CN106397590A - Single-domain antibody of heparin-binding epidermal growth factor and application thereof - Google Patents

Single-domain antibody of heparin-binding epidermal growth factor and application thereof Download PDF

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CN106397590A
CN106397590A CN201610808368.XA CN201610808368A CN106397590A CN 106397590 A CN106397590 A CN 106397590A CN 201610808368 A CN201610808368 A CN 201610808368A CN 106397590 A CN106397590 A CN 106397590A
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domain antibody
single domain
heparin
epidermal growth
seq
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CN106397590B (en
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吕鹏
陈克平
潘晔
屠志刚
林锋
聂志超
王沁芸
廉彩霞
阮玲玲
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Shanghai Touch Research Medical Technology Co ltd
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Jiangsu University
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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
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • 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
    • C07K2317/565—Complementarity determining region [CDR]
    • 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
    • C07K2317/567—Framework region [FR]
    • 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
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen

Abstract

The invention relates to a single-domain antibody of a heparin-binding epidermal growth factor and the application thereof, and belongs to the field of the biological medicine or the biological pharmaceutical technology. According to the technical scheme of the invention, firstly, a strain of single-domain antibody that is high in affinity to HB-EGF is obtained based on the phage antibody library panning and sequencing technology, and then the single-domain antibody is obtained through the expression and purification process of an escherichia coli expression system. Meanwhile, the purified single-domain antibody is applied to the ELISA detection of the HB-EGF and the content detection of the HB-EGF in a sample. The single-domain antibody can inhibit the infiltration and the migration of ovarian cancer cells. The single-domain antibody is easy in mass production, lower in cost and more stable. Therefore, the single-domain antibody has important application value in ELISA and the inhibition of tumors.

Description

A kind of single domain antibody of heparin-binding epidermal growth factors and its application
Technical field
The invention belongs to biomedical or biological pharmacy technical field, it is related to a kind of list of heparin-binding epidermal growth factors Domain antibodies and its application.
Background technology
Heparin-binding epidermal growth factors (heparin-binding EGF-like growth factor, HB-EGF) A 22KD, the glycosylated protein of O-, belong to a member in EGF family, to fibroblast, smooth muscle cell and on Chrotoplast etc. has mitogenesis effect.HB-EGF participates in a lot of physiological process, also relevant with many pathological processes, such as wound Healing, eyelid is formed, Embryonic limb bud cell, heart development；Also relevant with many pathological processes, such as atherosclerosiss, smooth muscle increases Life, brain injury and tumor occur.
It is relevant with the generation development of kinds cancer that increasing evidence shows that the exception of HB-EGF signal path is adjusted, its In the mechanism of main cause cancer have the transfer promoting tumour growth, angiogenesis and tumor and invasion and attack, so suppression or in Activity with HB-EGF is to treat the measure of a lot of cancers.Malignant ovary carninomatosis people's ascites promotes ability of cell proliferation than optimum ovum The promotion ability of cell proliferation of nest tumour patient and normal person's ascites is much higher, and their activity simply use EGFR and HB-EGF antibody just can be suppressed.Cell survival rate in malignant ovary carninomatosis people's ascites with respect to rear both also greatly improve, And it is similarly subjected to the suppression of HB-EGF antibody.HB-EGF antibody reduces Malignant glioma cells, multiple myeloma cells Propagation；Identification sHB-EGF simultaneously has the antibody Y-142 of high affinity to suppress ovarian cancer cell line SKOV3, breast carcinoma to it The generation of VEGF of HB-EGF induction and angiogenesis in cell line T47D, colon carcinoma cell line SW480, and inhibition ratio The effect of EGFR antibody cetuximab and diphtheria toxin, diphtherotoxin non-toxic mutant CRM197 is good.
The target therapeutic agent of the ovarian cancer in research and development and clinical research mainly has targeting receptor blocking agent, tumor at present Angiogenesis inhibitor and be directed to ovarian cancer, VEGF（vascular endothelial growth factor, VEGF）, EGF-R ELISA（Epidermal growth factor receptor, EGFR）And substrate The monoclonal antibody of metalloproteases or chimeric antibody, but do not have large-scale clinical practice.
Additionally, clinical trial certificate, the expression of HB-EGF increases clinical prognosis with patient and anti-chemotherapy formed close Cut is closed.HB-EGF is possible to adjust anti-apoptotic effect such as ERK- and Akt- signal path, or promotees apoptosis effect JNK- and p38- signal path, thus lead to the generation of cell chemoresistant.Because the expression of HB-EGF and maturation can By lysophosphatidic acid（Lysophosphatidic acid, LPA) activation specific regulation and control, and sHB-EGF is as excreted factor It is easily detected, therefore HB-EGF is considered as the biomarker assisting in anti-LPA treatment meanss, in the market The ELISA kit of You Duo company can carry out the detection of HB-EGF.
No matter being that in clinic or scientific research ELISA kit, HB-EGF antibody used is all derived from traditional polyclone at present And monoclonal antibody, or human mouse chimeric antibody, these Antibody stability are poor, and production cost is high, for clinical treatment and detection All limit.Belgian scientist reports in Nature first within 1993：One of camel blood lacks the antibody of light chain Can be combined with target high-affinities such as antigens as normal antibody, and unlike single-chain antibody（scFV）Equally easily assemble. This antibody only comprises a weight chain variable district and two conventional CH2, CH3 area, and prokaryotic expression VHH area out has well Structural stability and binding activity, molecular weight is less, only the 1/10 of normal antibody, so VHH（Single domain antibody）Also referred to as Nanobody（Nano antibody）.By single domain antibody VHH expression in phage surface, through the screening of " absorption-eluting-amplification " process And be enriched with specific antibody, here it is phage antibody library technique.This is one kind extremely efficient antibody expression, screening system, So producing single domain antibody with its screening, the cycle is shorter, and more preferably, solubility is high for stability, and available Escherichia coli fermentation produces, and becomes This is lower, has broad prospects.
Content of the invention
It is an object of the invention to provide a kind of single domain antibody for heparin-binding epidermal growth factors, provide this list simultaneously The coded sequence of domain antibodies and this single domain antibody detect in preparation and suppress the application of tumor.
For achieving the above object, the invention provides a kind of single domain antibody of heparin-binding epidermal growth factors（Dab）, its VHH chain includes framework region FR and complementary determining region CDR, and described framework region FR includes SEQ ID NO：FR1 shown in 1, SEQ ID NO：FR2 shown in 2, SEQ ID NO：FR3 shown in 3, SEQ ID NO：FR4 sequence fragment shown in 4；
Described complementary determining region CDR includes SEQ ID NO：CDR1 shown in 5, SEQ ID NO：CDR2 shown in 6, SEQ ID NO：The sequence fragment of the CDR3 shown in 7；
Preferably, the VHH chain of the single domain antibody of described heparin-binding epidermal growth factors, it has SEQ ID NO：Shown in 8 Aminoacid sequence.
The present invention also provides a kind of single domain antibody of heparin-binding epidermal growth factors, and it is for heparin binding epidermal life The single domain antibody of long factor epi-position, including having SEQ ID NO：The VHH chain of aminoacid sequence shown in 8.
Present invention also offers a kind of DNA molecular, it encodes the list of heparin-binding epidermal growth factors of the present invention The protein of domain antibodies VHH chain or the protein of heparin-binding epidermal growth factors single domain antibody of the present invention.
Preferably, described DNA molecular, has SEQ ID NO：9 DNA sequence.
The present invention also provides a kind of expression vector, and described carrier is that the restructuring that can express single domain antibody of the present invention carries Body
PET28a-Dab, described carrier comprises SEQ ID NO：DNA sequence shown in 9.
The present invention also provides a kind of host cell, and described cell contains expression vector of the present invention, has expression originally Invention
The ability of described single domain antibody.
Present invention also offers described heparin-binding epidermal growth factors single domain antibody is used for detecting heparin binding epidermal The purposes of somatomedin, described purposes is single domain antibody in preparation detection heparin-binding epidermal growth factors ELISA kit Application.
Present invention also offers described heparin-binding epidermal growth factors single domain antibody be used for anticancer migration and The application of infiltration, and the application in preparing the medicine of anticancer migration and infiltration.
Described sequence information is as follows：
SEQ ID NO. 1：MKKLLFAIPLVVPFYAAQPAMAQVQLLESGGGLVQPGGSLRLSCAAS
SEQ ID NO. 2：MGWVRQAPGKGLEWVSS
SEQ ID NO. 3：YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC
SEQ ID NO. 4：WGQGTLVTVSSAAAEQKLISEEDLNSAAHYTDIEMNRLGKGAA
SEQ ID NO. 5：GVIFITYD
SEQ ID NO. 6：INTNNGST
SEQ ID NO. 7：ATGGWYGHKRLDSAHLRS
SEQ ID NO. 8：
MKKLLFAIPLVVPFYAAQPAMAQVQLLESGGGLVQPGGSLRLSCAASGVIFITYDMGWVRQAPGKGLEWVSSI NTNNGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATGGWYGHKRLDSAHLRSWGQGTLVTVSSAAA EQKLISEEDLNSAAHYTDIEMNRLGKGAA
SEQ ID NO. 9：
ATGAAAAAATTATTATTCGCAATTCCTTTAGTGGTACCTTTCTATGCGGCCCAGCCGGCCATGGCCCAGGTGC AGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGAGTTATC TTTATCACTTACGATATGGGCTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTATCAAGCATTAATACCAA TAACGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGT ATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTATTGCGCGACTGGGGGTTGGTATGGGCATAAG AGGCTGGATTCCGCGCACTTGAGGTCTTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCGCGGCCGCAGAACAAAA ACTCATCTCAGAAGAGGATCTGAATTCGGCCGCACATTATACAGACATAGAGATGAACCGACTTGGAAAGGGGGCCG CATAG
Compared with prior art, advantages of the present invention is as follows：
Obtain the single domain antibody gene order for heparin-binding epidermal growth factors using phage selection technology screening, by this Gene goes in escherichia coli, thus establish can be in the single domain antibody strain of E. coli.The list that purification obtains Domain antibodies can combine epidermal growth factor by heparin-binding, and can suppress migration and the infiltration of ovarian cancer cell in vitro.Should Invention heparin-binding epidermal growth factors single domain antibody, size is less, good stability, easily transports, and is also easy in vivo penetrate carefully Born of the same parents；It is easily achieved large-scale production, process takes short, low cost, operation is easy, has broad application prospects.
Brief description
Fig. 1 is phage single domain antibody storehouse screening process figure, and wherein A is with HB-EGF as target molecule, uses phage single domain Antibody
Storehouse is combined with pre-fixed target molecule；B washes unconjugated phage off；C trypsin by specifically bind Phage cuts down from target molecule；D carries out next round screening by after the Phage amplification eluting；E is according to A, B, C, D Carry out 3 wheels and wash in a pan sieve, be finally enriched to the phage with target molecule specific binding the acquisition nucleotide sequence that is sequenced；In figure,Table Show phage,Represent phage single domain antibody storehouse,Represent HB-EGF target molecule,Represent and divide with target The phage of son specific binding.
Fig. 2 is the euzymelinked immunosorbent assay (ELISA) with phage（ELISA）The ideograph of the identification single positive colony of specificity；Wherein 1 It is that HB-EGF is coupled in ELISA Plate, 2 is single domain antibody, 3 is Mus anti-Myc antibody, and 4 is goat anti-mouse horse radish peroxide Enzymic-labelled antibody, 5 is horseradish peroxidase nitrite ion TMB；The colourless circle in the left side represents non-chromogenic substrate, the right black circles Represent and developed the color after Catalyzed Synthesis By Peroxidase.
Fig. 3 is the HB-EGF single domain antibody of expression, through the SDS-PAGE after Protein L nucleophilic chromatography purification； Wherein swimming lane M is protein molecular standard；Swimming lane 1 expresses bacterium full bacterium lysate for HB-EGF single domain antibody；Swimming lane 2 is uncombined The outflow sample of Protein L affinity media；Swimming lane 3,4,5,6 is to use level pad（20 mM Na2HPO4,0.15 M NaCl,pH 7.0）Washing medium flows out sample；Swimming lane 7,8 is to use elution buffer（0.1 M glycine, pH 2.5）Eluting The single domain antibody getting off.
Fig. 4 is the ELISA testing result that biotinylated HB-EGF single domain antibody is used for HB-EGF.HB-EGF+DAb table Show with HB-EGF as antigen, single domain antibody DAb is anti-as one；SKOV3+DAb represents with ovarian cancer cell SKOV3 lysate work For antigen, single domain antibody DAb is anti-as one；DAb represents the negative control being added without that antigen is directly added into DAb.
Fig. 5 is the infiltration inhibition test result for ovarian cancer SKOV3 cell for the HB-EGF single domain antibody；In figure a is comparison Group（Not reagent adding）, b is experimental group（Add the good HB-EGF single domain antibody of purification in 200 μ L embodiments 3（Dab）, final concentration of 30μg/mL）.
Fig. 6 is infiltration inhibition test quantitative result that HB-EGF single domain antibody is for ovarian cancer SKOV3 cell.
Fig. 7 is the inhibition of metastasis result of the test to ovarian cancer cell SKOV3 for the HB-EGF single domain antibody.
Fig. 8 is the impact broken line graph to SKOV3 cell scratch experiment migration rate for the single domain antibody.
Specific embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail.
Embodiment 1：Single domain antibody screening for heparin-binding epidermal growth factors
(1) 100 μ g/mL are encoded the Partial Protein of heparin-binding epidermal growth factors aminoacid 63-149 position（P6018, purchase From in Yanuofa Biotechnology Co., Ltd.）It is coated in the immune pipe of Nunc with PBS, 4 DEG C stand overnight.
（2）Add 5mL 5% skim milk within second day, room temperature is closed 2 hours；
（3）1mL single domain antibody phage display library is added after 2 hours（6001_hDAb, purchased from Britain Source Biosciences company）, it is incubated 1 hour under room temperature；
（4）Use PBST（PBS, SN330-2 containing 0.05% polysorbas20, purchased from Nanjing Sheng Xing Bioisystech Co., Ltd）Wash 20 Time；
（5）Add 100 μ g/mL trypsin（T1426, purchased from sigma company）Incubated at room 1 hour, the Trypsin of addition Enzymatic solution needs, full of entirely immunity pipe, to obtain the phage with HB-EGF specific binding, and infect the big of exponential phase Enterobacteria TG1（6001_hDAb, purchased from Source Biosciences company of Britain）, produce and under purified phage is used for One wheel screening；
（6）In continuous screening process, positive colony is constantly enriched with, thus reached washing in a pan sieve antibody using display technique of bacteriophage The purpose of HB-EGF specificity single domain antibody in storehouse.
Embodiment 2：Screen specificity list positive colony with the ELISA of phage
（1）In the phage clone of enrichment from embodiment 1, select 96 single bacterium colonies and be inoculated in the ammonia benzyl containing 100 μ g/mL The TB culture medium of penicillin（Potassium dihydrogen phosphate containing 2.3g in 1L TB culture medium, 12.52g dipotassium hydrogen phosphate, 12g peptone, 24g Yeast extract, 4mL glycerol）In, to exponential phase, increase the IPTG of concentration 1mM, 28 DEG C of overnight incubation；
（2）Culture supernatant transfers to the Partial Protein through heparin-binding epidermal growth factors aminoacid 63-149 position（P6018, Purchased from Yanuofa Biotechnology Co., Ltd.）In coated elisa plate（This hole is sample well, and control wells are to add PBS to substitute Culture supernatant）, place 1 hour at room temperature；
（3）Wash away uncombined antibody with PBST, add an anti-mouse anti-myc 9E10 antibody（Anti- myc Mus resist, purchase From Santa Cruz biotech company）, place 1 hour at room temperature；
（4）PBST washes away unconjugated antibody, adds two anti-anti-mouse HRP conjugated secondary Antibody (sheep anti-Mouse horseradish peroxidase-labeled two resists, purchased from Wuhan three eagle bio tech ltd), under room temperature Place 1 hour；
（5）PBST washes away unconjugated antibody, adds horseradish peroxidase substrate TMB（Purchased from KPL company）30 points of reaction Clock, adds hydrochloric acid, reading under 450nm.Sample well OD is judged to positive colony for more than 3 times more than control wells.
（6）Positive colony is cultivated in the LB fluid medium containing 100 μ g/mL extraction plasmid to be sequenced.
According to sequence alignment program Vector NTI and IMGT analysis software gene order, parse its FR region and CDR region domain, and obtain single domain antibody VHH chain amino acid sequence.
Embodiment 3：Single domain antibody is in colibacillary expression and purification
（1）The single domain antibody nucleotide sequence that sequencing analysis are obtained is cloned into expression vector pET28a（Purchased from Life Technology company）, form the prokaryotic expression recombiant plasmid pET28a-Dab that can express single domain antibody, and it be correct to be sequenced Recombinant plasmid transformed to expression bacterium BL21（DE3）（Purchased from Life Technology company）In, it is coated on containing 100 μ g/mL On the LB solid culture plate of kanamycin, 37 DEG C of overnight incubation；
（2）Picking individual colonies are seeded in the LB culture fluid that 3mL contains kanamycin, 37 DEG C of overnight incubation；
（3）Inoculation 2mL overnight to 200mL LB culture medium, cultivate for 37 DEG C and reach 0.5 to OD value by strain, addition IPTG, 28 DEG C Overnight incubation；
（4）Bacterium, bacterial cell disruption are received in centrifugation, through Protein L affinity column purifying protein, use elution buffer（0.1 M glycine, pH 2.5）Eluting single domain antibody.Fig. 3 is the HB-EGF single domain antibody of expression, chromatographs pure through Protein L nucleophilic SDS-PAGE after change, Fig. 3 swimming lane M is protein molecular standard；Swimming lane 1 splits for the HB-EGF single domain antibody expression full bacterium of bacterium Solution thing；Swimming lane 2 is the outflow sample of uncombined Protein L affinity media；Swimming lane 3,4,5,6 is to use level pad（20 mM Na2HPO4,0.15 M NaCl,pH 7.0）Washing medium flows out sample；Swimming lane 7,8 is to use elution buffer（0.1 M glycine, pH 2.5）The single domain antibody eluting.Fig. 3 shows that the HB-EGF single domain antibody screening can pass through large intestine Bacillus system great expression, and purification is carried out by L albumen, the purity of the single domain antibody albumen being obtained is more than 70%.By this Method obtains antibody and simply obtains a large amount of target antibodies, with low cost.
Embodiment 4：Application single domain antibody ELISA detection HB-EGF analysis
（1）The biotinylation of HB-EGF single domain antibody：Single domain antibody good for purification is measured concentration.With DMF by BNHS（H1759, Purchased from sigma company）It is made into 1mg/mL solution, then the NaHCO with 0.1mol/L pH9.03Single domain antibody is diluted to 1mg/ ML, by BNHS：Antibody volume ratio 1：8～1：15 concussions mix, and room temperature stands 3～4 hours, 4 DEG C of dialysed overnight.
（2）The HB-EGF multi-resistance that will buy（LS-C166803/44318, Lifespan, the U.S.）It is coated in ELISA Plate, It is separately added into HB-EGF antigen, ovarian cancer SKOV3 cell lysate again and is not added with any material as negative control, room temperature is transferred Put 1 hour；
（3）Washed with PBST, and add step（1）The biotinylation HB-EGF single domain antibody obtaining；
（4）PBST washes away uncombined antibody, adds the streptavidin of HRP labelling（016-030-084, purchased from Jackson ImmunoResearch Laboratiories company）Anti- as two, it is incubated 1 hour；
（5）PBST washs, and adds tmb substrate（Purchased from KPL company）Reaction 30 minutes, adds sulphuric acid, reads OD at 450nm Value.
Result is as shown in figure 4, in figure HB-EGF+DAb represents with HB-EGF as antigen, single domain antibody DAb is anti-as one； , as antigen, single domain antibody DAb is anti-as one for SKOV3+DAb expression ovarian cancer cell SKOV3 lysate；DAb represents and is not added with Enter the negative control that antigen is directly added into DAb.The single domain antibody that result display purification of the present invention obtains can be used for ELISA method Detection HB-EGF antigen and similar SKOV3 cell lysate so contain the HB-EGF content in the sample of HB-EGF.
Embodiment 5：Cell migration assay
（1）Prepare ovarian cancer cell SKOV3 suspension：Use PBS（SN330-2, purchased from Nanjing Sheng Xing Bioisystech Co., Ltd） Cleaning SKOV3 cell, siphons away PBS, plus trypsin（Sigma-Aldrich company, the U.S.）Digestion, with containing 10% hyclone And the RMPI-1640 culture medium of Pen .- Strep（It is purchased from Gbico company）Piping and druming mixes, and terminates digestion, and centrifugation siphons away Supernatant, with the RMPI-1640 culture medium re-suspended cell containing 10% hyclone and Pen .- Strep, dilutes corresponding multiple Afterwards, adjustment cell density is to 2 × 105Individual/mL, is counted to cell using blood counting chamber, adjustment cell density to 2 × 105 Individual/mL.
（2）Inoculating cell：Cell suspension piping and druming after adjusting mixes, and respectively takes 200 μ L to be separately added into pure in embodiment 3 The HB-EGF single domain antibody changed is as experimental group（Final concentration of 30 μ g/mL）And reagent adding, as comparison, is not added to Transwell cell（Purchased from Corning company）In, finally add 500 μ L in cell bottom and contain 10% hyclone and penicillium sp The RMPI-1640 culture medium of element-streptomycin, according to normal cell culture rule culture, routine observation.
（3）Result counts：Press from both sides out cell with tweezers, gently wipe the attached cell remaining in upper interior with cotton swab.Use again 0.1% crystal violet（Chemical Reagent Co., Ltd., Sinopharm Group）Dyeing 15-20 minute, washes 3 times with PBS.Then take a clean load glass Piece, clear water is dripped on surface, Transwell cell is placed on and is just clearly visible the thin of the attachment of room side under cell counterdie on clear water Born of the same parents, with micro- sem observation and taking pictures, select the 5-10 visual field to take pictures at random.Finally collect pictures, statistical analysiss.
Experimental result such as Fig. 5, Fig. 6, room under Transwell cell are counted, for ovarian cancer cell SKOV3, warp Cross three independent experiments, by matched group (control) and experimental group（DAb）The cell number of migration carries out percentage ratio relative analyses, Result shows that the number of the SKOV3 cell permeable Transwell cell that single domain antibody was processed significantly reduces, and shows single domain antibody The infiltration of gonad cell SKOV3 can be significantly reduced, there is statistical significance.Fig. 6 is by the quantitative result of Fig. 5 resulting number, Can significantly find out that single domain antibody acts on to the inhibition of metastasis of ovarian cancer cell SKOV3 notable, its gap has statistics meaning Justice（P<0.01）, show that the HB-EGF antibody that we screen can significantly suppress the infiltration of ovarian cancer cell SKOV3.
Embodiment 6：Cell scratch experiment
（1）Plant plate：After T25 bottle SKOV3 cell culture and passing on well, abandon original fluid, use 2mL PBS, 0.5mL pancreas Enzymic digestion, plus 2mL contains 10% hyclone and the RMPI-1640 culture medium piping and druming of Pen .- Strep mixes, 146g is centrifuged 5 points Clock, abandons supernatant, plus the RMPI-1640 culture medium piping and druming containing 10% hyclone and Pen .- Strep mixes, and takes 0.5mL to EP Pipe, draws 10 μ L and counting on cell counting count board with rifle.Every hole 4000 cells of kind, mix in V-shaped groove.Plant to 24 orifice plates In, experimental group（DAb）To final concentration of 100 μ g/mL, matched group is not added with single domain antibody to middle addition single domain antibody, and normal culture is thin Born of the same parents.
（2）Cut：Draw straight line with 10 μ L white pipette tips perpendicular to hole surface, drawing two straight lines is in crosswise as far as possible.Draw Start to take pictures（0h）, often cross 6h later and just take once photograph, till cut heals.Finally gather picture, statistical analysiss.
After treatment, respectively in 0h, 6h, 12h, 24h, 30h and 36h gather picture to SKOV3 cell, through three times solely After vertical experiment, each group migration distance is carried out statistical analysiss.Result such as Fig. 7, shown in Fig. 8, the SKOV3 after single domain antibody is processed is thin Born of the same parents are slack-off compared with the migration of untreated control group, show that HB-EGF single domain antibody can suppress ovarian cancer cell SKOV3 to a certain extent Migration.Fig. 8 is the quantitative result of Fig. 7 resulting number, is that statistical summaries analysis obtains single domain after repeating three biologys The impact broken line graph to SKOV3 cell scratch experiment migration rate for the antibody, shows that the HB-EGF antibody screening can suppress swollen The migration of oncocyte SKOV3.
The above be only the preferred embodiment of the present invention it should be pointed out that：Ordinary skill people for the art For member, under the premise of without departing from present invention spirit and principle, some improvements and modifications can also be made, these improvements and modifications Also should be regarded as protection scope of the present invention.
SEQUENCE LISTING
<110>Jiangsu University
<120>A kind of single domain antibody of heparin-binding epidermal growth factors and its application
<130>A kind of single domain antibody of heparin-binding epidermal growth factors and its application
<160> 9
<170> PatentIn version 3.3
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Met Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ala
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Ala Gln Pro Ala Met Ala Gln Val Gln Leu Leu Glu Ser Gly Gly Gly
20 25 30
Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser
35 40 45
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Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser
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Ser
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Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
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Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
20 25 30
Thr Ala Val Tyr Tyr Cys
35
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Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ala Ala Glu Gln
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Lys Leu Ile Ser Glu Glu Asp Leu Asn Ser Ala Ala His Tyr Thr Asp
20 25 30
Ile Glu Met Asn Arg Leu Gly Lys Gly Ala Ala
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Gly Val Ile Phe Ile Thr Tyr Asp
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Ala Thr Gly Gly Trp Tyr Gly His Lys Arg Leu Asp Ser Ala His Leu
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Met Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ala
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Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
35 40 45
Val Ile Phe Ile Thr Tyr Asp Met Gly Trp Val Arg Gln Ala Pro Gly
50 55 60
Lys Gly Leu Glu Trp Val Ser Ser Ile Asn Thr Asn Asn Gly Ser Thr
65 70 75 80
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
85 90 95
Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
100 105 110
Thr Ala Val Tyr Tyr Cys Ala Thr Gly Gly Trp Tyr Gly His Lys Arg
115 120 125
Leu Asp Ser Ala His Leu Arg Ser Trp Gly Gln Gly Thr Leu Val Thr
130 135 140
Val Ser Ser Ala Ala Ala Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu
145 150 155 160
Asn Ser Ala Ala His Tyr Thr Asp Ile Glu Met Asn Arg Leu Gly Lys
165 170 175
Gly Ala Ala
<210> 9
<211> 540
<212> DNA
<213>Artificial sequence
<400> 9
atgaaaaaat tattattcgc aattccttta gtggtacctt tctatgcggc ccagccggcc 60
atggcccagg tgcagctgtt ggagtctggg ggaggcttgg tacagcctgg ggggtccctg 120
cgtctctcct gtgcagcctc cggagttatc tttatcactt acgatatggg ctgggtccgc 180
caggctccag ggaagggtct agagtgggta tcaagcatta ataccaataa cggtagcaca 240
tactacgcag actccgtgaa gggccggttc accatctccc gtgacaattc caagaacacg 300
ctgtatctgc aaatgaacag cctgcgtgcc gaggacaccg cggtatatta ttgcgcgact 360
gggggttggt atgggcataa gaggctggat tccgcgcact tgaggtcttg gggtcaggga 420
accctggtca ccgtctcgag cgcggccgca gaacaaaaac tcatctcaga agaggatctg 480
aattcggccg cacattatac agacatagag atgaaccgac ttggaaaggg ggccgcatag 540

Claims (10)

1. a kind of VHH chain of the single domain antibody of heparin-binding epidermal growth factors, including framework region FR and complementary determining region CDR, It is characterized in that, described framework region FR includes FR2, SEQ ID shown in FR1, SEQ ID NO. 2 shown in SEQ ID NO. 1 NO. the FR4 sequence fragment shown in FR3 and SEQ ID NO. 4 shown in 3；
Described complementary determining region CDR includes CDR2 and SEQ shown in CDR1, SEQ ID NO. 6 shown in SEQ ID NO. 5 CDR3 sequence fragment shown in ID NO. 7.
2. as claimed in claim 1 a kind of VHH chain of the single domain antibody of heparin-binding epidermal growth factors it is characterised in that The VHH chain of described single domain antibody has the aminoacid sequence shown in SEQ ID NO. 8.
3. a kind of single domain antibody of heparin-binding epidermal growth factors is it is characterised in that described single domain antibody is for heparin knot Close epidermal growth factor epi-position, including the VHH chain with aminoacid sequence shown in SEQ ID NO. 8.
4. a kind of DNA molecular is it is characterised in that described DNA molecular encodes the Heparin-binding described in claim 1-2 any one Heparin-binding epidermal growth factors list described in the protein of VHH chain of the single domain antibody of epidermal growth factor or claim 3 The protein of domain antibodies.
5. DNA molecular according to claim 4 is it is characterised in that described DNA molecular has shown in SEQ ID NO. 9 DNA sequence.
6. a kind of expression vector is it is characterised in that described carrier is the restructuring load that can express single domain antibody described in claim 3 Body pET28a-Dab, described carrier comprises the DNA sequence shown in SEQ ID NO. 9.
7. a kind of host cell, it is characterised in that described cell contains the expression vector described in claim 6, has expression power Profit requires the ability of single domain antibody described in 3.
8. a kind of heparin-binding epidermal growth factors single domain antibody described in claim 3 is used for detecting heparin binding epidermal growth The purposes of the factor.
9. purposes according to claim 6 is it is characterised in that described purposes is single domain antibody in preparation detection Heparin-binding Application in epidermal growth factor ELISA kit.
10. a kind of heparin-binding epidermal growth factors single domain antibody described in claim 3 prepare anticancer migration and Application in the medicine of infiltration.
CN201610808368.XA 2016-09-08 2016-09-08 Single-domain antibody of heparin-binding epidermal growth factor and application thereof Active CN106397590B (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109336976A (en) * 2018-09-27 2019-02-15 暨南大学 The nano antibody of anti-human EGFR and its application
CN109535252A (en) * 2018-12-17 2019-03-29 江苏莱森生物科技研究院有限公司 A kind of anti-HB-EGF monoclonal antibody and preparation method thereof
CN109580959A (en) * 2018-12-17 2019-04-05 江苏莱森生物科技研究院有限公司 A kind of ELISA kit detecting heparin-binding epidermal growth factor

Citations (2)

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Publication number Priority date Publication date Assignee Title
CA2654572A1 (en) * 2006-06-06 2007-12-13 Kyowa Hakko Kirin Co., Ltd. Monoclonal antibody capable of binding to heparin-binding epidermal growth factor-like growth factor
CN105837665A (en) * 2016-05-16 2016-08-10 江苏大学 Polypeptide with specificity inhibiting HB-EGF promoting tumor cell migration and infiltration

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2654572A1 (en) * 2006-06-06 2007-12-13 Kyowa Hakko Kirin Co., Ltd. Monoclonal antibody capable of binding to heparin-binding epidermal growth factor-like growth factor
CN105837665A (en) * 2016-05-16 2016-08-10 江苏大学 Polypeptide with specificity inhibiting HB-EGF promoting tumor cell migration and infiltration

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109336976A (en) * 2018-09-27 2019-02-15 暨南大学 The nano antibody of anti-human EGFR and its application
CN109336976B (en) * 2018-09-27 2021-04-02 暨南大学 Anti-human EGFR nano antibody and application thereof
CN109535252A (en) * 2018-12-17 2019-03-29 江苏莱森生物科技研究院有限公司 A kind of anti-HB-EGF monoclonal antibody and preparation method thereof
CN109580959A (en) * 2018-12-17 2019-04-05 江苏莱森生物科技研究院有限公司 A kind of ELISA kit detecting heparin-binding epidermal growth factor
CN109580959B (en) * 2018-12-17 2020-03-31 江苏莱森生物科技研究院有限公司 ELISA kit for detecting heparin-binding epidermal growth factor

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