CN108997501A - Anti- VWF protein monoclonal antibody and application thereof - Google Patents
Anti- VWF protein monoclonal antibody and application thereof Download PDFInfo
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Abstract
The present invention relates to field of biotechnology, disclose a kind of hybridoma cell strain (deposit number is CGMCC No.15594), and the monoclonal antibody OTI9F3 that thus hybridoma cell strain generates.The invention further relates to monoclonal antibody OTI9F3 to prepare the application in the immune detection tool for detecting VWF albumen, application of the immunohistochemical kit and monoclonal antibody OTI9F3 of the OTI9F3 containing monoclonal antibody in the kit that preparation is used for marked tumor.Monoclonal antibody of the present invention can in conjunction with VWF protein-specific, and with other intracellular albumen no cross reactions, significantly improve specificity, the accuracy and reliability of the detection of VWF protein immunization.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to can specific bond VWF albumen monoclonal antibody OTI9F3, produce
The cell strain and the diagnostic method of the application antibody and purposes of the raw monoclonal antibody.
Background technique
VWF, that is, vWF ELISA is a kind of poly plasma glycoprotein, can combine platelet glycoprotein Ib, sugar
Protein I Ib/IIIa, collagen and heparin, adherency of the mediating platelet to vascular lesions sites.VWF is mainly by endothelial cell
It is synthesized with megacaryocyte, is present in blood plasma, in the stick tubulose corpusculum in blood platelet in α-particle and endothelial cell.VWF is for only
Blood is extremely important with thrombosis, and the genetic defect of VWF structure and modification will lead to von Willebrand disease (VWD), this is human body
In the most common congenital hemorrhagic disease.In other words VWF level raising show it is related with acute coronary thrombosis, can
A clinical risk marker as atherosclerosis.
VWF is one of identification tumor endothelial cell (or megacaryocyte) the most useful marker.Studies have shown that VWF can make
It plays a role in inflammation for the site of sticking of many WBC sub-populations.Immunohistochemistry (IHC) disease is clinically commonly used at present
The expression situation of albumen in reason experiment detection tumour cell, however the core of IHC experiment is the monoclonal of binding proteins specific
Antibody, the superiority and inferiority of performance directly decide the sensitivity and specificity entirely detected.Therefore, a kind of binding specificity is developed
The higher monoclonal antibody for VWF albumen has great importance to IHC detection VWF expression.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of monoclonal antibody of the higher VWF albumen of binding specificity,
And its preparing the application in the immune detection tool for detecting VWF albumen.
The present invention provides a kind of hybridoma cell strains, and it is commonly micro- to be preserved in China Committee for Culture Collection of Microorganisms
Bio-Centers (referred to as CGMCC), the deposit date is on April 26th, 2018, deposit number was CGMCC No.15594.
It is thin by above-mentioned hybridoma the present invention also provides a kind of monoclonal antibody OTI9F3 for specifically binding VWF albumen
Born of the same parents' strain generates.
Monoclonal antibody of the present invention the preparation method is as follows:
(1) building of recombinant expression carrier: according to VWF ORF nucleotide sequence (VWF ORF nucleotide sequence such as SEQ ID
Shown in NO.1,8442bp；VWF amino acid sequence is as shown in SEQ ID NO.2)
Design primer PCR amplification VWF ORF 2292bp to 3789bp bit sequence, gene two sides introduce limitation respectively
Property restriction enzyme site SgfI and MluI, be inserted into expression vector pFASTbac-C-DDK, construct VWF recombinant expression plasmid
pFASTbac-DDK-rVWF。
(2) expression and purification of VWF recombinant protein: VWF recombinant expression plasmid is transfected into SF9 cell, in the culture of acquisition
Reset and add into SF9 cell, then cracking centrifugation obtains supernatant, and DDK affinity chromatography column purification obtains the VWF recombinant protein of purifying.
(3) screening and preparation of monoclonal antibody: Balb/c mouse is immunized using the VWF recombinant protein of above-mentioned purifying, is taken
Mouse spleen cells are merged with SP2/0 cell, and limiting dilution assay obtains monoclonal, and it is thin that ELISA method screens positive hybridoma
Born of the same parents obtain the hybridoma cell strain that can secrete anti-VWF specific antibody, are named as OTI9F3, subtype identification IgG2b；Pass through
It collects ascites and prepares antibody, VWF monoclonal antibody OTI9F3 is obtained by affinity chromatography column purification.Pass through Western respectively
Blot, immunohistochemical experiment verify the sensitivity and specificity of the monoclonal antibody.
The present invention also provides monoclonal antibody OTI9F3 in preparing the immune detection tool for detecting VWF albumen
Using.
Specifically, the immune detection tool is kit, chip or test paper.
In the particular embodiment, the present invention provides a kind of immunologic combined detection reagent kit, including above-mentioned monoclonal are anti-
Body OTI9F3 can detect the expression situation of VWF in histocyte.
The present invention also provides application of the said monoclonal antibody in the kit that preparation is used for marked tumor.Wherein institute
State the tumour being proliferated and the expression of VWF is closely related that tumour specifically refers to tumour cell, including but not limited to colon cancer, forefront
The associated tumor tissues such as gland cancer and breast cancer.
Compared with prior art, the present invention provides a kind of hybridoma cell strain (deposit number CGMCC
No.15594), and the thus monoclonal antibody OTI9F3 that hybridoma cell strain generates.The present invention also provides monoclonal antibodies
OTI9F3 is preparing the application in the immune detection tool for detecting VWF albumen, the immune group of the OTI9F3 containing monoclonal antibody
Change the application of kit and monoclonal antibody OTI9F3 in the kit that preparation is used for marked tumor.List of the present invention
Clonal antibody can in conjunction with VWF protein-specific, and with other intracellular albumen no cross reactions, significantly improve VWF albumen
Specificity, the accuracy and reliability of immune detection, it is true to reflect VWF protein expression level in tumour cell, it can be applied to tie
The expression of VWF in the associated tumor tissues such as intestinal cancer, prostate cancer and breast cancer.
Preservation information
The classification naming of hybridoma cell strain OTI9F3 for preservation are as follows: the hybridoma of VWF mouse monoclonal antibody
Strain；
Depositary institution's full name: China Committee for Culture Collection of Microorganisms's common micro-organisms center；
Depositary institution's abbreviation: CGMCC；
Depositary institution address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica；
Preservation date: on April 26th, 2018；
Deposit number: CGMCC No.15594.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described.
Fig. 1 shows the design of 1 cloning site of embodiment as schemed, and wherein shading part is the area ORF；
Fig. 2 shows 2 Restructure VWF albumen Western blot testing result figure of embodiment, with anti-DDK antibody test recombination
Expression of the VWF albumen in SF9 lysate, swimming lane are that the SF9 cell pyrolysis liquid of conversion pFASTbac-DDK-rVWF plasmid is anti-
Former testing result；
Fig. 3 shows 2 Restructure VWF protein SDS-PAGE result figure of embodiment, with anti-DDK affinity chromatography column purification Restructure VWF egg
White, albumen after purification passes through SDS-PAGE glue electricity arteries and veins, coomassie brilliant blue staining；
Fig. 4 shows that embodiment 3 identifies complete VWF (Full length VWF, VWF-FL) egg with monoclonal antibody OTI9F3
White Western blot testing result figure.Left side swimming lane is the cell pyrolysis liquid for transfecting pCMV6-Entry；Right lanes are to turn
Contaminate the cell pyrolysis liquid of pCMV6-VWF-FL；
Fig. 5 shows that 4 formalin of embodiment is fixed, (primary antibody is that VWF is mono- to the human pancreas cancer ImmunohistochemistryResults Results figure of paraffin embedding
Clonal antibody OTI9F3)；
Fig. 6 shows that 4 formalin of embodiment is fixed, (primary antibody is VWF Dan Ke to the human bladder ImmunohistochemistryResults Results figure of paraffin embedding
Grand antibody OTI9F3)；
Fig. 7 shows that 4 formalin of embodiment is fixed, (primary antibody is that VWF is mono- to human tonsil's ImmunohistochemistryResults Results figure of paraffin embedding
Clonal antibody OTI9F3)；
Specific embodiment
Below in conjunction with the embodiment of the present invention, technical scheme in the embodiment of the invention is clearly and completely described,
Obviously, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based in the present invention
Embodiment, every other embodiment obtained by those of ordinary skill in the art without making creative efforts, all
Belong to the scope of protection of the invention.
The building of embodiment 1, VWF recombinant expression plasmid
It is with the plasmid RC218497 (ORF8442bp containing VWF) obtained from Biotechnology Co., Ltd of Aureal Dongyuan County of the U.S.
Template, design primer simultaneously introduce restriction enzyme site SgfI and MluI respectively, are cloned into expression vector pFASTbac-C-DDK, establish
VWF recombinant expression plasmid.Cloning site design is as shown in Figure 1.
The expression and purification of embodiment 2, VWF recombinant protein
1, DH10BAC is convertedTME.coli cell: it is light that Plasmid DNA is added after 100ul competent cell is placed on ice to melt
It mixes, then 42 DEG C of heat shock 90s after ice bath 30min are continued ice bath 1-2min.The fresh nothing of 900ul is added in super-clean bench
Anti- LB culture medium takes appropriate bacterium solution to be spread evenly across on antibiotic LB plate, will cultivate after 37 DEG C of shaking tables are incubated for 4 hours
Ware is inverted in overnight incubation in 37 DEG C of constant incubators.Conservation extracts recombinant plasmid.
2, transfect SF9 cell: after the SF9 cell count of culture plus suitable cell is into 6 orifice plates；Take 100 μ L
Sf900TM-III SFM (serum-free and antibiotic) is added 8 μ LPEI MegaTran1.0 and mixes into small centrifuge tube；Dissolve 5 μ
The recombinant plasmid of l purifying is in 100 μ l Sf900TM-III SFM culture mediums；The plasmid diluted and the transfection agents diluted are mixed
It is even, it is incubated at room temperature 20 minutes；Recombinant plasmid and transfection agents mixed liquor add in cell, mix gently.27 DEG C of wet box are incubated for, until
Lesion phenomenon generates；Centrifugation is collected supernatant and is saved, and cell precipitation adds lysis buffer to crack, using the table of WB identification Restructure VWF
It reaches, result figure 2.
3, lytic cell: the supernatant that appropriate transfection process is collected is added SF9 cell, 27 DEG C incubated cell 48-96 hours；
Cell suspension is collected, 600rpm is centrifuged 5min, and supernatant is kept in dark place, and cell precipitation adds appropriate lysis buffer (to use preceding addition
Protease inhibitors PI and PMSF) it is resuspended；Ultrasonication, 200HZ are crushed 3s, interval 3s, 10 circulations；4 DEG C, 15000rpm,
It is centrifuged 30min, 0.45 μm of filter filters off precipitating, obtains lysate.
4, DDK affinity chromatography column purification: with 0.45 μM, the lysate supernatant after 33mm pvdf membrane filter filter centrifugation is simultaneously
It is transferred to 15mL pipe, the Beads 1mL mixed is added, is put into after sealing in 360 degree of vortex mixers, is combined 2 hours in 4 DEG C；It takes out
15mL pipe, lysate is poured into BIO-RAD chromatographic column, and catch and penetrate liquid, and liquid sampling WB detection is penetrated after drop is most；With cracking
Buffer rinses column material 1-2 times, is rinsed Beads 3 times with TBST after drop is most, is washed after dripping to the greatest extent with 0.1M Glycine pH3.5 again
De-, 200 μ L, drop are not collected to the greatest extent for the first time, second and third each 500 μ L, the 4th 250 μ L are collected to a 1.5mL centrifuge tube
In, and it is rapidly added NaH2PO4(pH=11.0) it is neutralized to pH7.0 or so, glycerol is added to final concentration of 10% in every pipe,
Tween-80 to final concentration of 0.1%.Restructure VWF albumen after purification is identified with SDS-PAGE, sees Fig. 3.
By Fig. 2 result as it can be seen that transfection pFASTbac-DDK-rVWF plasmid SF9 cell pyrolysis liquid in WB detection after
There is apparent band at 60kD, molecular weight is consistent with expected molecular size range.Show Restructure VWF protein expression in cell.
By Fig. 3 result as it can be seen that the albumen of purifying has an apparent specific band in SDS-PAGE Jiao66kDChu, molecular weight and pre-
Phase molecular size range is consistent.Show to have obtained the preferable VWF recombinant protein of purity.
The preparation and screening of embodiment 3, VWF monoclonal antibody
According to the VWF protein fragments of the standard method purifying of recombination generation, to Balb/c mouse, (it is real that tonneau China is tieed up in Beijing
Test zoo technical Co., Ltd) it is immunized.The specific method is as follows:
1, animal immune: purified VWF antigen is emulsified with complete Freund's adjuvant, and 6- is immunized using subcutaneous injection method
8 week old Balb/c mouse, immunizing dose are 30 μ g/, and progress is immune for the second time after two weeks at interval, newborn with incomplete Freund's adjuvant
Change, immunizing dose is 20 μ g/.It is immune that tail blood is taken to measure serum titer with ELISA method gradient dilution afterwards twice；It is true according to result
It is fixed whether booster immunization, choose the highest mouse of antibody titer and carry out cell fusion.
2, cell fusion: myeloma cell uses the sp2/0 in the source Balb/c, and logarithmic growth phase is in when fusion；It takes
Immune mouse spleen, is made lymphocyte single cell suspension；Myeloma cell is mixed with mouse spleen lymphocyte with 1:5-1:10,
37 DEG C 50%PEG (PH 8.0) 1mL is added dropwise, incomplete culture medium terminate liquid and remaining terminate liquid is added, after supernatant is abandoned in centrifugation
HAT culture medium be added suspend and mix, MC constant volume to 50mL is dispensed into 3.5cm culture dish, is put in wet box, be placed in 37 DEG C,
5%CO2It is cultivated in constant incubator.
3, screen and clone: fusion selected cell clone in 7-10 days, carried out ELISA survey using VWF purification of recombinant proteins
Examination.Mark cell strain number.Limiting dilution, 5-6 days measurement ELISA values after each limiting dilution, picking are carried out to positive hole cell
The OD280 positive is worth higher monoclonal hole and carries out limiting dilution, until ELISA measures 96 orifice plates, hardened fruit is the positive entirely.Picking
The positive is worth high monoclonal singling.It is OTI9F3 that it, which corresponds to fusion plate cell strain,.
4, the preparation and purification of ascites monoclonal antibodies: the male Balb/c mouse peritoneal of 10-12 week old injects 0.5ml norphytane,
Every mouse is injected intraperitoneally with 1mL syringe and wash the monoclonal cell suspension being resuspended through PBS after a week, cell dosage for 5 ×
106/ only, every strain antibody makes a call to 2 mouse.Ascites, centrifuging and taking supernatant are collected after mouse ascites accumulation, affinity chromatography carries out abdomen
Water purifying selects corresponding column material according to antibody subtype, and the monoclonal antibody that cell strain OTI9F3 is generated is IgG2b, using protein G into
Row purifying.Monoclonal antibody concentration mensuration after purification, WB are detected, dispense, are frozen at -20 DEG C.Wherein WB testing result is shown in Fig. 4.
By Fig. 4 result as it can be seen that OTI9F3 can be good at identifying the VWF full-length proteins that 293T cell is overexpressed, molecular weight
It is consistent with expected molecular size range, show that monoclonal antibody OTI9F3 specifically can detect complete VWF egg by Westernblot
It is white.
Embodiment 4, the immunohistochemistry that monoclonal antibody OTI9F3 is primary antibody detect
(1), experimental method:
1, human pancreas cancer, human bladder, the human tonsil's tissue for taking formalin fixed carry out paraffin embedding, use
Finesse histotome is sliced, and tissue thickness is 6 μm.
2, dewaxing and aquation: analyzing pure 3 × 10min of dimethylbenzene, dehydrated alcohol 3 × 10min, 95% ethyl alcohol 5min, and 85%
Ethyl alcohol 5min, 75% ethyl alcohol 5min, deionized water impregnate 3min × 3 time
3, antigen retrieval buffers (0.01M, pH6.0 sodium citrate buffer) pressure cooker hot high pressure is added and repairs 3min, to height
When pressure pot temperature is down to about 90 DEG C, pressure cooker is opened, takes out sample, then cooled to room temperature.Deionized water impregnates 3min
× 3 times.
4, using 3% hydrogen peroxide deactivation tissue endogenous peroxydase, it is stored at room temperature 10min.Deionized water is impregnated
5min × 3 time.
5, confining liquid (+5% Normal Goat Serum of PBS+5% skimmed milk power) is added, 37 DEG C of incubation 60min.
6, confining liquid is removed, is not rinsed, is added VWF monoclonal antibody (OTI9F3), thinner ratio: 1:150 is carried out dilute using confining liquid
It releases.It is placed in wet box, 37 DEG C of incubation 60min.PBST (0.1%Tween-20) is washed 2 times, washs 5min every time.PBST
(0.02%Tween-20) is washed 1 time, washs 5min every time.
7,1,37 DEG C of reagent of Polink- kit 2 (Catlog No.D37-15) incubation 10-20 minutes is added dropwise.Use PBS
Washing 3 times, each 5min.2,37 DEG C of reagent of Polink-2 kit (Catlog No.D37-15) incubation 10-20 minutes is added dropwise,
It is washed 3 times using PBS, each 5min.
8, it develops the color using DAB solution (Zhong Shan Golden Bridge ZLI-9019), develop the color 3~10min.Distill water washing.
9, haematoxylin redyeing nucleus 2min, distilled water rinsing, the differentiation of 1% hydrochloric acid.Distilled water rinses 3 times, is stored at room temperature
1min。
10, it is dehydrated and transparent: 75% ethyl alcohol 5min, 100% ethyl alcohol 5min x 3 times, 85% ethyl alcohol 5min, 95% ethyl alcohol
5min, 100% 3 × 5min of ethyl alcohol；3 × 5min of dimethylbenzene, neutral gum mounting.
11, microscopy is shown in Fig. 5-7.
(2), experimental result:
By Fig. 5-7 result as it can be seen that human pancreas cancer, human bladder, human tonsil tissue in the visible extracellular dyeing of specificity.Knot
Fruit and VWF positioning in the cell and tissue expression specificity are consistent, show that monoclonal antibody OTI9F3 can be used for immuning tissue
The level of chemical detection VWF albumen.
<110>Wuxi Origene Bio-tech Co., Ltd.
<120>anti-VWF protein monoclonal antibody and application thereof
<210> 1
<211>8442
<212> DNA
<213>artificial sequence
<400> 1
ATGATTCCTGCCAGATTTGCCGGGGTGCTGCTTGCTCTGGCCCTCATTTTGCCAGGGACCCTTTGTGCAGAAG
GAACTCGCGGCAGGTCATCCACGGCCCGATGCAGCCTTTTCGGAAGTGACTTCGTCAACACCTTTGATGGGAGCATG
TACAGCTTTGCGGGATACTGCAGTTACCTCCTGGCAGGGGGCTGCCAGAAACGCTCCTTCTCGATTATTGGGGACTT
CCAGAATGGCAAGAGAGTGAGCCTCTCCGTGTATCTTGGGGAATTTTTTGACATCCATTTGTTTGTCAATGGTACCG
TGACACAGGGGGACCAAAGAGTCTCCATGCCCTATGCCTCCAAAGGGCTGTATCTAGAAACTGAGGCTGGGTACTAC
AAGCTGTCCGGTGAGGCCTATGGCTTTGTGGCCAGGATCGATGGCAGCGGCAACTTTCAAGTCCTGCTGTCAGACAG
ATACTTCAACAAGACCTGCGGGCTGTGTGGCAACTTTAACATCTTTGCTGAAGATGACTTTATGACCCAAGAAGGGA
CCTTGACCTCGGACCCTTATGACTTTGCCAACTCATGGGCTCTGAGCAGTGGAGAACAGTGGTGTGAACGGGCATCT
CCTCCCAGCAGCTCATGCAACATCTCCTCTGGGGAAATGCAGAAGGGCCTGTGGGAGCAGTGCCAGCTTCTGAAGAG
CACCTCGGTGTTTGCCCGCTGCCACCCTCTGGTGGACCCCGAGCCTTTTGTGGCCCTGTGTGAGAAGACTTTGTGTG
AGTGTGCTGGGGGGCTGGAGTGCGCCTGCCCTGCCCTCCTGGAGTACGCCCGGACCTGTGCCCAGGAGGGAATGGTG
CTGTACGGCTGGACCGACCACAGCGCGTGCAGCCCAGTGTGCCCTGCTGGTATGGAGTATAGGCAGTGTGTGTCCCC
TTGCGCCAGGACCTGCCAGAGCCTGCACATCAATGAAATGTGTCAGGAGCGATGCGTGGATGGCTGCAGCTGCCCTG
AGGGACAGCTCCTGGATGAAGGCCTCTGCGTGGAGAGCACCGAGTGTCCCTGCGTGCATTCCGGAAAGCGCTACCCT
CCCGGCACCTCCCTCTCTCGAGACTGCAACACCTGCATTTGCCGAAACAGCCAGTGGATCTGCAGCAATGAAGAATG
TCCAGGGGAGTGCCTTGTCACAGGTCAATCACACTTCAAGAGCTTTGACAACAGATACTTCACCTTCAGTGGGATCT
GCCAGTACCTGCTGGCCCGGGATTGCCAGGACCACTCCTTCTCCATTGTCATTGAGACTGTCCAGTGTGCTGATGAC
CGCGACGCTGTGTGCACCCGCTCCGTCACCGTCCGGCTGCCTGGCCTGCACAACAGCCTTGTGAAACTGAAGCATGG
GGCAGGAGTTGCCATGGATGGCCAGGACGTCCAGCTCCCCCTCCTGAAAGGTGACCTCCGCATCCAGCATACAGTGA
CGGCCTCCGTGCGCCTCAGCTACGGGGAGGACCTGCAGATGGACTGGGATGGCCGCGGGAGGCTGCTGGTGAAGCTG
TCCCCCGTCTATGCCGGGAAGACCTGCGGCCTGTGTGGGAATTACAATGGCAACCAGGGCGACGACTTCCTTACCCC
CTCTGGGCTGGCGGAGCCCCGGGTGGAGGACTTCGGGAACGCCTGGAAGCTGCACGGGGACTGCCAGGACCTGCAGA
AGCAGCACAGCGATCCCTGCGCCCTCAACCCGCGCATGACCAGGTTCTCCGAGGAGGCGTGCGCGGTCCTGACGTCC
CCCACATTCGAGGCCTGCCATCGTGCCGTCAGCCCGCTGCCCTACCTGCGGAACTGCCGCTACGACGTGTGCTCCTG
CTCGGACGGCCGCGAGTGCCTGTGCGGCGCCCTGGCCAGCTATGCCGCGGCCTGCGCGGGGAGAGGCGTGCGCGTCG
CGTGGCGCGAGCCAGGCCGCTGTGAGCTGAACTGCCCGAAAGGCCAGGTGTACCTGCAGTGCGGGACCCCCTGCAAC
CTGACCTGCCGCTCTCTCTCTTACCCGGATGAGGAATGCAATGAGGCCTGCCTGGAGGGCTGCTTCTGCCCCCCAGG
GCTCTACATGGATGAGAGGGGGGACTGCGTGCCCAAGGCCCAGTGCCCCTGTTACTATGACGGTGAGATCTTCCAGC
CAGAAGACATCTTCTCAGACCATCACACCATGTGCTACTGTGAGGATGGCTTCATGCACTGTACCATGAGTGGAGTC
CCCGGAAGCTTGCTGCCTGACGCTGTCCTCAGCAGTCCCCTGTCTCATCGCAGCAAAAGGAGCCTATCCTGTCGGCC
CCCCATGGTCAAGCTGGTGTGTCCCGCTGACAACCTGCGGGCTGAAGGGCTCGAGTGTACCAAAACGTGCCAGAACT
ATGACCTGGAGTGCATGAGCATGGGCTGTGTCTCTGGCTGCCTCTGCCCCCCGGGCATGGTCCGGCATGAGAACAGA
TGTGTGGCCCTGGAAAGGTGTCCCTGCTTCCATCAGGGCAAGGAGTATGCCCCTGGAGAAACAGTGAAGATTGGCTG
CAACACTTGTGTCTGTCGGGACCGGAAGTGGAACTGCACAGACCATGTGTGTGATGCCACGTGCTCCACGATCGGCA
TGGCCCACTACCTCACCTTCGACGGGCTCAAATACCTGTTCCCCGGGGAGTGCCAGTACGTTCTGGTGCAGGATTAC
TGCGGCAGTAACCCTGGGACCTTTCGGATCCTAGTGGGGAATAAGGGATGCAGCCACCCCTCAGTGAAATGCAAGAA
ACGGGTCACCATCCTGGTGGAGGGAGGAGAGATTGAGCTGTTTGACGGGGAGGTGAATGTGAAGAGGCCCATGAAGG
ATGAGACTCACTTTGAGGTGGTGGAGTCTGGCCGGTACATCATTCTGCTGCTGGGCAAAGCCCTCTCCGTGGTCTGG
GACCGCCACCTGAGCATCTCCGTGGTCCTGAAGCAGACATACCAGGAGAAAGTGTGTGGCCTGTGTGGGAATTTTGA
TGGCATCCAGAACAATGACCTCACCAGCAGCAACCTCCAAGTGGAGGAAGACCCTGTGGACTTTGGGAACTCCTGGA
AAGTGAGCTCGCAGTGTGCTGACACCAGAAAAGTGCCTCTGGACTCATCCCCTGCCACCTGCCATAACAACATCATG
AAGCAGACGATGGTGGATTCCTCCTGTAGAATCCTTACCAGTGACGTCTTCCAGGACTGCAACAAGCTGGTGGACCC
CGAGCCATATCTGGATGTCTGCATTTACGACACCTGCTCCTGTGAGTCCATTGGGGACTGCGCCTGCTTCTGCGACA
CCATTGCTGCCTATGCCCACGTGTGTGCCCAGCATGGCAAGGTGGTGACCTGGAGGACGGCCACATTGTGCCCCCAG
AGCTGCGAGGAGAGGAATCTCCGGGAGAACGGGTATGAGTGTGAGTGGCGCTATAACAGCTGTGCACCTGCCTGTCA
AGTCACGTGTCAGCACCCTGAGCCACTGGCCTGCCCTGTGCAGTGTGTGGAGGGCTGCCATGCCCACTGCCCTCCAG
GGAAAATCCTGGATGAGCTTTTGCAGACCTGCGTTGACCCTGAAGACTGTCCAGTGTGTGAGGTGGCTGGCCGGCGT
TTTGCCTCAGGAAAGAAAGTCACCTTGAATCCCAGTGACCCTGAGCACTGCCAGATTTGCCACTGTGATGTTGTCAA
CCTCACCTGTGAAGCCTGCCAGGAGCCGGGAGGCCTGGTGGTGCCTCCCACAGATGCCCCGGTGAGCCCCACCACTC
TGTATGTGGAGGACATCTCGGAACCGCCGTTGCACGATTTCTACTGCAGCAGGCTACTGGACCTGGTCTTCCTGCTG
GATGGCTCCTCCAGGCTGTCCGAGGCTGAGTTTGAAGTGCTGAAGGCCTTTGTGGTGGACATGATGGAGCGGCTGCG
CATCTCCCAGAAGTGGGTCCGCGTGGCCGTGGTGGAGTACCACGACGGCTCCCACGCCTACATCGGGCTCAAGGACC
GGAAGCGACCGTCAGAGCTGCGGCGCATTGCCAGCCAGGTGAAGTATGCGGGCAGCCAGGTGGCCTCCACCAGCGAG
GTCTTGAAATACACACTGTTCCAAATCTTCAGCAAGATCGACCGCCCTGAAGCCTCCCGCATCACCCTGCTCCTGAT
GGCCAGCCAGGAGCCCCAACGGATGTCCCGGAACTTTGTCCGCTACGTCCAGGGCCTGAAGAAGAAGAAGGTCATTG
TGATCCCGGTGGGCATTGGGCCCCATGCCAACCTCAAGCAGATCCGCCTCATCGAGAAGCAGGCCCCTGAGAACAAG
GCCTTCGTGCTGAGCAGTGTGGATGAGCTGGAGCAGCAAAGGGACGAGATCGTTAGCTACCTCTGTGACCTTGCCCC
TGAAGCCCCTCCTCCTACTCTGCCCCCCGACATGGCACAAGTCACTGTGGGCCCGGGGCTCTTGGGGGTTTCGACCC
TGGGGCCCAAGAGGAACTCCATGGTTCTGGATGTGGCGTTCGTCCTGGAAGGATCGGACAAAATTGGTGAAGCCGAC
TTCAACAGGAGCAAGGAGTTCATGGAGGAGGTGATTCAGCGGATGGATGTGGGCCAGGACAGCATCCACGTCACGGT
GCTGCAGTACTCCTACATGGTGACTGTGGAGTACCCCTTCAGCGAGGCACAGTCCAAAGGGGACATCCTGCAGCGGG
TGCGAGAGATCCGCTACCAGGGCGGCAACAGGACCAACACTGGGCTGGCCCTGCGGTACCTCTCTGACCACAGCTTC
TTGGTCAGCCAGGGTGACCGGGAGCAGGCGCCCAACCTGGTCTACATGGTCACCGGAAATCCTGCCTCTGATGAGAT
CAAGAGGCTGCCTGGAGACATCCAGGTGGTGCCCATTGGAGTGGGCCCTAATGCCAACGTGCAGGAGCTGGAGAGGA
TTGGCTGGCCCAATGCCCCTATCCTCATCCAGGACTTTGAGACGCTCCCCCGAGAGGCTCCTGACCTGGTGCTGCAG
AGGTGCTGCTCCGGAGAGGGGCTGCAGATCCCCACCCTCTCCCCTGCACCTGACTGCAGCCAGCCCCTGGACGTGAT
CCTTCTCCTGGATGGCTCCTCCAGTTTCCCAGCTTCTTATTTTGATGAAATGAAGAGTTTCGCCAAGGCTTTCATTT
CAAAAGCCAATATAGGGCCTCGTCTCACTCAGGTGTCAGTGCTGCAGTATGGAAGCATCACCACCATTGACGTGCCA
TGGAACGTGGTCCCGGAGAAAGCCCATTTGCTGAGCCTTGTGGACGTCATGCAGCGGGAGGGAGGCCCCAGCCAAAT
CGGGGATGCCTTGGGCTTTGCTGTGCGATACTTGACTTCAGAAATGCATGGTGCCAGGCCGGGAGCCTCAAAGGCGG
TGGTCATCCTGGTCACGGACGTCTCTGTGGATTCAGTGGATGCAGCAGCTGATGCCGCCAGGTCCAACAGAGTGACA
GTGTTCCCTATTGGAATTGGAGATCGCTACGATGCAGCCCAGCTACGGATCTTGGCAGGCCCAGCAGGCGACTCCAA
CGTGGTGAAGCTCCAGCGAATCGAAGACCTCCCTACCATGGTCACCTTGGGCAATTCCTTCCTCCACAAACTGTGCT
CTGGATTTGTTAGGATTTGCATGGATGAGGATGGGAATGAGAAGAGGCCCGGGGACGTCTGGACCTTGCCAGACCAG
TGCCACACCGTGACTTGCCAGCCAGATGGCCAGACCTTGCTGAAGAGTCATCGGGTCAACTGTGACCGGGGGCTGAG
GCCTTCGTGCCCTAACAGCCAGTCCCCTGTTAAAGTGGAAGAGACCTGTGGCTGCCGCTGGACCTGCCCCTGCGTGT
GCACAGGCAGCTCCACTCGGCACATCGTGACCTTTGATGGGCAGAATTTCAAGCTGACTGGCAGCTGTTCTTATGTC
CTATTTCAAAACAAGGAGCAGGACCTGGAGGTGATTCTCCATAATGGTGCCTGCAGCCCTGGAGCAAGGCAGGGCTG
CATGAAATCCATCGAGGTGAAGCACAGTGCCCTCTCCGTCGAGCTGCACAGTGACATGGAGGTGACGGTGAATGGGA
GACTGGTCTCTGTTCCTTACGTGGGTGGGAACATGGAAGTCAACGTTTATGGTGCCATCATGCATGAGGTCAGATTC
AATCACCTTGGTCACATCTTCACATTCACTCCACAAAACAATGAGTTCCAACTGCAGCTCAGCCCCAAGACTTTTGC
TTCAAAGACGTATGGTCTGTGTGGGATCTGTGATGAGAACGGAGCCAATGACTTCATGCTGAGGGATGGCACAGTCA
CCACAGACTGGAAAACACTTGTTCAGGAATGGACTGTGCAGCGGCCAGGGCAGACGTGCCAGCCCATCCTGGAGGAG
CAGTGTCTTGTCCCCGACAGCTCCCACTGCCAGGTCCTCCTCTTACCACTGTTTGCTGAATGCCACAAGGTCCTGGC
TCCAGCCACATTCTATGCCATCTGCCAGCAGGACAGTTGCCACCAGGAGCAAGTGTGTGAGGTGATCGCCTCTTATG
CCCACCTCTGTCGGACCAACGGGGTCTGCGTTGACTGGAGGACACCTGATTTCTGTGCTATGTCATGCCCACCATCT
CTGGTCTACAACCACTGTGAGCATGGCTGTCCCCGGCACTGTGATGGCAACGTGAGCTCCTGTGGGGACCATCCCTC
CGAAGGCTGTTTCTGCCCTCCAGATAAAGTCATGTTGGAAGGCAGCTGTGTCCCTGAAGAGGCCTGCACTCAGTGCA
TTGGTGAGGATGGAGTCCAGCACCAGTTCCTGGAAGCCTGGGTCCCGGACCACCAGCCCTGTCAGATCTGCACATGC
CTCAGCGGGCGGAAGGTCAACTGCACAACGCAGCCCTGCCCCACGGCCAAAGCTCCCACGTGTGGCCTGTGTGAAGT
AGCCCGCCTCCGCCAGAATGCAGACCAGTGCTGCCCCGAGTATGAGTGTGTGTGTGACCCAGTGAGCTGTGACCTGC
CCCCAGTGCCTCACTGTGAACGTGGCCTCCAGCCCACACTGACCAACCCTGGCGAGTGCAGACCCAACTTCACCTGC
GCCTGCAGGAAGGAGGAGTGCAAAAGAGTGTCCCCACCCTCCTGCCCCCCGCACCGTTTGCCCACCCTTCGGAAGAC
CCAGTGCTGTGATGAGTATGAGTGTGCCTGCAACTGTGTCAACTCCACAGTGAGCTGTCCCCTTGGGTACTTGGCCT
CAACTGCCACCAATGACTGTGGCTGTACCACAACCACCTGCCTTCCCGACAAGGTGTGTGTCCACCGAAGCACCATC
TACCCTGTGGGCCAGTTCTGGGAGGAGGGCTGCGATGTGTGCACCTGCACCGACATGGAGGATGCCGTGATGGGCCT
CCGCGTGGCCCAGTGCTCCCAGAAGCCCTGTGAGGACAGCTGTCGGTCGGGCTTCACTTACGTTCTGCATGAAGGCG
AGTGCTGTGGAAGGTGCCTGCCATCTGCCTGTGAGGTGGTGACTGGCTCACCGCGGGGGGACTCCCAGTCTTCCTGG
AAGAGTGTCGGCTCCCAGTGGGCCTCCCCGGAGAACCCCTGCCTCATCAATGAGTGTGTCCGAGTGAAGGAGGAGGT
CTTTATACAACAAAGGAACGTCTCCTGCCCCCAGCTGGAGGTCCCTGTCTGCCCCTCGGGCTTTCAGCTGAGCTGTA
AGACCTCAGCGTGCTGCCCAAGCTGTCGCTGTGAGCGCATGGAGGCCTGCATGCTCAATGGCACTGTCATTGGGCCC
GGGAAGACTGTGATGATCGATGTGTGCACGACCTGCCGCTGCATGGTGCAGGTGGGGGTCATCTCTGGATTCAAGCT
GGAGTGCAGGAAGACCACCTGCAACCCCTGCCCCCTGGGTTACAAGGAAGAAAATAACACAGGTGAATGTTGTGGGA
GATGTTTGCCTACGGCTTGCACCATTCAGCTAAGAGGAGGACAGATCATGACACTGAAGCGTGATGAGACGCTCCAG
GATGGCTGTGATACTCACTTCTGCAAGGTCAATGAGAGAGGAGAGTACTTCTGGGAGAAGAGGGTCACAGGCTGCCC
ACCCTTTGATGAACACAAGTGTCTGGCTGAGGGAGGTAAAATTATGAAAATTCCAGGCACCTGCTGTGACACATGTG
AGGAGCCTGAGTGCAACGACATCACTGCCAGGCTGCAGTATGTCAAGGTGGGAAGCTGTAAGTCTGAAGTAGAGGTG
GATATCCACTACTGCCAGGGCAAATGTGCCAGCAAAGCCATGTACTCCATTGACATCAACGATGTGCAGGACCAGTG
CTCCTGCTGCTCTCCGACACGGACGGAGCCCATGCAGGTGGCCCTGCACTGCACCAATGGCTCTGTTGTGTACCATG
AGGTTCTCAATGCCATGGAGTGCAAATGCTCCCCCAGGAAGTGCAGCAAGTGA//
<210> 2
<211>2813
<212> PRT
<213>artificial sequence
<400> 2
ORIGIN
MIPARFAGVLLALALILPGTLCAEGTRGRSSTARCSLFGSDFVNTFDGSMYSFAGYCSYLLAGGCQKRSFSII
GDFQNGKRVSLSVYLGEFFDIHLFVNGTVTQGDQRVSMPYASKGLYLETEAGYYKLSGEAYGFVARIDGSGNFQVLL
SDRYFNKTCGLCGNFNIFAEDDFMTQEGTLTSDPYDFANSWALSSGEQWCERASPPSSSCNISSGEMQKGLWEQCQL
LKSTSVFARCHPLVDPEPFVALCEKTLCECAGGLECACPALLEYARTCAQEGMVLYGWTDHSACSPVCPAGMEYRQC
VSPCARTCQSLHINEMCQERCVDGCSCPEGQLLDEGLCVESTECPCVHSGKRYPPGTSLSRDCNTCICRNSQWICSN
EECPGECLVTGQSHFKSFDNRYFTFSGICQYLLARDCQDHSFSIVIETVQCADDRDAVCTRSVTVRLPGLHNSLVKL
KHGAGVAMDGQDVQLPLLKGDLRIQRTVTASVRLSYGEDLQMDWDGRGRLLVKLSPVYAGKTCGLCGNYNGNQGDDF
LTPSGLAEPRVEDFGNAWKLHGDCQDLQKQHSDPCALNPRMTRFSEEACAVLTSPTFEACHRAVSPLPYLRNCRYDV
CSCSDGRECLCGALASYAAACAGRGVRVAWREPGRCELNCPKGQVYLQCGTPCNLTCRSLSYPDEECNEACLEGCFC
PPGLYMDERGDCVPKAQCPCYYDGEIFQPEDIFSDHHTMCYCEDGFMHCTMSGVPGSLLPDAVLSSPLSHRSKRSLS
CRPPMVKLVCPADNLRAEGLECAKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCVALERCPCFHQGKEYAPGETVK
IGCNTCVCRDRKWNCTDHVCDATCSTIGMAHYLTFDGLKYLFPGECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVK
CKKRVTILVEGGEIELFDGEVNVKRPMKDETHFEVVESGRYIILLLGKALSVVWDRHLSISVVLKQTYQEKVCGLCG
NFDGIQNNDLTSSNLQVEEDPVDFGNSWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVDSSCRILTSDVFQDCNKL
VDPEPYLDVCIYDTCSCESIGDCACFCDTIAAYAHVCAQHGKVVTWRTATLCPQSCEERNLRENGYECEWRYNSCAP
ACQVTCQHPEPLACPVQCVEGCHAHCPPGKILDELLQTCVDPEDCPVCEVAGRRFASGKKVTLNPSDPEHCQICHCD
VVNLTCEACQEPGGLVVPPTDAPVSPTTLYVEDISEPPLHDFYCSRLLDLVFLLDGSSRLSEAEFEVLKAFVVDMME
RLRISQKWVRVAVVEYHDGSHAYIGLKDRKRPSELRRIASQVKYAGSQVASTSEVLKYTLFQIFSKIDRPEASRIAL
LLMASQEPQRMSRNFVRYVQGLKKKKVIVIPVGIGPHANLKQIRLIEKQAPENKAFVLSSVDELEQQRDEIVSYLCD
LAPEAPPPTLPPHMAQVTVGPGLLGVSTLGPKRNSMVLDVAFVLEGSDKIGEADFNRSKEFMEEVIQRMDVGQDSIH
VTVLQYSYMVTVEYPFSEAQSKGDILQRVREIRYQGGNRTNTGLALRYLSDHSFLVSQGDREQAPNLVYMVTGNPAS
DEIKRLPGDIQVVPIGVGPNANVQELERIGWPNAPILIQDFETLPREAPDLVLQRCCSGEGLQIPTLSPAPDCSQPL
DVILLLDGSSSFPASYFDEMKSFAKAFISKANIGPRLTQVSVLQYGSITTIDVPWNVVPEKAHLLSLVDVMQREGGP
SQIGDALGFAVRYLTSEMHGARPGASKAVVILVTDVSVDSVDAAADAARSNRVTVFPIGIGDRYDAAQLRILAGPAG
DSNVVKLQRIEDLPTMVTLGNSFLHKLCSGFVRICMDEDGNEKRPGDVWTLPDQCHTVTCQPDGQTLLKSHRVNCDR
GLRPSCPNSQSPVKVEETCGCRWTCPCVCTGSSTRHIVTFDGQNFKLTGSCSYVLFQNKEQDLEVILHNGACSPGAR
QGCMKSIEVKHSALSVELHSDMEVTVNGRLVSVPYVGGNMEVNVYGAIMHEVRFNHLGHIFTFTPQNNEFQLQLSPK
TFASKTYGLCGICDENGANDFMLRDGTVTTDWKTLVQEWTVQRPGQTCQPILEEQCLVPDSSHCQVLLLPLFAECHK
VLAPATFYAICQQDSCHQEQVCEVIASYAHLCRTNGVCVDWRTPDFCAMSCPPSLVYNHCEHGCPRHCDGNVSSCGD
HPSEGCFCPPDKVMLEGSCVPEEACTQCIGEDGVQHQFLEAWVPDHQPCQICTCLSGRKVNCTTQPCPTAKAPTCGL
CEVARLRQNADQCCPEYECVCDPVSCDLPPVPHCERGLQPTLTNPGECRPNFTCACRKEECKRVSPPSCPPHRLPTL
RKTQCCDEYECACNCVNSTVSCPLGYLASTATNDCGCTTTTCLPDKVCVHRSTIYPVGQFWEEGCDVCTCTDMEDAV
MGLRVAQCSQKPCEDSCRSGFTYVLHEGECCGRCLPSACEVVTGSPRGDSQSSWKSVGSQWASPENPCLINECVRVK
EEVFIQQRNVSCPQLEVPVCPSGFQLSCKTSACCPSCRCERMEACMLNGTVIGPGKTVMIDVCTTCRCMVQVGVISG
FKLECRKTTCNPCPLGYKEENNTGECCGRCLPTACTIQLRGGQIMTLKRDETLQDGCDTHFCKVNERGEYFWEKRVT
GCPPFDEHKCLAEGGKIMKIPGTCCDTCEEPECNDITARLQYVKVGSCKSEVEVDIHYCQGKCASKAMYSIDINDVQ
DQCSCCSPTRTEPMQVALHCTNGSVVYHEVLNAMECKCSPRKCSK
//
Claims (7)
1. a kind of monoclonal antibody, which is characterized in that it is in conjunction with vWF ELISA (VWF) protein-specific；It is described
Monoclonal antibody is generated by hybridoma cell strain OTI9F3, and deposit number is CGMCC No.15594.
2. a kind of hybridoma cell strain, deposit number is CGMCC No.15594.
3. monoclonal antibody as described in claim 1 is in preparation for detecting the application in VWF immune detection tool.
4. application according to claim 3, the immune detection tool is kit, chip or test paper.
5. a kind of immunologic combined detection reagent kit, including monoclonal antibody described in claim 1.
6. application of the monoclonal antibody as described in claim 1 in the kit that preparation is used for tagged tissue cell.
7. application according to claim 6, the histocyte is that (or macronucleus is thin for the vascular endothelial cell of kinds of tumors
Born of the same parents).
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| CN201811015871.5A CN108997501A (en) | 2018-09-01 | 2018-09-01 | Anti- VWF protein monoclonal antibody and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811015871.5A CN108997501A (en) | 2018-09-01 | 2018-09-01 | Anti- VWF protein monoclonal antibody and application thereof |
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Family
ID=64590842
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| CN201811015871.5A Pending CN108997501A (en) | 2018-09-01 | 2018-09-01 | Anti- VWF protein monoclonal antibody and application thereof |
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| Country | Link |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110624105A (en) * | 2019-09-24 | 2019-12-31 | 苏州大学 | Sequences of structurally sensitive polypeptide antigens of von Willebrand factor |
| CN113087803A (en) * | 2021-05-12 | 2021-07-09 | 苏州大学附属第一医院 | Monoclonal antibody SZ176 against human von willebrand factor leader peptide and application thereof |
| CN113307876A (en) * | 2021-05-12 | 2021-08-27 | 苏州大学附属第一医院 | Monoclonal antibody SZ175 of antihuman von willebrand factor leader peptide and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102532316A (en) * | 2010-12-24 | 2012-07-04 | 神州细胞工程有限公司 | Anti-vWF (von Willebrand factor) monoclonal antibody and application thereof |
| WO2017129630A1 (en) * | 2016-01-26 | 2017-08-03 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Anti-vwf d'd3 single-domain antibodies and polypeptides comprising thereof |
-
2018
- 2018-09-01 CN CN201811015871.5A patent/CN108997501A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102532316A (en) * | 2010-12-24 | 2012-07-04 | 神州细胞工程有限公司 | Anti-vWF (von Willebrand factor) monoclonal antibody and application thereof |
| WO2017129630A1 (en) * | 2016-01-26 | 2017-08-03 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Anti-vwf d'd3 single-domain antibodies and polypeptides comprising thereof |
Non-Patent Citations (2)
| Title |
|---|
| HANS ULRICHTS等: "Shielding of the A1 Domain by the D ‘D3 Domains of von Willebrand Factor Modulates Its Interaction with Platelet Glycoprotein Ib-IX-V.", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
| ISTVAN TORNAI等: "A Monoclonal Antibody Recognizes a von Willebrand Factor Domain within the Amino-terminal Portion of the Subunit that Modulates the Function of the Glycoprotein IB- and IIB/IIIA-binding Domains", 《J. CLIN. INVEST.》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110624105A (en) * | 2019-09-24 | 2019-12-31 | 苏州大学 | Sequences of structurally sensitive polypeptide antigens of von Willebrand factor |
| WO2021056609A1 (en) * | 2019-09-24 | 2021-04-01 | 苏州大学 | Sequence of structurally sensitive polypeptide antigen of von willebrand factor |
| CN113087803A (en) * | 2021-05-12 | 2021-07-09 | 苏州大学附属第一医院 | Monoclonal antibody SZ176 against human von willebrand factor leader peptide and application thereof |
| CN113307876A (en) * | 2021-05-12 | 2021-08-27 | 苏州大学附属第一医院 | Monoclonal antibody SZ175 of antihuman von willebrand factor leader peptide and application thereof |
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