice is anti-come the monoclonal for verifying the method preparation
The specificity and purposes of body.
Above-mentioned peptide based immunogens can be used for recombinant microorganism and transgenic animals or plant and its cell, make their conducts
Production plant produces fusion protein, coupling protein or conjugate containing above-mentioned peptide based immunogens, life is immunized in polymer, composition
Object prepares the antibody of anti-DPP-4 and generates the immune spleen lymphocyte of this antibody.
Further, also comprising acceptable carrier and adjuvant or pharmaceutically acceptable auxiliary material in immunology, including but
It is not limited to: heat shock protein HSP60/65, L-Asparaginasum, L-Asparaginasum-TTP, L-Asparaginasum-TTP-CETPC, Fc,
Bovine serum albumin(BSA) (BSA), keyhole limpet hemocyanin (KLH), polyethylene glycol, Freund's adjuvant, aluminium adjuvant, lactose, sucrose, grape
Sugar, starch, cellulose family (such as carboxymethyl cellulose, hypromellose), ethylene glycol, soya-bean oil, sesame oil etc., to be immunized
Biology prepares the antibody of anti-DPP-4 and generates the immune spleen lymphocyte of this antibody.
The beneficial effects of the present invention are:
The advantage B cell antigen epi-position ingredient of DPP-4 is single, and structure is simple, gets rid of the bad epitope in natural DPP-4
Influence, be immunoreacted it is with strong points, screening hybridoma it is simple.
Comprising adjuvant in a kind of specific molecular in Antigenic Peptide or peptide based immunogens of the invention, the Inner adjuvant is by IA2
(5) C-terminal of peptide fragment FEYQD connects by flexible peptide or link peptide with the N-terminal of Th2 epitope P2 peptide fragment and to form carrier peptides, is named as
IA-2 (5)-P2-1 (being abbreviated as IP), then pass through flexible peptide with B cell epitope or the C-terminal of synthetic peptide haptens (indicating with B)
Or link peptide is covalently attached, forming peptide based immunogens or Antigenic Peptide B-IA2 (5)-P2-1 (being abbreviated as B-IP), peptide based immunogens can lead to
It crosses chemical synthesis simply to obtain, the intramolecular carrier peptides in this structure can significantly increase the B cell epitope or synthesis of connection
The immunogenicity of peptide haptens, easily induction generate the antibody of Th2 type, and can successfully prepare monoclonal antibody, and hybridoma is thin
Born of the same parents' screening is simple, has most important theories value and broad prospect of application.
The immune spleen lymph for being used to prepare hybridoma using above-mentioned peptide based immunogens or the immune organism preparation of Antigenic Peptide is thin
Born of the same parents, and D41-KLH is obtained with B cell epitope and the KLH coupling of this person DPP-4, it is immunized with same method come Balb/c mouse
Compare, generates the antibody containing Th1 and Th2 type after D41-KLH is immune, and Th1 is slightly more, and D41-IA2 (5)-of the invention
It can also generate after P2-1 is immune greater than 10000 potency and Th2 antibody has comparative advantage and is higher than the Th2 antibody that D41-KLH is generated,
It is immune using B-IA2 (5)-P2-1 class peptide based immunogens such as 41-IA2 (5)-P2-1 on preparation Th2 antibody under same immune condition
The immune spleen lymphocyte that Balb/c mouse obtains has advantage, this immune spleen lymphocyte can be used for preparing hybridoma
Cell prepares Multiple Antibodies to prepare monoclonal antibody or by extracting immune spleen lymphocyte gene.Antigenic Peptide can be used for
Screen hybridoma cell clone.The Th2 hypotype monoclonal antibody mAb (being named as CPU-KD4) that this patent prepares can be with
Target antigen albumen containing this B cell epitope or peptide haptens such as DPP-4 specific bond, can develop specific detection reagent, can
To inhibit the enzymatic activity of target protein such as DPP-4, the significant Th1/Th2 dysequilibrium that adjusts, can be special by Th1 deviation Th2 transfer
Property identification combine inhibit DPP-4, can significantly increase GLP-1, insulin, reduce glucagon, internal blood glucose uric acid and flesh drop
Acid anhydride and four items of blood lipid tests adjust oxidative stress, adjust immunologic balance from Th1 toward Th2, reduce Th17, increase Treg, increase uric acid
And creatinine excretion, mitigate pancreas inflammatory, protects pancreas kidney, significantly increase oxidation resistance, significantly improve the morbidity disease of disease
Shape can prepare pharmaceutically acceptable drug for preventing and treating hyperlipidemia, AS, high lithemia disease and diabetes and complication, prevent and treat
Th1/Th2 immunologic balance is abnormal, Th17 is abnormal, uric acid and creatinine are abnormal, ephritis caused by oxidative stress disorder, gout, hepatopathy,
Hyperlipidemia, atherosclerosis, diabetes and complication, senile dementia, eye disease, the higher disease of the Th1 such as angiocarpy.According to this
Patent system is for synthetic peptide based immunogens, then prepares immune spleen lymphocyte, then prepares monoclonal antibody specific or specific antibody
Initiative way of thinking of theories and practical application in treatment metabolic disease are of great significance.
Preservation information
The classification naming of the hybridoma cell strain of DPP-4 mouse monoclonal antibody for preservation are as follows: hybridoma cell strain
CPULTM-4E9；
Depositary institution's title: China typical culture collection center；
Depositary institution's abbreviation: CCTCC；
Depositary institution address: Wuhan University, Wuhan, China city；
Preservation date: on October 24th, 2017；
Deposit number: CCTCC NO:C2017227.
Detailed description of the invention
In order to make the object of the invention, technical solution and advantage understand, are further retouched in detail in conjunction with attached drawing to the present invention
It states, in which:
Fig. 1 is that ELISA measurement Mouse Antisera potency after mouse is immunized in this Antigenic Peptide or peptide based immunogens.
Fig. 2 is monoclonal antibody mAb (CPU-KD4) SDS-PAGE electrophoresis of the present invention, and wherein M is molecular weight of albumen mark
Quasi- (kDa), swimming lane 1-6 are respectively antibody moiety after preparation and ProteinG column purification, and mouse resisting anteserum, former ascites, sun is immunized
Property hybridoma supernatant, octanoic acid-ammonium sulfate purified antibodies, ProteinG purified antibodies.
Fig. 3 is monoclonal antibody subtype identification figure of the present invention.
Fig. 4 is the Western Blot result of monoclonal antibody mAb (CPU-KD4) of the present invention and DPP-4 specific bond.
Fig. 5 is that monoclonal antibody of the present invention inhibits the active curve of DPP-4.
Fig. 6 is blood sugar decreasing effect of the monoclonal antibody of the present invention to the STZ diabetic mice induced
Fig. 7 is anti-trioxypurine, creatinine effect (Fig. 7 A of the monoclonal antibody of the present invention to the STZ diabetic mice induced
For serum uric acid variation, Fig. 7 B is the variation of urine uric acid, and Fig. 7 C is urine creatinine variation).
Fig. 8 is monoclonal antibody of the present invention to the total antioxidation of the STZ diabetic mice induced and drop MDA effect
(Fig. 8 A is total antioxidation effect, and Fig. 8 B is MDA variation).
Fig. 9 is that monoclonal antibody of the present invention examines spleen cell ELISA after the treatment of the STZ diabetic mice induced
Survey result (Fig. 9 C of cell factor result (Fig. 9 A is IL-10, and Fig. 9 B is IFN-γ) and flow cytometer detection Th17 and Treg expression
For Treg expression, Fig. 9 D is Th17 expression).
Figure 10 is monoclonal antibody of the present invention to mitigation pancreas inflammatory effect after the treatment of the STZ diabetic mice induced
Fruit (the inflammatory pathologies analysis chart that Figure 10 is HE dyeing detection pancreas).
Specific embodiment
Following embodiment further illustrates the contents of the present invention, but should not be construed as limiting the invention.Without departing substantially from
In the case where spirit of that invention and essence, to modifications or substitutions made by the method for the present invention, step or condition, the present invention is belonged to
Range.
Unless otherwise specified, the conventional means that technological means used in embodiment is well known to those skilled in the art.
Embodiment 1: the preparation of immune peptide based immunogens or Antigenic Peptide:
By the C-terminal of IA2 (5) peptide fragment FEYQD (amino acid sequence is shown in SEQ ID NO.1) and Th2 epitope P2 peptide fragment (amino
Acid sequence is shown in SEQ ID NO.2) N-terminal connect by flexible peptide or link peptide and to form carrier peptides, be named as IA2 (5)-P2-1
(being abbreviated as IP) (is indicated, D41 is a segment polypeptide sequence of DPP-4 again with the B cell epitope D41 of people DPP-4 with B
88VFLENSTFDE97, totally 10 amino acid residues, amino acid sequence are shown in SEQ ID NO.3) C-terminal pass through flexible peptide or connection
Peptide is covalently attached, and forming peptide based immunogens B-IA2 (5)-P2-1, (this patent is D41-IA2 (5)-P2-1, and amino acid sequence is shown in SEQ
ID NO.4), peptide based immunogens or Antigenic Peptide are all made of the synthesis of FMOC solid-phase synthesis, and HPLC detects purity > 90%.B is used in analysis
Cell epitope D41 is synthesized using FMOC solid-phase synthesis, and HPLC detects purity > 95%, and B cell epitope D41 is carried with tradition again
Body protein KLH or BSA are coupled to form D41-BSA, D41-KLH.
Embodiment 2: the preparation of animal immune and hybridoma
The preparation of animal immune and immune spleen lymphocyte: above-mentioned Antigenic Peptide is helped with QuickAntibody-Mouse5W
Agent emulsification, mixes with 0.9% injection physiological saline 1: 1,6-8 week old BALB/c mouse is immunized using intramuscular injection method, exempts from
Epidemic disease dosage is 100 μ g/, and interval carries out second with identical immunization method and dosage after two weeks and is immunized.Immune eye rear twice
Socket of the eye takes blood to measure serum titer with ELISA method gradient dilution, chooses the immune spleen lymphocyte of the highest mouse of antibody titer
Carry out cell fusion.Conclusion: above-mentioned Antigenic Peptide generates higher potency (being greater than 1: 10000) after mouse is immunized, and can be used to list
The preparation (Fig. 1) of clonal antibody.
Cell fusion: myeloma cell uses the Sp2/0 in BALB/c mouse source, and logarithmic growth phase is in when fusion；It takes
Above-mentioned immune mouse spleen, is made lymphocyte single cell suspension；Immune mouse spleen lymphocyte and myeloma cell are with 1:
5-1: 10 mixing, are added dropwise 37 DEG C 50%PEG (pH8.0) 1ml, and incomplete culture medium and remaining terminate liquid is added, and are centrifuged on abandoning
The suspension of HAT culture medium is added after clear to mix, constant volume to 50ml is dispensed into 96 porocyte culture plates, is placed in 37 DEG C, 5%CO2It is permanent
It is cultivated in warm incubator.
Screening and clone: fusion selected cell clone in 7-10 days, used the B cell of the DPP-4 of the coupling BSA of purifying
Epitope peptide and Antigenic Peptide carry out ELISA test, mark cell strain number.Limiting dilution is carried out to positive hole cell, it is limited dilute every time
Latter 5-6 days measurement ELISA values are released, the picking OD450 positive is worth higher monoclonal hole and carries out limiting dilution, until ELISA is measured
Hardened fruit is the positive to 96 orifice plates entirely, and the picking positive is worth high monoclonal single plant.
Embodiment 3: monoclonal antibody mAb (being named as CPU-KD4) preparation and purifying
The preparation of mouse monoclonal antibody ascites: using the method for producing monoclonal antibody in animal body.Every Balb/c mouse abdomen
Chamber injects sterilized liquid paraffin 0.5ml, after 7 days, every mouse peritoneal injection 1 × 106A mouse hybridoma cell.It is seen after 7 days
The mouse ascites condition of production is examined, if abdomen obviously expands, ascites can be extracted.Contain red blood cell, cell debris, fiber in ascites
Fibrin clot and lipid.Sedimentation cell, then high speed centrifugation removal cell residue and finely ground particle substance are first removed with low-speed centrifugal,
Fibrin clot and lipid are taken out with 0.2 μm of miillpore filter.
Octanoic acid-ammonium sulfate precipitation method: the ascites of preliminary purification slightly mentions immunoglobulin with octanoic acid-ammonium sulfate precipitation method, sinks
Dialysis of forming sediment removes ammonium sulfate.
ProteinG column chromatographic purifying antibody: octanoic acid-ammonium sulfate precipitation method can recycle most monoclonal antibody, further use
ProteinG binding antibody measures the protein content in each collecting pipe, merges protein pipe；PBS dialysis, thereafter according to the steady of antibody
It is qualitative, it is added preservative (0.020g/L Sodium azide), sets 4 DEG C or freezen protective.
Embodiment 4: the nature examination of monoclonal antibody mAb (being named as CPU-KD4)
Embodiment 4-1: antibody purity detection: using the purity of monoclonal antibody after SDS-PAGE method purification Identification, such as
Shown in Fig. 2, only there are two electrophoretic bands in monoclonal antibody, illustrates monoclonal antibody purity is high.The result shows that monoclonal is anti-
Body molecular weight about 160KDa is made of the light chain of two treaty 55kDa heavy chains and two treaty 25kDa.
Embodiment 4-2: Subclass of antibody identification: using indirect elisa method, is reflected using the antibody of the various Ig hypotypes of anti-mouse
The Ig hypotype for the antibody that fixed above-mentioned hybridoma generates, IgG1 signal is most strong as the result is shown, monoclonal antibody hypotype of the present invention
For IgG1 (Fig. 3).
Embodiment 4-3: antibody Western blot detection: the DPP-4 of pre-dyed albumen Marker and purifying purchase are carried out
12% SDS-PAGE separation.After electrophoresis, remove running gel be put into transfer equilibration buffer in balance 10-15min.It cuts
Good filter paper, which is also placed in transfering buffering liquid, to be infiltrated.Running gel is overlayed on the three layers of filter paper completed, by nitrocellulose
Film is placed on running gel, and remaining 3 filter paper is successively overlayed on nitrocellulose, and it is slow that transfer box loading is filled transfer
In the transfer instrument of fliud flushing, anode is leaned in film side, and cathode is leaned in gel side, and 30V transfer is stayed overnight in 4 DEG C of chromatography cabinets.Film is taken out, by nitric acid fibre
It ties up plain film and is put into Ponceau S dyestuff and decolourize in water after dyeing 5min, make to decolourize completely.Film is put into and fills 5ml confining liquid
Small plate in shake 1h.Confining liquid is removed, the monoclonal antibody 1h that 5ml has been diluted with confining liquid is added, film is taken out, is put into
It in larger plate, is washed 4 times with the TTBS of 200ml, film is moved into small plate, 5ml is added with confining liquid and dilutes horseradish peroxidating
The secondary antibody 1h of object enzyme label.It is put into larger plate, is washed 4 times with the TTBS of 200ml, each 10-15min.Film is taken out to be put into now
Develop the color 10-15min in the DAB developing solution matched.Taking-up is dried, and is taken pictures.
Conclusion: monoclonal antibody of the present invention only specifically binds dipeptidyl peptidase 4 (DPP-4) albumen, and with other albumen
No cross reaction (Fig. 4).
Embodiment 4-4: antibody in vitro DPP-4 maximum inhibition identification
DPP-4 is purchased from foreign biomolecule company (import packing).Inhibit DPP4 enzymatic activity inhibiting rate test experience principle be
Chromogenic assay.DPP4 hydrolyzes Gly-Pro-pNA, generates pNA (yellow), and pNA has characteristic absorption peak at 405nm, passes through
Microplate reader measures absorbance value at 405nm, and carrying out the active height of reaction enzymes, (i.e. each group Serum Antibody is to DPP4 inhibition level
Size).Specific experiment step are as follows: 1. are incubated for serum, buffer, enzyme 30 minutes respectively under 37 degrees Celsius；2. according to enzyme,
96 orifice plates are added in the sequence of buffer, substrate.Overall reaction system is 100ul, is divided into blank control group (0.26mmol/L substrate
5ul；Tris-Hcl buffer 95ul), negative control group (0.5U/L DPP4 10ul；0.26mmol/L substrate 5ul； Tris-
Hcl buffer 85ul), positive controls (0.5U/L DPP4 10ul；0.26mmol/L substrate 5ul；5 times of diluted each group blood
Clear 10ul；Tris-Hcl buffer 75ul), positive blank group (0.26mmol/L substrate 5ul；5 times of diluted each group serum
10ul；Tris-Hcl buffer 85ul).3.37 DEG C of constant incubators react 60min, measure the absorbance at 405nm.4. ratio
Compared with the value of each group OD405, or calculate inhibiting rate.
Calculating of the antibody to DPP-4 inhibiting rate
The calculation formula of inhibiting rate is as follows: inhibiting rate (%)=(OD negative control-OD blank control)-(OD inhibitor-
The blank control of OD inhibitor)/(OD negative control-OD blank control) x 100%.
As shown in figure 5, antibody mAb (CPU-KD4) has significant external inhibition DPP-4 enzymatic activity effect, half inhibiting rate
IC50For 21.6 μ g/mL.
Embodiment 5: the antibody sequencing of the mouse monoclonal antibody CPU-KD4 of anti-DPP-4:
The sequencing in the area VH and VL of monoclonal antibody CPU-KD4 and the preparation method of CDR region:
It is as follows that key step is sequenced:
(1) TakaRa RNA extraction agent box extracts the RNA of hybridoma cell strain CPULTM-4E9.
(2) by the RNA extracted, by 5 ' RACE reverse transcription cDNA, then light chain heavy chain PCR amplification variable region gene is carried out
(PCR instrument model: Thermo Fisher 2720), separately designs heavy chain and light chain amplimer, PCR as the result is shown kappa and
IgG primer amplification goes out target stripe, shows that light chain is kappa chain, heavy chain is IgG chain.
(3) the corresponding PCR product of recycling (J-E Beckman centrifuge), respectively and after the flat company of PUC 57/EcoRV digestion carrier
Bacterial examination obtains heavy chain and light-chain variable region gene clones and carries out heavy chain variable region VHWith light chain variable region VLNucleotide sequence is surveyed
It is fixed.
Using the sequence of seqman software analysis measurement, non-functional antibody gene is rejected, is analyzed with IMGT and snapgene
The heavy chain variable region V of protein sequence acquisition monoclonal antibody CPU-KD4HWith light chain variable region VLSequence.
(4) by the V of the monoclonal antibody CPU-KD4 of seaman analysisHAnd VLThe sequence in area reuses IMGT on-line analysis
Respectively obtain antibody VHAnd VLCDR region sequence in area.
The diabetes model of embodiment 6:STZ induction is established and monoclonal antibody CPU-KD4 treatment modelling effect measurement:
The foundation of the diabetes model of embodiment 6-1:STZ induction
4 week old male C 57 BL/6 J mouses (are purchased from Yangzhou University's comparative medicine center, credit number: SCXR (Soviet Union)
2012-0004.) with 0.1M pH4.4 citrate buffer configuration STZ concentration be 7.5mg/ml, by 50mg/kg dosage abdominal cavity
Injection, one time a day, continuous injection 5 times, the 3rd, 7, the 14 day measurement blood glucose value after modeling, with fasting blood-glucose twice in succession >=
11.1mmol/L is into mould standard.After vein gives monoclonal antibody medicine (25mg/kg) 1h of above-mentioned preparation once a week, in small
The blood sampling of rathole vena orbitalis posterior clump, whole blood are centrifuged 3min in 2500r/min, and upper serum is placed in -70 DEG C of refrigerators and saves after centrifugation
It is spare.
Embodiment 6-2: treatment diabetes STZ animal pattern experiment:
1 grouping experiment: 30 STZ induced diabetes model mices are randomly divided into 4 groups, every group 10, are respectively as follows: model group
(i.e. solvent group) gives PBS buffer solution, and Normal group is normal mouse group, and mAb is antibody group weekly administration 1 time, and dosage is
500 μ g//times, positive drug group Xi Gelieting are oral administration 1 time daily.Dosage is 350 μ g//times.In model success
(be subject to blood glucose >=11.1mmol/L) is administered on the 2nd week for the first time afterwards, and weekly administration later 1 time, by 13 weeks.
2. blood sugar test: after diabetic mouse model is established, being administered into the 13rd week, the method blood glucose of blood is taken using docking
The blood glucose of instrument results of regular determination mouse.After the C57BL/6J male mice of STZ modeling gives antibody, the change of blood sugar situation of mouse
As shown in Figure 6.The experimental results showed that antibody CPU-KD4 can significantly reduce blood glucose in diabetic mice, there is pole compared with solvent group
Significant difference (* * P < 0.01).
3. insulin assay in sugar tolerance experiment
After the 9th time immune, the glucose tolerance situation of administration group and control group mice is measured.Every group takes 6 mouse,
The blood glucose value of every mouse is near this group of average blood sugar value.After mouse is deprived of food but not water 16h, every mouse peritoneal injection dosage
For the glucose solution of 2g/Kg.The concentration of glucose solution is 400mg/mL, and the mouse injection dosage of weight 20g is 0.1mL.
0min after injectable dextrose monohydrate, 15min, 30min, 60min, 120min, 180min, 240min, 300min take blood using tail vein
Mode measure the blood glucose value of mouse.And blood is taken in 15min after having injected glucose, ELISA measures serum insulin level,
Specific experimental procedure is carried out according to kit specification, and rough operating process is as follows: 1,50 μ L standard items are added in every hole, or
5 times of diluted serum of person.2,100 μ L ELIAS secondary antibodies, 37 DEG C of incubation 60min after preservative film winding are added in every hole.3,Wash
Solution expires hole board-washing 4 times.4,50 μ L substrate As and 50 μ L substrate Bs are added in every hole, mix gently, 37 DEG C are protected from light incubation
15min.5, every hole is added in 50 μ L terminate liquid (2mol/L H2SO4) (color is by Lan Bianhuang) 6,15min in 450nm wavelength
Measure OD value.7, sample insulin level is calculated by standard curve.As the result is shown: in 500ug/ antibody administration group serum
Insulin is apparently higher than Placebo group, as a result has significant difference (* * P < 0.01), body blood sugar regulation ability significantly changes
It is kind to improve.
3. uric acid detects: vena ophthalmica is stored at room temperature after waiting for blood to solidify from blood is taken after drug treatment, 4000 r/min centrifugation
10min separates serum afterwards twice, and -70 DEG C freeze.It is horizontal using phosphotungstic acid reduction method measurement uric acid in serum (SUA), according to south
Capital is built up corresponding reagent box specification and is measured.As a result as shown in Figure 7 A, antibody administration group has significant anti-trioxypurine effect,
There is extremely significant difference (* * P < 0.01) compared with solvent group.
4. the uric acid creatinine of urine detects: urine is collected, detects uric acid and creatinine content result as shown in Fig. 7 B and 7C,
Antibody administration group has significant increase urine uric acid (* * * P < 0.001) and creatinine (* * P < 0.01) effect, with solvent group
Compared to extremely significant difference, illustrate that antibody can increase uric acid creatinine excretion.
5. after treatment in mice serum total antioxidant capacity (T-AOC) measurement
After high lithemia Establishment of mouse model, 1h vena ophthalmica is stored at room temperature from blood is taken to blood after last time is administered weekly
After solidification, 4000r/min centrifugation 10min separates serum afterwards twice, and -70 DEG C freeze, and measures blood according to corresponding reagent box specification
Total antioxidant capacity (T-AOC) is horizontal in clear and MDA is horizontal.Respectively as shown in figs. 8 a and 8b, antibody administration group can be apparent
The oxidative stress for mitigating body has extremely significant difference (* * P < 0.01) compared with model group.
6. cytokines measurement
After the observation phase, each group mouse boosting cell is taken to be cultivated, centrifuge cell culture plate after 72h retains in each group
Clear culture solution.Detect the cytokine levels such as IFN-γ and IL-10 respectively with ELISA kit.Concrete operations are made by kit
It is carried out with specification.The results show that compared with model group, antibody administration group mouse inflammatory factor IFN-γ (Th1 cytokines
Representing, Fig. 9 B) level is decreased obviously, and IL-10 (Th2 cytokines represent, Fig. 9 A) is significantly raised, has extremely significant difference
(* * P < 0.01), and Th1 and Th2 passes through the performance of the cell factors such as secretion of gamma-IFN and IL-10 respectively respectively in Mouse spleen cells
From immune response, after illustrating antibody administration, immuno-modulatory type is changed into Th2 type by Th1 type in Mice Body.
7. being identified for spleen cell Th17, Treg content analysis after MAb immunotherapy model mice；
At the end of zoopery, experimental group and the fresh spleen of control animals tissue are taken, conventional treatment prepares spleen cell,
It is marked, washs according to flow cytometer showed conventional method, detect Treg (Fig. 9 C) and Th17 (Fig. 9 D) amount.As the result is shown: Dan Ke
Treg cell concentration dramatically increases after grand antibody mediated immunity treatment model mice, and Th17 is significantly reduced.
8. pancreas HE dyeing and pathological observation after treatment
At the end of observation, each group mice pancreatic is taken, fixes 24~72h through 10% formalin solution, it is conventional to draw materials, it takes off
Water, paraffin embedding, film-making (4 μ m-thicks slice), hematoxylin-eosin (HE) dyeing.As shown in Figure 10, compared with model group mouse,
The pancreas inflammatory degree of antibody administration group mouse significantly mitigates, and illustrates that the pancreas of the Antibody on Mouse has preferable anti-inflammatory and protects
Shield effect.
The effect of 7. monoclonal antibody CPU-KD4 of embodiment treatment type 1 diabetes NOD mouse:
Embodiment 7-1.NOD mouse type 1 diabetes model.
Female NOD mice age of mouse 4~5 weeks, weight 18-20g, is provided by Fukang Biotechnology Co., Ltd, Beijing China
SCXK (capital) 2014-0004.
The experiment of embodiment 7-2. animal immune
1. model foundation: 20 diabetic mices are randomly divided into 2 groups, and every group 10, it is (molten to be respectively as follows: placebo group
Agent control group, negative control group), antibody administration group, once a week vein give above-mentioned preparation monoclonal antibody medicine (25mg/kg)
Afterwards, it takes a blood sample in mouse orbit rear vein beard, whole blood is centrifuged 3min in 2500r/min, and upper serum is placed in -70 DEG C of ice after centrifugation
It is saved backup in case, model group (i.e. solvent group) gives PBS buffer solution.
2. antibody group treats blood glucose in diabetic mice and uric acid and creatinine and MDA detection: model success (with blood glucose >=
Subject to 13.19mmol/L) weekly administration 1 time afterwards, it is administered continuously by 34 weeks.Diabetic mice docking is taken into blood before being immunized every time,
It takes and detects mouse blood sugar level with blood sugar monitoring instrument when blood.The results are shown in Table 1, the results showed that, compared with solvent group, antibody group
Blood glucose in diabetic mice and uric acid and creatinine and MDA are significantly reduced, there is statistical difference.It can develop for treating diabetes
And complication.
1 monoclonal antibody CPU-KD4 of table treats blood glucose, uric acid creatinine and the MDA detection of NOD type 1 diabetes NOD mouse
Conclusion: antibody of the present invention is prepared into after adjusting peptide or Antigenic Peptide or the immune mouse of peptide based immunogens by small molecule immune
The monoclonal antibody CPU-KD4 for the single B cell epitope D41 of DPP-4 arrived, purity and specificity with higher, and produce
The effect of the raw efficient enzymatic activity for inhibiting DPP-4, significantly reducing blood glucose has significant drop blood to diabetic mice simultaneously
Clear uric acid creatinine and four items of blood lipid tests increase uric acid creatinine excretion, alleviate oxidative stress, mitigate pancreas inflammatory, protection pancreas kidney,
The effect that Th1 changes to Th2 immuno-modulatory type is adjusted, can be used for diabetes especially type 1 diabetes significant effect and substantially can
Blood glucose normal value is kept, the death rate is reduced, also to gout, hepatopathy, height caused by uric acid creatinine dyslipidemia, oxidative stress disorder
Blood lipid, atherosclerosis, diabetes and complication, senile dementia, eye disease, cardiovascular disease and Th1 type immunological regulation are inclined
High related disease etc. is upper to have important application value；The antibody can develop the reagent of detection DPP-4 in combination with DPP-4 simultaneously.
Finally, it is stated that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although passing through ginseng
According to certain preferred embodiments of the invention, invention has been described, but those skilled in the art should manage
Solution, can make various changes, without departing from defined by the appended claims to it in the form and details
The spirit and scope of the present invention.
Claims (10)
1. the hybridoma cell strain of the monoclonal antibody of one plant of anti-dipeptidyl peptidase 4 (DPP-4) of secretion, classification naming are as follows: hybridization
Tumor cell strain CPULTM-4E9, the Chinese Typical Representative culture that Wuhan University, Wuhan, China city is preserved on October 24th, 2017 are protected
Hiding center, deposit number are CCTCC NO:C2017227.
2. being named as by the monoclonal antibody mAb for the anti-dipeptidyl peptidase 4 that hybridoma cell strain described in claim 1 is secreted
CPU-KD4。
3. the preparation method of spleen lymphocyte is immunized in the technology of preparing of monoclonal antibody CPU-KD4 as claimed in claim 2:
It is the B cell epitope D41 (amino acid sequence is shown in SEQ ID NO.3) and intramolecular carrier peptides (ID containing SEQ with dipeptidyl peptidase 4
NO.1 and SEQ ID NO.2,2 peptide connection method multiplicity) it is coupled to prepare small molecule antigens peptide or peptide based immunogens D41-IA2
(5)-P2-1 (amino acid sequence is shown in SEQ ID NO.4) and by synthesizing quick and convenient acquisition, small molecule antigens peptide is not required to be coupled
BSA or KLH can generate strong immune response directly as immunogen immune mouse, and immune animal can be used for preparing immune spleen
Lymphocyte, this immune spleen lymphocyte can be used for preparing hybridoma to prepare monoclonal antibody or extract immune spleen
Lymphocyte gene prepares Multiple Antibodies by commonly using antibody library or genetic engineering, and Antigenic Peptide can be used for screening hybridoma
Clone.
4. preparing hybridoma cell strain by hybridoma technology according to the immune spleen lymphocyte that claim 3 obtains
CPULTM-4E9, then the monoclonal antibody CPU-KD4 of anti-dipeptidyl peptidase 4 is prepared, this antibody is Th2 type antibody, is in mouse
IgG1 hypotype, energy specific recognition combine and inhibit 4 activity of dipeptidyl peptidase, can increase GLP-1 and insulin, reduce the high blood of pancreas
Sugared element, hypoglycemic, uric acid, creatinine and four items of blood lipid tests promote uric acid creatinine excretion, adjust oxidative stress, adjust immunologic balance by
Th1 tends to Th2, reduces Th17, increases Treg, mitigates pancreas inflammatory, protects liver kidney.
5. the monoclonal antibody can be developed as two peptidyls according to the application of monoclonal antibody described in claim 2 and 4
The detection reagent of peptase 4.
6. the monoclonal antibody can prepare pharmaceutically acceptable according to the application of monoclonal antibody described in claim 2 and 4
Drug for preventing and treating diabetes and complication, prevention and treatment Th1/Th2 immunologic balance is abnormal, Th17 is abnormal, blood glucose and uric acid and flesh
Ephritis caused by acid anhydride dyslipidemia and oxidative stress disorder, gout, hepatopathy, hyperlipidemia, atherosclerosis, diabetes and concurrent
Disease, senile dementia, eye disease, the higher disease of the Th1 such as angiocarpy.
7. -4 hybridoma cell strain CPULTM-4E9 obtained and the monoclonal antibody CPU-KD4 of secretion can according to claim 1
Heavy chain variable region V is obtained with sequencing technologiesHSequence and light chain variable region VLSequence and its VH3 CDR regions sequence and VL3
The sequence of a CDR region, above-mentioned sequence include amino acid sequence and nucleotide sequence and its application based on above-mentioned sequence.
8. amino acid sequence according to claim 7 or nucleotide sequence obtain it is standby obtain recombinant microorganism and transgenic animals or
Plant and its cell and as production plant's producer gene or antibody or for being transformed.
9. spleen lymphocyte is immunized to prepare antibody in preparation immunogene and preparation according to the purposes of claim 7 and 8, it is used for
Develop various pharmaceutically acceptable drugs or composition or conjugate or polymer.
10. preparing peptide based immunogens according to the claims, then immune spleen lymphocyte is prepared, then prepares specific Dan Ke
The initiative way of thinking of theories and practical application of grand antibody or specific antibody in treatment metabolic disease.
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