CN108774287A - A kind of VEGF-B monoclonal antibodies and kit - Google Patents
A kind of VEGF-B monoclonal antibodies and kit Download PDFInfo
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- CN108774287A CN108774287A CN201810683274.3A CN201810683274A CN108774287A CN 108774287 A CN108774287 A CN 108774287A CN 201810683274 A CN201810683274 A CN 201810683274A CN 108774287 A CN108774287 A CN 108774287A
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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
- C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
-
- 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
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/475—Assays involving growth factors
Abstract
The invention discloses a kind of VEGF-B monoclonal antibodies, the amino acid sequence such as VH of heavy chain:Shown in 2VWE_B, the amino acid sequence such as VL of light chain:Shown in AF099134_1.There is the VEGF-B monoclonal antibodies higher affinity, high specificity to have good biological activity in vitro.The VEGF-B protein detection kits of VEGF-B monoclonal antibodies of the present invention and the how anti-foundation of VEGF-B can be used as to the auxiliary diagnosis and dynamic monitoring tool of liver cancer.
Description
Technical field
The invention belongs to biotechnologies, and in particular to a kind of VEGF-B monoclonal antibodies and the examination of VEGF-B Protein Detections
Agent box.
Background technology
Vascular endothelial growth factor (vascular endothelial growth factor, VEGF) is a kind of with height
Spend a kind of dimer cationic glycoproteins of bioactivity.VEGF families include VEGF-A, VEGF-B, VEGF-C, VEGF-D,
VEGF-E, VEGF-F and placenta growth factor (Placental growth factor, PlGF).People's VEGF-B genes are located at dye
On colour solid 11q13, overall length 4kb has 7 exons, by 6 introne intervals.The promoter region of its promoter region and VEGF-A
There are some serious differences, this may lead to its differential responses to physiology sexual stimulus.The promoter region of the two all includes one
The binding site of a islands CpG and transcription factor Spl, Ap-2.VEGF-B promoter regions include the sites Egr-1, but lack VEGF-A
The sites HIF-1a and Ap-1 having.Therefore anoxic, cold etc. can induce the environmental stimulus deficiency of Yin and energy of VEGF-A expression
Induce the molecule of VEGF-A expression such as：Growth factor, prostaglandin E2 and steroid hormone not can induce VEGF-B expression.
Two different turns generated due to two kinds of different montage modes of coding VEGF-B mRNA are had now been found that
Record, i.e. VEGF-B167 and VEGF-B186, contain 167,186 amino acid residues respectively.The molecular weight of VEGF-B167 albumen point
Not Wei 21kD, the different dimerization of heterodimer VEGF-B167VEGF165 that disulfide bond be connect can be formed with the monomer of VEGF
Body cannot be secreted into extracellularly when lacking heparin, and the homodimer of VEGF165 can be freely secreted into extracellularly, thus be seen
Come, VEGF-B167 plays decisive role to the secretion of heterodimer, and the formation of VEGFVEGF-B167 heterodimers can
The bioavilability of VEGF can be controlled.VEGF-B can stimulate thymidine incorporation Human umbilical vein endothelial cells or bovine capillary endothelial
In cell DNA, prompts expression quantity of the VEGF-B167 to liver cancer by cancer to have increasing trend, illustrate VEGF-B167 in the process
Effect it is more important compared with VEGF-B186；VEGF-B167 high expressing promotings are formed into portal vein tumor thrombus and metastases；VEGF-B167
Low expression patient's prognosis is preferable, and postoperative overall survival and disease free survival are higher.
The blood vessel of adult human liver is very abundant, the side of blood vessel be the connected sinus hepaticus of endothelial cell be spaced with gap come and
Sinus hepaticus in liver cancer tissue is different from normal liver tissue, be no longer by discontinuous flat endothelial cell cover, but by even
Continuous endothelial cell conventional capillary structure made of surrounding.Therefore, angiogenesis function is more typical in liver cancer.
There are positivity up-regulation effects to angiogenesis by VEGF-B, should also have certain effect for the angiogenesis of liver cancer.VEGF-
What VEGF families and its acceptor molecule biological function including B were studied deepens continuously, and VEGF-B is detected in diseases such as liver cancer
It plays a positive role in clinical treatment monitoring.VEGF-B plays the role of Angiogensis same as VEGF-A again, therefore combines inspection
VEGF-A, VEGF-B are surveyed, the non-tumour-specific interference in the VEGF-A detection positives can be reduced, detects and assists in clinical tumor
There is clinical meaning in diagnosis.
The research of labelling immunoassay technology and application development are rapid nearly ten years, are widely used to biomedical basis
Theoretical research and each field of clinical disease diagnosis.Method for detecting serological index is mainly immune including radioactive isotope
Analysis, enzyme linked immunosorbent assay and chemiluminescence immune assay.These methods can both can also be used as really as Primary Screening Test
Recognize experiment, wherein chemoluminescence method has many advantages, such as to detect that the range of linearity is wide, detecting instrument is simple and convenient to operate.
Its specificity of the similar product of liver cancer clinical monitoring and affinity etc. promote possible, base there is also further
In this, we have developed VEGF-B assay kits (chemoluminescence method) and preparation method thereof.
Invention content
In order to overcome it is existing in the above problem have higher the present invention provides a kind of VEGF-B monoclonal antibodies
Affinity has good biological activity in vitro；Development prospect is wide.
A kind of VEGF-B monoclonal antibodies, which is characterized in that the heavy chain variable amino acid sequence of the antibody is：Pro
Val Ser Gln Pro Asp Ala Pro Gly His Gln Arg Lys Val Val Ser Trp Ile Asp Val
Tyr Thr Arg Ala Thr Cys Gln Pro Arg Glu Val Val Val Pro Leu Thr Val Glu Leu
Met Gly Thr Val Ala Lys Gln Leu Val Pro Ser Cys Val Thr Val Gln Arg Cys Gly
Gly Cys Cys Pro Asp Asp Gly Leu Glu Cys Val Pro Thr Gly Gln His Gln Val Arg
Met Gln Ile Leu Met Ile Arg Tyr Pro Ser Ser Gln Leu Gly Glu Met Ser Leu Glu
Glu His Ser Gln Cys Glu Cys Arg Pro Lys Lys Lys Asp Ser Ala Val Lys Pro Asp
Ser Pro Arg Pro Leu Cys Pro Arg Cys Thr Gln His His Gln Arg Pro Asp Pro Arg
Thr Cys Arg Cys Arg Cys Arg Arg Arg Ser Phe Leu Arg Cys Gln Gly Arg Gly Leu
Glu Leu Asn Pro Asp Thr Cys Arg Cys Arg Lys Leu Arg Arg；
Chain variable region amino acid sequence is：Trp Ile Asp Val Tyr Ala Arg Ala Thr Cys Gln
Pro Arg Glu Val Val Val Pro Leu Asn Met Glu Leu Met Gly Thr Val Ala Lys Gln
Leu Val Pro Ser Cys Val Thr Val Gln Arg Cys Gly Gly Cys Cys Pro Asp Asp Gly
Leu Glu Cys Val Pro Thr Gly Gln His Gln Val Arg Met Gln Ile Leu Met Ile。
Further, the present invention also provides the multinuclears for encoding above-mentioned light chain variable region and heavy chain variable amino acid sequence
Thuja acid.
Further, the present invention also provides the recombinant dna expression vector for including above-mentioned polynucleotides, the recombinant DNAs
Include coding VEGF-B monoclonal antibody heavies variable region, light chain variable region and the DNA sequences with antibody constant region in expression vector
Row.
Further, the present invention provides the detection kits for including above-mentioned VEGF-B monoclonal antibodies.The kit system
Preparation Method is：It is reaction support with Nunc chemiluminescence reaction plates, VEGF-B monoclonal antibodies is diluted to 0.5mPBS
2.5ug/ml is coated with the holes 100ul/ into chemiluminescence blank plate, at 4 DEG C after 24 hours, with 0.9%Nacl board-washings 3 times, is added
Enter the 0.5mol PBS closings containing 1%BSA, is dried after 12 hours at 4 DEG C.
The present invention has the following advantages：
(1) there is VEGF-B monoclonal antibodies of the invention higher affinity, high specificity to have in vitro good
Biological activity.
(2) include VEGF-B monoclonal antibodies detection kit detection method is easy, accuracy is strong, at low cost.
(3) by VEGFb167 and VEGF-B it is how anti-establish VEGF-B protein detection kits can be used as diagnosis for liver cancer and
Clinical monitoring tool.
Description of the drawings
Fig. 1 is VEGFa167 antibody dosages-reaction system schematic diagram.
Fig. 2 is VEGFa167 antibody quality controlled serums detection scatter plot.
Specific implementation mode
It is better understood the present invention by means of following specific implementation modes, however, these specific implementation modes are only used for
It illustrates the present invention, is not necessarily to be construed as limitation of the present invention.
Embodiment 1
One, the preparation of VEGF-B antigens：
(1) according to the sequence design pair of primers of people VEGF-B (U52819.1) in GenBank databases, sense primer is
vP：5 '-CATGAGCCCTCTGCTCCGCCGCCTGCT-3 ' introduce Xho I restriction enzyme sites；Downstream primer is vP：5'-
CCCAGTGGGGGAACAAAGAGGAG-3 ' introduces EcoR I restriction enzyme sites, is extracted from human liver cell by RT-PCR method
Total serum IgE, then reverse transcription is at cDNA, and using cDNA as template, VEGF-B gene specifics are carried out with Pyrobest archaeal dna polymerases
Amplification.PCR product after amplification carries out gel recycling, VEGF-B genes and the pPIC9K plasmids of recycling carry out respectively Xho I and
EcoR I digestions, the target fragment after the connection recycling of T4 ligases, connection product convert DH5 α competent cells, pass through ammonia benzyl
(1mg/mL) antibody screen selects positive colony, and body takes plasmid and identified through digestion and sequencing.
(2) Pichia pastoris GS115 of the pPIC9K-VEGF-B expression vectors through Stu I linearisations, electrotransformation to competence
(his4), the transformant of MD plate screening height copy of the coating containing G418, waits for that bacterium colony is grown, selects monoclonal bacterial strain and carry out PCR
PCR results are positive clone with plasmid by identification
PPIC9-Kal/GS115 (His4) conversions are to Pichia pastoris, and high-density cells 5G4 is sent out in 7.5L culture tanks
Ferment, with the formaldehyde inducement VEGF expression-B of 1%-2%.Fermented supernatant fluid is through Phenyl Sephadex G-25 Gel filtrations
Analysis, Heparin Sepharose FF heparin affinity chromatographies, Sephacryl S-100 gel permeation chromatographies obtain sufficient people
VEGF-B albumen, expression 50mg/L carry out SDS-PAGE electroresis appraisals, VEGF-B purity of protein 98% after purification.
Two, mostly anti-VEGF-B preparation and label
(1) it uses male New Zealand rabbit as immune animal, first uses 10mg BCG vaccines injection stimulation animal, start after a week
It is immune.Using VEGF-B antigens as immunogene；Immune pattern is subcutaneously injected using foot injection and back 4-6 points, and assistant is immunized
Agent uses Freund's complete adjuvant and incomplete Freund's adjuvant, and animal is immunized in four times, takes VEGF-B antigen 1s mg by equivalent every time
Freund's complete adjuvant and antigenic solution suck respectively it is 30-60 minutes fully emulsified in two syringes after metapedes be subcutaneously injected, often
Minor tick two weeks.Ear vein blood examination is taken to survey potency, CLIA reaches 1:50000 carry out strength arterial blood letting, centrifuging and taking serum.It utilizes
DEAE ion-exchange purifications, gained antibody-solutions are up to 98% or more.
(2) how anti-VEGF-B is dilutes 1mg/ml with 0.5mol PBSPH7.5, configures EZ-LinkTM
21338 biotin reagent 10mM of Sulfo-NHS-LC-LC-Biotin article No.s marks more anti-and biotins, molar ratio
1:20；Room temperature reaction 1 hour, PD10 desalting columns cross column, collect and preserve.
Three, the preparation of VEGF-B monoclonal antibodies
(1) mouse immune：The 5G4 cells of expression are stablized in culture, are resuspended with PBS (pH7.4) after centrifugation, and 3 females are immunized
BALA/c mouse, every BALB/c mouse are subcutaneously injected 5 × 106Cell, continuous 3 times, every minor tick 2 weeks, after the 4th is immune
7d detects antiserum titre with CLIA methods, takes the highest mouse of potency, intrasplenic injection 1 × 105A cell booster immunization, after 3d
Mouse spleen is taken, is ground, and splenocyte is counted for use.
(2) cell fusion and Antibody preparation：Extracting spleen cell presses cell count 6 with bone marrow cell Sp2/0:1 ratio fusion,
PEG1200 is induced, and the 96 orifice plate cultures that the feeder layer completed is added to after fusion change liquid, CLIA with half amount of HAT culture mediums after a week
Method screens positive cell strain.Positive hybridoma cell strain is screened with indirect CLIA methods.After 3 effective dilution method clonings,
Select I plant 98% or more VEGF-BAb continuous releases positive rates hybridoma 3A9 and expand culture prepare be injected intraperitoneally it is small
Mouse.I weeks before inoculation hybridoma, mouse peritoneal injects 500 μ L atoleines.Hybridoma is collected by centrifugation when inoculation, uses
Endless full nutrient solution suspends and mixing, and cell number is adjusted to 1 × 109/ L, every mouse peritoneal inject 500 μ L, mouse abdomen after 1 week
Portion's obvious tumefaction after sterilizing lower abdomen skin, extracts ascites, and gained ascites 3000r/min collects supernatant.
(3) GE company proteinG are selected to purify column purification ascites；Purification column is balanced with 20mMPB buffer solutions, ascites is added
Loading is eluted with PH2.70.1mol glycine HCI buffers, is received with the EP pipes containing PH8.71moltris buffer solutions
Collection, 0.05mMPB dialysis, concentration freeze.
Four, monoclonal antibody application screening
(1) monoclonal antibody that coating preceding method prepares dilutes antibody to 1-5ug/ml with 0.5mPBS.100ul is every
Hole is coated with into chemiluminescence blank plate, and after 4 DEG C are stayed overnight, 0.9%Nacl is washed 3 times, is added and is sealed per hole 150ul containing 1%BSA
Close, 4 DEG C overnight after dry it is spare.
(2) optimal monoclonal antibody is screened：Dilution prepare antigen dilute in proportion 8 gradients (0,10,50,100,200,400,
1000,2000pg/m), sequentially add in the aforementioned Chemiluminescent plate prepared, per hole 50ul, add biotinylation it is mostly anti-and
It is marked with the Streptavidin of horseradish peroxidase, 37 DEG C incubate 1h, and PBST is washed 5 times, and Chemoluminescent substrate is added, keeps away
Light reaction detects luminous value；The linearly dependent coefficient R values for comparing calibration object under different monoclonal antibody coating Chemiluminescent plates, with VEGF-B
A concentration of abscissa X values, RLU values are ordinate Y value.Final to select with good linear, the best monoclonal of evaluation index is anti-
Body VEGFb167.The grand antibody of monoclonal antibody should meet S0RLU values < 100 simultaneously as the evaluation criterion of sandwich method CLIA detection methods,
S5/S0 (P/N) is maximum, and 30 quality controlled serum recall rates are more than the conditions such as 90%.
VEGFa167 antibody dosages-reaction system schematic diagram is as shown in Figure 1.
It is as shown in Figure 2 that VEGFa167 antibody quality controlled serums detect scatter plot.
Five, monoclonal antibody gene is transferred and is prepared in hybridoma
(1) Trizol reagents are used to extract 5 × 106Hybridoma 3A9 total serum IgE, then using OligodT as primer, AMV
37 DEG C of 15min of reverse transcriptase reverse transcription temperature, synthesis obtain cDNA.To synthesize cDNA as template, primer for RNA sequence and
Gene-specific primer GSP carries out nested PCR amplification, and the condition of PCR is：95 DEG C of denaturation 5min, 94 DEG C of 30s, 55 DEG C of 30s,
72 DEG C of 1min, totally 35 cycles, finally extend 10min then at 72 DEG C.
(2) PCR product is identified through 1% agarose gel electrophoresis, cuts and recycle purpose gel-tape, transfers purpose
Gene is connected in pGEM-T carriers, and the positive recombinant plasmid of EcoRI digestions identification, the measurement for carrying out DNA sequence dna obtains respectively
VL and VH gene orders.
VH (weight chain variabl area sequence)
VL (light-chain variable sequence)
Heavy chain and chain variable region gene sequence will be obtained and the constant region of antibody carries out codon optimization and synthesizes together,
The light chain of acquisition and heavy chain gene are cloned into pMD18-T expression by the heavy chain and light chain for obtaining complete antibody gene respectively respectively
In carrier.Plasmid extracts the amount of 100-150ug respectively, and removes endotoxin (< 1EU/mg), by 2 plasmids 1 of acquisition:1 corotation
In the competence TG1 cells for contaminating the suspension of 50ml volumes, transfection reagent uses PEI, and the ratio of plasmid and PEI are 1:2.Transfection
After 3-7 days, cell conditioned medium is collected, whether SDS-PAGE detection antibody correctly expresses, and ProteinA column purifications are closed after concentration
The VEGF-B antibody VEGFb167 of lattice, purity are more than 98%.
Six, VEGF-B detects the preparation of CLIA kits
(1) monoclonal antibody is coated with：It is reaction support, the optimal monoclonal antibody that will be filtered out with Nunc chemiluminescence reaction plates
VEGFb167 is diluted to 2.5ug/ml with 0.5mPBS.Be coated with into chemiluminescence blank plate with the holes 100ul/, 4 degree 24 hours
Afterwards, with the holes 0.9%Nacl 300ul/ board-washing 3 times, the 0.5mol PBS/ hole 150ul closings containing 1%BSA are added, it is 4 degree 12 small
When dry it is spare.
(2) it is loaded：Dilution prepare VEGF-B antigens dilute in proportion 6 gradients (0,10,50,100,200,400,
800pg/m) and test serum sample, each holes 50ul/ are added in the coating plate of coating VEGFb167, and the holes 50ul/ add biology
Elementization VEGF-B it is mostly anti-and be marked with the Streptavidin of horseradish peroxidase (0.5mPBS, 1:10000 dilutions), 37 DEG C of temperature
1h is educated, PBST is washed 5 times, removes anti-and Streptavidin-horseradish peroxidase more than unbonded VEGF.
(3) luminol-holes peroxide chemical luminous substrate 50ul/ are added, utilize TZD-CL-200S chemiluminescence immunoassays
Analyzer detects chemiluminescence intensity (RLU).
Seven, the performance of VEGF-B detection kits
(1) analytical performance of VEGF-B detection kits
Repeatability：The variation within batch coefficient (CV) of VEGF-B detection kits is not more than 10%；Interassay coefficient of variation (CV)
No more than 15%；
Sensitivity for analysis：Kit minimum detectability is not more than 30pg/mL；
Analysis specificity：A certain concentration specificity substance (FGF2, EGF) testing result is not more than 50pg/mL；
The range of linearity：In 0-800pg/mL concentration ranges, linearly dependent coefficient r is not less than 0.9900.
(2) clinical performance of VEGF-B detection kits
Carry out 155 clinical sample detections, 83 normal persons, 72 liver cancer patients.Kit normal reference value 0-
115pg/ml, kit testing result are as follows：
Coincidence rate and its confidence interval
Crosstab
It counts
Coincidence rate and its confidence interval
Symmetry magnitude
A. without assuming empty hypothesis.
B. assume empty hypothesis using asymptotic standard error.
Consistency coefficient Kappa (K) value is：0.986。
Serum in patients with primary hepatic VEGF-B levels increase, and detection liver cancer positive coincidence rate is combined up to 98.61%
VEGF-A detections can be as the effective means of diagnosis for liver cancer and clinical monitoring.
SEQUENCE LISTING
<110>The Zhejiang bio tech ltd Zhong Yi
<120>A kind of VEGF-B monoclonal antibodies and kit
<130>
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 167
<212> PRT
<213>VEGF-B monoclonal antibody heavies variable region
<400> 1
Pro Val Ser Gln Pro Asp Ala Pro Gly His Gln Arg Lys Val Val Ser
1 5 10 15
Trp Ile Asp Val Tyr Thr Arg Ala Thr Cys Gln Pro Arg Glu Val Val
20 25 30
Val Pro Leu Thr Val Glu Leu Met Gly Thr Val Ala Lys Gln Leu Val
35 40 45
Pro Ser Cys Val Thr Val Gln Arg Cys Gly Gly Cys Cys Pro Asp Asp
50 55 60
Gly Leu Glu Cys Val Pro Thr Gly Gln His Gln Val Arg Met Gln Ile
65 70 75 80
Leu Met Ile Arg Tyr Pro Ser Ser Gln Leu Gly Glu Met Ser Leu Glu
85 90 95
Glu His Ser Gln Cys Glu Cys Arg Pro Lys Lys Lys Asp Ser Ala Val
100 105 110
Lys Pro Asp Ser Pro Arg Pro Leu Cys Pro Arg Cys Thr Gln His His
115 120 125
Gln Arg Pro Asp Pro Arg Thr Cys Arg Cys Arg Cys Arg Arg Arg Ser
130 135 140
Phe Leu Arg Cys Gln Gly Arg Gly Leu Glu Leu Asn Pro Asp Thr Cys
145 150 155 160
Arg Cys Arg Lys Leu Arg Arg
165
<210> 2
<211> 67
<212> PRT
<213>VEGF-B monoclonal antibody light chain variable regions
<400> 2
Trp Ile Asp Val Tyr Ala Arg Ala Thr Cys Gln Pro Arg Glu Val Val
1 5 10 15
Val Pro Leu Asn Met Glu Leu Met Gly Thr Val Ala Lys Gln Leu Val
20 25 30
Pro Ser Cys Val Thr Val Gln Arg Cys Gly Gly Cys Cys Pro Asp Asp
35 40 45
Gly Leu Glu Cys Val Pro Thr Gly Gln His Gln Val Arg Met Gln Ile
50 55 60
Leu Met Ile
65
<210> 3
<211> 27
<212> DNA
<213>Artificial sequence
<400> 3
catgagccct ctgctccgcc gcctgct 27
<210> 4
<211> 23
<212> DNA
<213>Artificial sequence
<400> 4
cccagtgggg gaacaaagag gag 23
Claims (5)
1. a kind of VEGF-B monoclonal antibodies, which is characterized in that the heavy chain variable amino acid sequence of the antibody is：Pro
Val Ser Gln Pro Asp Ala Pro Gly His Gln Arg Lys Val Val Ser Trp Ile Asp Val
Tyr Thr Arg Ala Thr Cys Gln Pro Arg Glu Val Val Val Pro Leu Thr Val Glu Leu
Met Gly Thr Val Ala Lys Gln Leu Val Pro Ser Cys Val Thr Val Gln Arg Cys Gly
Gly Cys Cys Pro Asp Asp Gly Leu Glu Cys Val Pro Thr Gly Gln His Gln Val Arg
Met Gln Ile Leu Met Ile Arg Tyr Pro Ser Ser Gln Leu Gly Glu Met Ser Leu Glu
Glu His Ser Gln Cys Glu Cys Arg Pro Lys Lys Lys Asp Ser Ala Val Lys Pro Asp
Ser Pro Arg Pro Leu Cys Pro Arg Cys Thr Gln His His Gln Arg Pro Asp Pro Arg
Thr Cys Arg Cys Arg Cys Arg Arg Arg Ser Phe Leu Arg Cys Gln Gly Arg Gly Leu
Glu Leu Asn Pro Asp Thr Cys Arg Cys Arg Lys Leu Arg Arg；
Chain variable region amino acid sequence is：Trp Ile Asp Val Tyr Ala Arg Ala Thr Cys Gln Pro
Arg Glu Val Val Val Pro Leu Asn Met Glu Leu Met Gly Thr Val Ala Lys Gln Leu
Val Pro Ser Cys Val Thr Val Gln Arg Cys Gly Gly Cys Cys Pro Asp Asp Gly Leu
Glu Cys Val Pro Thr Gly Gln His Gln Val Arg Met Gln Ile Leu Met Ile。
2. a kind of polynucleotides of coding light chain variable region and heavy chain variable amino acid sequence described in claim 1.
3. a kind of recombinant dna expression vector including polynucleotides as claimed in claim 2, the recombinant dna expression vector
In include coding VEGF-B monoclonal antibody heavies variable region, light chain variable region and the DNA sequence dna with antibody constant region.
4. a kind of detection kit including VEGF-B monoclonal antibodies described in claim 1.
5. the preparation method of detection kit described in claim 4, which is characterized in that with Nunc chemiluminescence reaction plates be reaction
VEGF-B monoclonal antibodies are diluted to 2.5ug/ml with 0.5mPBS, are coated with to chemiluminescence blank with the holes 100ul/ by support
In plate, at 4 DEG C after 24 hours, with 0.9%Nacl board-washings 3 times, the 0.5mol PBS closings containing 1%BSA are added, 12 at 4 DEG C
It is dried after hour.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810683274.3A CN108774287A (en) | 2018-06-28 | 2018-06-28 | A kind of VEGF-B monoclonal antibodies and kit |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810683274.3A CN108774287A (en) | 2018-06-28 | 2018-06-28 | A kind of VEGF-B monoclonal antibodies and kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108774287A true CN108774287A (en) | 2018-11-09 |
Family
ID=64030464
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|---|---|---|---|
| CN201810683274.3A Withdrawn CN108774287A (en) | 2018-06-28 | 2018-06-28 | A kind of VEGF-B monoclonal antibodies and kit |
Country Status (1)
| Country | Link |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1441849A (en) * | 2000-05-17 | 2003-09-10 | 路德维格癌症研究所 | Methods for detecting for presence of tumor cells and for screening for anti-tumor agents |
| CN107537026A (en) * | 2017-08-31 | 2018-01-05 | 中山大学中山眼科中心 | VEGF B application |
-
2018
- 2018-06-28 CN CN201810683274.3A patent/CN108774287A/en not_active Withdrawn
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1441849A (en) * | 2000-05-17 | 2003-09-10 | 路德维格癌症研究所 | Methods for detecting for presence of tumor cells and for screening for anti-tumor agents |
| CN107537026A (en) * | 2017-08-31 | 2018-01-05 | 中山大学中山眼科中心 | VEGF B application |
Non-Patent Citations (3)
| Title |
|---|
| LEONARD ET AL.: "ACCESSION：2VWE_B，Chain B, Crystal Structure Of Vascular Endothelial Growth Factor-B In Complex With A Neutralizing Antibody Fab Fragment", 《GENBANK》 * |
| MANDRIOTA ET AL.: "ACCESSION：AAG29746，Vascular endothelial growth factor-B, partial [Bos taurus]", 《GENBANK》 * |
| PHILIP LEONARD ET AL.: "Crystal Structure of Vascular EndothelialGrowth Factor-B in Complex with a Neutralising Antibody Fab Fragment", 《J.MOL.BIOL.》 * |
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Application publication date: 20181109 |