CN108635579A - Anti-human bFGF nano antibodies are preparing the application in treating melanoma drug - Google Patents
Anti-human bFGF nano antibodies are preparing the application in treating melanoma drug Download PDFInfo
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Abstract
The invention discloses anti-human bFGF nano antibodies to prepare the application in treating melanoma drug.The nano antibody of the anti-recombinant human bfgf has many advantages, such as small molecular weight, high specificity, affinity height, high temperature resistant；Meanwhile required dosage is low, is significantly inhibited to the proliferation of melanoma cells, and can inhibit the growth and migration of melanoma cells.The preparation method of the anti-human bFGF nano antibodies is simple, and purity after purification can reach 90% or more.The present invention provides new drug candidate for the treatment of bFGF related neoplasms diseases, has a good application prospect.
Description
Technical field
The invention belongs to biotechnology, more particularly to anti-human bFGF nano antibodies are preparing treatment melanoma drug
In application.
Background technology
Melanoma is a kind of tumour that grade malignancy is high, is quickly grown, and the death rate is high, unwise to radiation and chemotherapy
Sense, except the melanoma that early stage melanoma in situ can be shifted by surgical resection, late period is then difficult to cure.One
In group malignant melanoma cell system, researcher has found each unique expressed bFGF (basic fibroblast of cell line
Growth factor), PDGF-A, PDGF-B, TGF-β, two kinds or four kinds, five kinds of cell factors in TGF- α, but it is all thin
Born of the same parents system all expressed bFGFs, illustrate that expression of the bFGF in melanoma cells has generality.From the atypical increasing of normal epithelial
It is raw, arrive the malignant mela noma shifted again to carcinoma in situ early invasive carcinoma, expression intensities of the bFGF in cell is successively
It increases.The study found that FGFR1 (the FGFR being mutated using antisense nucleic acid technique and overexpression：Fibroblast growth factor
Receptor) bFGF accesses are interfered, the proliferation of melanoma cells and internal tumour can be inhibited to occur, to further relate to bFGF be
Promote the great influence factor of melanoma occurrence and development.Target spots one of of the bFGF/FGFR as antineoplaston, at present still
There are not the therapeutic bFGF antibody of commercialization, the research and development of bFGF targeted drugs still to have broad space.
The inhibition neonate tumour blood vessel and antineoplastic action of bFGF monoclonal antibodies have been obtained for domestic and foreign scholars confirmation and
Certainly.Massoglia in 1987 etc. has started the beginning of bFGF monoclonal antibodies research, and Reilly in 1989 etc. is prepared for the first time in having
With active bFGF monoclonal antibodies, Akira Hori in 1991 etc. report that bFGF monoclonal antibodies can inhibit tumour under given conditions for the first time
Growth, Anouma in 1999 recognize it with inside and outside antitumor activity.The research of domestic bFGF monoclonal antibodies starts from 1998, Xiang Jun
The bFGF monoclonal antibodies of thrifty equal preparations can promote cell Proliferation, report within 2010 that prepared mouse bFGF monoclonal antibodies have again and inhibit
The effect of HUVEC angiogenesis, and humanized's bFGF monoclonal antibodies have the function of angiogenesis inhibiting.Although existing at present resist
The research papers and patent report of the small molecular antibodies such as bFGF conventional antibodies and Fab antibody, but it is prepared in these reports
Antibody be all from mouse or rabbit, for the classical IgG classes antibody and its derivative containing heavy chain and light chain, do not come from camel
The correlative study report of section animal, the only novel antibodies (namely nano antibody) of heavy chain.
Invention content
The shortcomings that it is an object of the invention to overcome the prior art and deficiency, it is anti-to provide a kind of nanometer of anti-recombinant human bfgf
Body is preparing the application in treating melanoma drug.
The purpose of the invention is achieved by the following technical solution：
A kind of nano antibody of anti-recombinant human bfgf is preparing the application in treating melanoma drug；The anti-recombination
The nano antibody of people bFGF includes by framework region FR (Framework Region) and complementary determining region CDR
The Variable domain of (Complementarity-determining Regions) composition, the Variable domain packet
Containing CDR1, CDR2 and CDR3 selected from the following：
(1) CDR1 shown in any amino acid sequence in No.1~4 SEQ ID,
(2) CDR2 shown in any amino acid sequence in No.5~8 SEQ ID；
(3) CDR3 shown in any amino acid sequence in No.9~12 SEQ ID.
The Variable domain includes selected from any one of following CDR1, CDR2 and CDR3：
(1) shown in CDR2 and SEQ ID No.9 shown in CDR1 shown in SEQ ID No.1, SEQ ID No.5
CDR3；
(2) CDR2 and SEQ ID shown in CDR1 shown in SEQ ID No.2, SEQ ID No.5 or SEQ ID No.6
CDR3 shown in No.9 or SEQ ID No.10；
(3) shown in CDR2 and SEQ ID No.11 shown in CDR1 shown in SEQ ID No.3, SEQ ID No.7
CDR3；
(4) shown in CDR2 and SEQ ID No.12 shown in CDR1 shown in SEQ ID No.4, SEQ ID No.8
CDR3。
Described framework region FR FR1, FR2, FR3 and the FR4 selected from the group below：
(1) FR1 shown in any amino acid sequence in No.13~15 SEQ ID；
(2) FR2 shown in any amino acid sequence in No.16~19 SEQ ID；
(3) FR3 shown in any amino acid sequence in No.20~24 SEQ ID；
(4) FR4 shown in any amino acid sequence in No.25~27 SEQ ID.
The nano antibody of the anti-recombinant human bfgf, amino acid sequence appointing in amino acid sequence as shown
Meaning is a kind of (NO.28~33 SEQ ID)；It is specific as follows：
The nano antibody of the anti-recombinant human bfgf include and any amino acid sequence shown in NO.28~33 SEQ ID
Row have at least 90%, preferably at least 95%, the more preferably at least amino acid sequence of 99% sequence identity.
Any amino acid sequence as shown in NO.28~33 SEQ ID passes through the either addition one or several of substitution, missing
The amino acid sequence of a amino acid.
The substitution preferably compared with any amino acid sequence shown in NO.28~33 SEQ ID comprising one or
Multiple amino acid substitutions, preferably conserved amino acid replace.For example, including 1,2,3,4,5,6,7,8,9 or 10 conserved amino acid
Substitution.
The nucleotide sequence of the amino acid sequence as shown in NO.28~33 SEQ ID is encoded successively such as SEQ ID NO.34
Shown in~39.
The nano antibody by 342 bases, 336 bases or 369 base compositions, 114 amino acid of coding,
112 amino acid or 123 amino acid are made of 4 framework regions (FR) of antibody and 3 complementary determining regions (CDR)：Nano antibody
CDR1 encode 10 amino acid, CDR2 encode 7 amino acid, CDR3 encode 11 amino acid；Or CDR1 encodes 10 amino
Acid, CDR2 encode 7 amino acid, and CDR3 encodes 9 amino acid；Or CDR1 encodes 10 amino acid, CDR2 encodes 8 amino
Acid, CDR3 encode 19 amino acid；3 CDR regions are specific sequence.
The drug can be pharmaceutical composition, including pharmaceutically acceptable carrier.
The drug can also be applied in combination therapy, i.e., be combined with other medicaments.For example, combination therapy can wrap
Include at least one other antitumor drugs of nano antibody joint of the anti-recombinant human bfgf of the present invention.
The present invention has the following advantages and effects with respect to the prior art：
The nano antibody of anti-recombinant human bfgf provided by the invention has that molecular weight is small, high specificity, affinity are strong, resistance to height
Temperature has apparent inhibiting effect to the proliferation of melanoma cells.Therefore, the nanometer of anti-recombinant human bfgf provided by the invention
Antibody can provide new drug candidate for the treatment of bFGF related neoplasms diseases.
Description of the drawings
Fig. 1 is that the wheel of bFGF nano antibodies phage library 3 eluriates result figure.
Fig. 2 is the ELISA result figures of different bacteriophages antibody cloning；Wherein 1 is positive control, and 2 be negative control, 3~
86 be respectively that different phage antibodies is cloned.
Fig. 3 is the electrophoretogram of the SDS-PAGE of bFGF nano antibodies after purification；Wherein swimming lane 1 is protein molecular standard, swimming
Road 2~7 is the nano antibody obtained after 300 mMs of imidazole elutions elute, is followed successively by Nb1~Nb6.
Fig. 4 is bFGF nano antibodies and bFGF specific binding figures；Wherein 1 is positive control (the commercially available anti-bFGF IgG of mouse
Antibody is abbreviated as bFGF mAb), 2~7 indicate different nano antibody clone strains, are followed successively by Nb1~Nb6.
Fig. 5 is bFGF nano antibody thermostabilization analysis of experimental results figures；Wherein bFGF monoclonal antibodies are positive control, Nb1~Nb6
Indicate different nano antibody clone strains.
Fig. 6 is bFGF nano antibodies and the binding site result of study analysis chart of FGFR2；Wherein Nb1~Nb6 indicates different
Nano antibody clone strain.
Fig. 7 is bFGF nano antibodies and bFGF monoclonal antibody binding site result of study analysis charts；Wherein Nb1~Nb6 is indicated not
Same nano antibody clone strain.
Fig. 8 various concentrations Nb6 treated melanoma cells migrate analysis of experimental results figure；Wherein * * are indicated and bFGF
Group is compared, p<0.01.
Fig. 9 Nb6 influence interpretation of result figure to different melanoma cells Erk1/2 and Akt phosphorylation leveled time point；Its
Middle * * expressions are compared with 0h groups, p<0.01.
Figure 10 is bFGF nano antibodies treatment melanoma knurl photo figure；Wherein 1 is negative control group, and 2 be anti-bFGF
Nano antibody group.
Specific implementation mode
With reference to embodiment, the present invention is described in further detail, and embodiments of the present invention are not limited thereto.
The screening technique of the nano antibody of 1 anti-recombination human basic fibroblast growth factor (bFGF) of embodiment
1, the structure of nano antibody library gene
(1) with the recombination human basic fibroblast growth factor of a concentration of 0.2mg/mL, (bFGF sticks up god purchased from Beijing justice
State Bioisystech Co., Ltd) alpaca is immunized (purchased from U.S. Allele Biotechnology＆Pharmaceuticals
Co.), immune every time to mix the bFGF of 0.2mg in equal volume with Freund's adjuvant, it once every two weeks, is immunized 4 times altogether, except for the first time
It is remaining all (public purchased from Sigma using not formula incomplete adjuvant several times using complete Freund's adjuvant (being purchased from Sigma companies)
Department).Immunologic process moderate stimulation B cell expresses the single-chain antibody of antigentic specificity.
After (2) 4 times immune, alpaca peripheral blood lymphocytes 100mL is extracted, (is purchased from Trizol reagents
Invitrogen companies) extraction total serum IgE, (NEB public affairs are referred to using AMV First Strand cDNA Synthesis kits
Take charge of reagent kit product specification) reverse transcription synthesis cDNA.
(3) (reaction system is shown in Table 1, PCR conditions and is shown in Table 2) variable region progress PCR amplification of the design primer to heavy chain antibody.
PCR product through QIA quick PCR Purification kits (be purchased from QIAGEN companies) recovery purifying, obtain 330~
The alpaca heavy chain variable region PCR product of 370bp or so.
The pcr amplification reaction system of 1 alpaca single-chain antibody variable region of table
The PCR amplification condition of 2 alpaca single-chain antibody variable region of table
2, the structure of nano antibody phage antibody library
(1) (it is purchased from Allele using 20 μ g pComb3X of restrictive restriction endonuclease Sfi I (being purchased from NEB companies) digestion
Biotechnology and Pharmaceuticals companies) Vector for Phage Display and 10 μ g alpaca heavy chain variable regions PCR productions
T4DNA ligases (being purchased from NEB companies) two segments of connection are used in combination in object.Connection product electrotransformation to electricity is turned into competent cell
In XL1-Blue (being purchased from Allele Biotechnology and Pharmaceuticals companies), rear five additions respectively
The bacterium solution in cup is resuspended in 1mL SOC culture mediums (being purchased from NEB companies) to electric revolving cup, and 5mL is cultivated bacterium solution in 37 DEG C of shaking tables,
250rpm cultivates 1h.
(2) 10mL SB culture medium (preparation methods are added to 5mL culture bacterium solutions：Weigh 8g MOPs, 24g peptones, 16g
Yeast extract is settled to 800mL, and sterilize 20min sterilizings in 121 DEG C；Tetracycline containing 10mg/L in culture medium) and 3 μ L ammonia benzyls
Penicillin storing liquid (100g/L ampicillins) takes after mixing 1 μ L culture solutions to dilute 100 times, is spread evenly across LB tablets and (contains
100mg/L ampicillins), detect storage capacity.By remaining culture solution in 37 DEG C, 275rpm cultivates 1h.4.5 are added into culture solution
μ L ampicillins storing liquid (100g/L ampicillins) continues to cultivate 1h.2mL helper phages are added into culture solution
VCSM13 (is purchased from Allele Biotechnology and Pharmaceuticals companies；Titre is 1012~1013pfu/
ML), addition SB culture mediums (tetracycline containing 10mg/L) are to 200mL, and 92.5 μ L ampicillins storing liquid (100g/L ammonia are added
Parasiticin), in 37 DEG C, 275rpm cultivates 2h.400 μ L kanamycins storing liquids are added into culture solution, and (that is mould for 35g/L cards
Element), overnight incubation.
(3) next day, by the bacterium solution being incubated overnight in 4 DEG C, 3000g centrifuges 15min.Supernatant is transferred to containing 4% (w/v)
In the 50mL centrifuge tubes of PEG-8000 and 3% (w/v) NaCl, vortex mixing, and it is incubated 30min in 4 DEG C, for precipitating phagocytosis
Body.15min is centrifuged under the conditions of 4 DEG C, 15 000g.Supernatant is abandoned, phages are resuspended with 2mL TBS.It, will be upper under the conditions of 4 DEG C
Clear maximum (top) speed centrifuges 5min, is used in combination in 0.22 μm of filter filtering supernatant to sterile EP tube, this is anti-bFGF nano antibodies
The original library of bacteriophage.
3, the screening of phage antibody library
(1) bFGF antigens (being purchased from the Divine Land Yi Qiao Bioisystech Co., Ltd) are diluted to 2 holes μ g/, are coated in 96 hole enzymes
Version is marked, 4 DEG C are coated with overnight.Next day, with 5% skim milk, (it is molten that the skim milk of 2.5g is dissolved completely in 50mL 0.05%PBST
In liquid) after 37 DEG C of closings 1 hour, by phage library preparation solution (and the bacteriophage of anti-bFGF nano antibodies that step 2 obtains
Original library, 1014Pfu/mL it) is added and is coated in the hole of antigen, 37 DEG C are incubated 2 hours.XL1-blue bacterium solutions (OD=0.8 is added
~1), 100 holes μ L/ are incubated at room temperature 15min.The step is repeated as many times, and reaches 2mL until obtaining and being infected bacterium solution.2mL is felt
The bacterium solution of dye is added in 6mL SB culture mediums (tetracycline containing 10mg/L), and is added into 1.6 μ L ampicillin storing liquids
(100g/L ampicillins).1 μ L of infected bacterium solution are taken, 100 times is diluted, is applied to LB tablets (tetracycline containing 10mg/L),
It is incubated overnight in 37 DEG C, measures antibody library titre.By 8mL culture solutions in 37 DEG C, 275rpm cultivates 1h, rear that 2.4 μ L ammonia benzyls are added
Penicillin storing liquid (100g/L ampicillins), is further cultured for 1h.1mL helper phage VCSM13 are added into culture solution, and
SB culture mediums (tetracycline containing 10mg/L) are added to final volume 100mL, add 92 μ L ampicillins storing liquid (100g/L ammonia benzyls
Penicillin).By 100mL culture solutions in 37 DEG C, 275rpm cultivates 1.5~2h.200 μ L kanamycins storing liquid (35g/L cards are added
That mycin), in 37 DEG C, 275rpm is incubated overnight.Next day, purified phage are used for the elutriation of next round, identical elutriation process weight
Multiple 3 wheel.As a result such as Fig. 1 is shown, during constantly screening, positive colony is constantly enriched with, and is bitten to reach to utilize
Phage display technique sieves the purpose for taking antibody library moderate resistance bFGF specific antibodies.
(2) 84 positive colonies are selected from the culture dish that the 3rd wheel is eluriated, is inoculated in and (contains containing 1mL SB culture mediums
10mg/L tetracyclines, 100mg/L ampicillins), culture solution is placed in by the negative control using helper phage as this experiment
37 DEG C of shaking table culture 5h.The VCSM13 helper phages in 10 holes μ L/ are added afterwards, are further cultured for 1.5~2h.2 μ L kanamycins are added
Storing liquid (35g/L kanamycins), in 37 DEG C of incubator overnight cultures.Next day, by culture solution in 4 DEG C, 3000g centrifuges 15min.It takes
4%PEG-8000 the and 3%NaCl mixed solutions of 5 times of concentration of 800 μ L supernatants and 200 μ L of addition are incubated 30min in 4 DEG C.It will
For culture solution in 4 DEG C, 15 000g centrifuge 15min.Supernatant is abandoned, 200 μ L TBS are added, and gently concussion is uniform.50 μ L suspensions are added
In 96 orifice plates for entering to be coated with the holes 200ng/ bFGF antigens.Using the mixed liquor of arbitrary 3 hole sample as positive control, bitten with auxiliary
Thalline is the negative control of this experiment, blank controls of the PBS as this experiment.96 orifice plates are incubated 1h in 37 DEG C.Afterwards with
The amount in the holes 100ng/ is incubated Anti-M13 antibody 1 hour.The TMB developing solutions (being purchased from NEB companies) in 100 holes μ L/ are added, and are incubated
15min.Light absorption value at 450nm is measured, the clone strain that experimental group OD values are 3 times of helper phage group OD values or more is defined as sun
Property clone strain.As a result such as Fig. 2, it is positive clone strain to share 69 clone strains, and positive ratio reaches 82%.Select 16 plants of positives at random
Clone carries out plasmid extraction, and send to Sangon Biotech (Shanghai) Co., Ltd. and be sequenced.By DNA sequencing result
It is analyzed with Bioedit, the identical clone strain of amino acid sequence is considered as same clone strain, and the strain that amino acid sequence is different
Be considered as different clone strains, finally share 6 plants of different nano antibodies of amino acid sequence, be respectively designated as Nb1, Nb2, Nb3, Nb4,
Nb5 and Nb6, coding nucleotide sequence is as shown in NO.34~39 SEQ ID.
4, the expression and purifying of bFGF nano antibodies
(1) PCR expansions are carried out to the nano antibody target gene on pComb 3x carriers with a group-specific primers (being shown in Table 3)
(reaction system is shown in Table 4, PCR conditions and is shown in Table 5) for increasing.
3 nano antibody target gene amplimer of table
(underlining is the Hind III and Nde I restriction enzyme sites of addition)
4 nano antibody target gene PCR amplification system of table
The 5 nano antibody target gene pcr amplification reaction time of table
Above-mentioned PCR product is pure through QIA quick PCR Purification kits (being purchased from QIAGEN companies) recycling
Change, obtains the nano antibody PCR product of 330~370bp or so.(it is purchased from Thermo using restrictive restriction endonuclease Hind III
Company) and Nde I (be purchased from Thermo companies) digestion expression vector pET22b (being purchased from Novagen companies) and nano antibody purpose
Gene is used in combination T4 DNA ligases to connect two segments.By connection product conversion expression type host strain BL21 (DE3) (purchased from life
Work bioengineering (Shanghai) limited liability company) in, it is coated on containing LB solid mediums (containing 100mg/L ampicillins)
Plate on, 37 DEG C overnight.
(2) it selects single bacterium colony to be seeded in 5mL LB culture solutions (containing 100mg/L ampicillins), 37 DEG C of shaking table trainings
It supports overnight.Inoculation 1mL is stayed overnight in strain to 400mL LB culture mediums (containing 100mg/L ampicillins) afterwards, 37 DEG C of shaking table trainings
It supports, as OD=0.6~1, IPTG (final concentration of 0.5mmol/L), 18 DEG C of shaking table culture 20h is added.Second day, 4 DEG C,
6000rpm centrifuges 15min and receives bacterium.Thalline is resuspended with PBS, ultrasonication 10min.8000rpm centrifuges 30min, and supernatant is
Expression product crude extract.By crude extract through nickel column ion affinity chromatography antibody purification albumen.To obtain the antibody of high-purity, use
Imidazole gradient elution method, low concentration imidazole elution (100mmol/L) is for washing away miscellaneous band, high concentration imidazole elution
(300mmol/L) can finally prepare purity up to 90% or more albumen for eluting destination protein.As a result see Fig. 3, from left to right
It is respectively：Swimming lane 1 is standard protein molecule, and swimming lane 2~7 is followed successively by Nb1~Nb6 of the elution of 300mmol/L imidazoles
Protein sample.The results show that nano antibody passes through this after purification, purity can reach 90% or more.
2 anti-bFGF nano antibodies physicochemical property of embodiment and biological activity test
1, the specificity analysis of anti-bFGF nano antibodies
BFGF, aFGF, KGF, EGF, TNF α, the VEGF that the holes 50ng/ are added into 96 hole elisa Plates (are purchased from Beijing justice to stick up
Divine Land Bioisystech Co., Ltd), BSA, milk, in 4 DEG C coating overnight.Next day, with 5% skim milk closing ELISA Plate 2h
Afterwards, positive control bFGF mAb (being purchased from sigma companies), the blank control PBS in a concentration of holes 100ng/ are separately added into and is resisted
BFGF nano antibodies, 37 DEG C of incubation 1h.Anti- Flag (the being purchased from Sigma companies) tag antibody in the holes 100ng/ is added afterwards, is incubated 1h.
The TMB developing solutions in 100 holes μ L/ are added, are incubated 10min.It is finally terminated and is reacted with 2.29% sulfuric acid, measure light absorption value at 450nm.
Compare the antibody gram of 3 times of antigen light absorption value or more with other with the absorbance value of corresponding antigen bFGF with anti-bFGF nano antibodies
It is grand to be defined as the positive.
As a result as Fig. 4 is indicated, there are specific binding capacity is strong and other 7 right by 6 plants of anti-bFGF nano antibodies and bFGF
It is weaker according to the binding ability of antigen.
2, the affinity determination of anti-bFGF nano antibodies
Using the affinity of the anti-bFGF nano antibodies of surface plasma resonance biosensor assay.By 0.4mol N-
With 20 μ L/min after ethyl-N '-(dimethylamino-propyl) carbodiimides and the isometric mixing of 0.1mol n-hydroxysuccinimides
Flow velocity be passed through in instrument chip surface activated.BFGF albumen is diluted to the 0.2mol acetate buffer solutions of pH 4.2
After 2mg/mL, chip surface is flowed through.Fixed amount is passed through PBS buffer solution after reaching aequum.It is passed through 1mol ethanol amines (pH 8.5)
Solution 7min inactivates remaining ester bond.The anti-bFGF of various concentration (3.125,6.25,12.5,25,50,100,200nM) is received
Meter Kang Ti is passed through with the flow velocity of 20 μ L/min in instrument.The affinity of anti-bFGF nano antibodies is calculated by instrument software.As a result
As shown in table 6, there is the nano antibody of anti-bFGF high-affinity, highest affinity to reach 0.69nM.Remaining anti-bFGF nanometers anti-
Body affinity constant is respectively 11.32nM, 317.60nM, 5.37nM, 12.37nM and 10.32nM.
The nano antibody affinity constant of 6 anti-bFGF of table
| Nb1 | Nb2 | Nb3 | Nb4 | Nb5 | Nb6 | |
| KD(nM) | 11.32 | 317.60 | 5.37 | 0.69 | 12.37 | 10.32 |
3, the measurement of thermal stability
The obtained nano antibody (Nb1~Nb6) of screening is respectively placed in 25 DEG C, 37 DEG C, 60 DEG C, 90 DEG C of four kinds of different temperatures
Under each 10min, 30min, 60min, 120min and 180min；Sigma (is purchased from, mouse source is anti-with bFGF monoclonal antibodies simultaneously
Body) it is used as positive control.Different temperatures, different time treated sample are collected, they are combined with bFGF with ELISA method detection
Property, i.e., after the processing of different temperatures different time, whether antibody also has the ability combined with bFGF.Testing result Fig. 5 shows
Compared with untreated nano antibody, after 25 DEG C and 37 DEG C processing, nano antibody relative activity is held in 90% or more,
And after 60 DEG C of processing, nano antibody relative activity is declined, but remains at 80% or more.But by 90 DEG C of processing
Afterwards, nano antibody relative activity is remarkably decreased, and relative activity is reduced to 0% when wherein Nb5 processing 60min, and Nb6 processing
Relative activity is just down to 0% after 180min, other several groups of nano antibody relative activities drop to 0% in 120min.This with
BFGF monoclonal antibodies as positive control are compared to having a clear superiority, although bFGF monoclonal antibodies are at 25 DEG C, 37 DEG C, 60
Relative activity under DEG C three kinds of different temperatures is 90% or more, but after 90 DEG C of processing, its relative activity is in 10min
Inside just have fallen to 0%.Therefore, illustrate the relatively common mouse monoclonal antibody of nano antibody, the stability in high-temperature process
Better.
4, competion experiment
(1) the nano antibody Nb of the Inhibition ELISA detection present invention is usedbFGF(Divine Land biology skill is stuck up with FGFR2 purchased from justice
Art Co., Ltd) competitive binding bFGF (be purchased from the Divine Land Yi Qiao Bioisystech Co., Ltd) ability.This experiment will quantitative 100ng
Nano antibody and the FGFR2 of variable (0~1 μ g) be added in the tablet of coating 50ng bFGF and be incubated simultaneously, if with FGFR2
The raising of concentration, the content that nano antibody detected can be fewer and fewer, illustrate the combination table of nano antibody and FGFR2 with bFGF
The affinity of the consistent either close or nano antibody in position and bFGF are not as good as FGFR2 with the affinity of bFGF；If FGFR2 concentration
It gradually rises, the content of nano antibody does not change, then illustrates that nano antibody and the combination epitope of FGFR2 and same bFGF differ
The tightness degree that cause or nano antibody are combined with bFGF is higher than the tightness degree that FGFR2 is combined with bFGF.As a result such as Fig. 6, with
The increase of FGFR2 contents, Nb2 and Nb4 contents be always maintained at can steadily speculate the combination epitope of they and bFGF with
The affinity that the combination epitope of FGFR2 and bFGF is inconsistent or they are with the affinity of bFGF higher than FGFR2 and bFGF.
The binding force of tetra- plants of nano antibodies of Nb1, Nb3, Nb5 and Nb6 and bFGF show becoming of successively decreasing with the increase of FGFR2 concentration
Gesture, illustrate the combination epitope of four plants of nano antibodies and bFGF it is consistent or close with the combination epitope of FGFR2 and bFGF or it
Be less than the affinity of FGFR2 and bFGF with the affinity of bFGF.
(2) Inhibition ELISA is used to detect NbbFGF(it is purchased from Sigma with business mouse monoclonal antibody bFGF mAb (FB-8)
Company) competitive binding bFGF ability.Fig. 7 shows that increases of the Nb4 with bFGF mAb contents, content are always maintained at steadily, can
With speculate it and the combination epitope of bFGF and the combination epitope of bFGF mAb and bFGF is inconsistent or Nb4 and bFGF it is affine
Power is higher than the affinity of bFGF mAb and bFGF.The binding force of five plants of nano antibodies of Nb1, Nb2, Nb3, Nb5 and Nb6 and bFGF with
The increase of bFGF mAb concentration and show the trend successively decreased, illustrate the combination epitope of five plants of nano antibodies and bFGF with
The combination epitope of bFGF mAb and bFGF are consistent or close or the affinity of they and bFGF are less than bFGF mAb and bFGF
Affinity.
3 anti-bFGF nano antibodies of embodiment treat murine melanoma experiment
1, to the Inhibition test of melanoma cells proliferation
(1) by the B16 mouse melanoma cell line, B16-F10 and human melanoma cell of culture to exponential phase
A375 (being purchased from Shanghai cell institute) is digested with 0.25% pancreatin digestive juice, is then trained with the DMEM containing 10%FBS (fetal calf serum)
It is 3 × 10 to support base (being purchased from Gibco companies) and adjust its density4A cell/mL is inoculated in 96 orifice plates with 100 holes μ L/, changes afterwards for 24 hours
It is that DMEM of 100 holes μ L/ without serum is cultivated, in 37 DEG C, 5%CO2Under the conditions of 4~6h of hungry culture.
(2) positive control FGFR and sample nanometer are diluted with the DMEM culture mediums of the bFGF containing 0.2%FBS and 12.5ng/mL
Antibody, with 4 times of gradient dilutions since 16nmol/L, if each 2 multiple holes of 6 dilutions, to contain 0.2%FBS and 12.5ng/mL
The DMEM culture mediums of bFGF are as negative control, and the DMEM culture mediums containing 0.2%FBS are as blank control, in 37 DEG C, 5%CO2
Under the conditions of cultivate 48h.
(3) 10 holes μ L/ CCK-8 reagents (be purchased from Sigma companies) are added, in 37 DEG C, 5%CO2Under the conditions of be incubated 4h.
(4) light absorption value is measured at 450nm and 630nm with microplate reader.
(5) cell proliferation inhibiting rate (%)=[negative control group OD- experimental groups OD]/negative control group OD × 100%.
The results are shown in Table 7, when by 12.5ng/mL bFGF and positive control FGFR, sample NbbFGFIt is incubated with processing
When melanoma cells, FGFR and NbbFGFIt induces the proliferation of melanoma cells to have apparent inhibiting effect bFGF, and is in
Dose dependent.Each group NbbFGFTo the highest of B16, B16-F10 and A375 cell increment inhibiting rate respectively 25~46%,
31~43% and 24~36%, and the FGFR of same dose be to the highest of each strain cell increment inhibiting rate (27.10 ±
1.50) %, (22.10 ± 2.56) % and (25.31 ± 2.13) %.In 5 plants of NbbFGFIn, Nb1, Nb3 and Nb6 pairs of 3 plants of melanin
The increment inhibition of oncocyte is put up the best performance.Nb2 is when inhibiting B16-F10 cell Proliferations, highest inhibiting rate ratio FGFR
Group is high by 40%, and when inhibiting other two plants of melanoma cells, highest inhibiting rate and FGFR group indifferences.Nb5 is although right
The highest inhibiting rate of B16-F10 and A375 cell Proliferations is all significantly higher than FGFR groups, but its proliferation inhibition rate to B16 cells
But with FGFR group indifferences.Illustrate not homophyletic NbbFGFThe proliferation inhibiting effect of not homophyletic melanoma cells is had differences.
Positive control FGFR and each group NbbFGFHalf-inhibition concentration (IC50) be shown in Table 7.As it can be seen that compared with FGFR groups, remove
Nb2 is higher than FGFR groups, other Nb to the IC50 of A375bFGFFGFR groups are below to the IC50 of melanoma cells, illustrate with
FGFR is compared, NbbFGFCell Proliferation suppression is carried out to B16 mouse melanoma cell line, B16-F10 and human melanoma cell A375
The used time is made, required dosage is lower, but inhibiting rate higher.
7 Nb of tablebFGFTo the highest increment inhibiting rate and half-inhibition concentration (IC50) of melanoma cells
2, scratch experiment detects NbbFGFInfluence to melanoma cells migration
(1) 6 porocyte culture plates are taken, with marking pen in its bottom per two horizontal lines of parallel picture at hole.
(2) tumour cell needed for the DMEM culture medium regulation experiments containing 10%FBS (B16 mouse melanoma cell line,
B16-F10 and human melanoma cell A375) density to 3 × 105A cell/mL takes the holes 2mL/ to be added in 6 orifice plates, culture
For 24 hours orifice plate bottom 80% is paved with to cell monolayer.
(3) culture medium in 6 orifice plates is taken out to be used in combination with the 200 μ L pipette tips direction parallel scratches 2 vertical with back side horizontal line
PBS washing flat boards 2 times, with the cell of cleaning floating.
(4) with the DMEM culture medium dilute sample nano antibodies (Nb6) of the bFGF containing 0.2%FBS and 12.5ng/mL, from
16nmol/L starts with 4 times of gradient dilutions, if each 2 multiple holes of 3 dilutions, with 0.2%FBS+DMEM+12.5ng/mL bFGF
Culture medium is as a contrast.The place intersected to cell cut and back horizontal line with fluorescence microscope is taken pictures, after cell is set
In 37 DEG C, 5%CO2Under the conditions of cultivate 48h.
(5) it is taken pictures, is used with the fluorescence microscope same place that rear cell cut and back horizontal line intersect before administration
Image J softwares calculate the mobility of cell.Cell migration rate (%)=[scratch area after scratch area-administration before administration]/
Scratch area × 100% before administration.
The results are shown in Figure 8, and compared with bFGF groups, the Nb6 of three various dose groups can inhibit the life of cell at cut
Long and migration, and at dose-dependence.For B16 cells, the mobilities of tri- dosage groups of Nb6 be respectively (41.58 ±
6.06) % (* * p<0.01), (32.52 ± 5.78) % (* * p<And (27.23 ± 4.96) % (* * p 0.01)<0.01).With bFGF
Group is compared, and it is respectively 26.66%, 42.65% and 51.97% that they, which inhibit the inhibiting rate of cell migration,.B16-FI0 is high transfer
Melanoma cell strain, therefore, the mobility of the cell strain is above B16 cells and A375 cells, respectively (72.73 ±
6.03) % (* * p<0.01), (63.20 ± 7.54) % (* * p<And (55.38 ± 8.69) % (* * p 0.01)<0.01).With bFGF
Group is compared, and it is respectively 26.79%, 36.39% and 44.25% that they, which inhibit the inhibiting rate of cell migration,.A375 cells are behaved black
Pigment oncocyte, after Nb6 is handled, the mobility of the cell strain is similar to B16 cell strain mobilities, respectively (40.92 ±
5.85) % (* * p<0.01), (35.60 ± 4.29) % (* * p<And (28.83 ± 4.85) % (* * p 0.01)<0.01).With bFGF
Group is compared, and it is respectively 37.44%, 45.67% and 55.91% that they, which inhibit the inhibiting rate of cell migration,.Therefore, this description of test
Nb6 can inhibit the growth and migration of B16 mouse melanoma cell line, B16-F10 and human melanoma cell A375, and at dosage
Dependence.
3, Westerning Blotting detect NbbFGFInfluence to melanoma cells signal path
(1) the 0.25% pancreatin digestive juice of cell by culture to exponential phase digests, and then uses 10%FBS+DMEM
It is 3 × 10 that culture medium, which adjusts its density,4A cell/mL, takes the holes 2mL/ to be placed in 6 orifice plates, is changed to the holes 2mL/ afterwards for 24 hours and is free of serum
DMEM cultures, in 37 DEG C, 5%CO2Under the conditions of 4~6h of hungry culture.
(2) when phosphorylation time point is detected, with 0.2%FBS+DMEM+12.5ng/mL bFGF culture medium diluted samples
Product Nb6 to 4nmol/L, by cell in 37 DEG C, 5%CO2Under the conditions of culture 0,0.5,1,3h, collect cell at this 4 time points.
(3) cell is collected, 1000g centrifuges 1min.Supernatant is removed, thalline is resuspended with 1X PBS.The step is repeated twice.
(4) appropriate zooblast lysate (being purchased from Sigma companies) is added to specifications to cell precipitation, and presses volume
Than 1:Protease inhibitors PMSF (100mM) is added in 100 ratio, and mixing is placed on 30min on ice, 13,500g centrifugations
10min。
(5) supernatant is removed, albumen concentration is measured with BCA methods.
(6) albumen concentration is adjusted, ensures that the concentration of each sample is consistent.Appropriate 5X Loading are added into sample
Buffer is incubated 10min in 100 DEG C.
(7) sample is subjected to SDS-PAGE electrophoresis experiments, transferring film and closing is carried out after running glue.
(8) it is incubated primary antibody：0.05%TBST washs NC films 1 time, each 5min.According to required detection albumen (p-Erk1/2,
P-Akt, t-Erk1/2, t-Akt and β-action) specification, with 1:5000 dilution proportion antibody, after under the conditions of 4 DEG C,
NC films are incubated on 40rpm shaking tables with primary antibody to stay overnight.
(9) it is incubated secondary antibody：0.05%TBST washs NC films 6 times, each 5min.According to required detection albumen (namely with His
Label protein) specification, with 1:10000 dilution proportion antibody, after in being incubated NC films 1h with secondary antibody on room temperature 40rpm shaking tables.
(10) develop：0.05%TBST washs NC films 6 times, each 5min.Developing solution is sprayed to the NC films cleaned up, until
NC films moisten completely.Developed and taken pictures with the gel imaging system of BIO-RAD.As a result it is analyzed with Image J softwares
Processing.
(11) if need to also develop other antibody in same NC film, NC film 5min are cleaned with Stripping Buffer, after
5min X are cleaned with 0.05%TBST 5 times, then repeatedly step 3~5.As a result see Fig. 9.
It can be seen in figure 9 that when 4nmol/L Nb6 effects, it is thin that three plants of melanomas can be significantly inhibited in 0.5h
Expression (the * * p of intracellular p-Erk1/2 and p-Akt<0.01), but with the passage of action time, inhibit phosphorylation level by
It is decrescence weak.And t-Erk1/2 and t-Akt levels do not change with the variation of action time.
4, anti-bFGF nano antibodies treat murine melanoma zoopery
It is right in 18 C57BL/6J mouse (being purchased from Guangdong Medical Lab Animal Center, SPF grades, 6~7 week old are female)
The soft place's inoculum density of hind leg thigh lateral cutaneous is 5X 106The Murine melanoma B16 cells of a/mL are (purchased from the Chinese Academy of Sciences
Extra large cell bank), 0.1mL/ is only.When tumour average longest diameter is 1~3mm, have no tumour growth and longest diameter is eliminated
Mouse more than 5mm.Then mouse is randomly divided into negative control group, anti-bFGF nano antibodies group (Nb6), every group 8.Using
The mode of knurl surrounding subcutaneous administration is administered mouse.PBS control group gives 1 × PBS 0.1mL/ pcs/day, and anti-bFGF receives
Rice antibody group gives bFGF nano antibodies 20mg/kg/ days.Successive administration 14 times.Last dose puts to death mouse afterwards for 24 hours, and solution takes
Tumor mass is taken pictures.The results are shown in Figure 10, and analysis is found, the mouse knurl body of negative control group and anti-bFGF nano antibodies group
It is respectively (3.45 ± 1.73) g and (0.96 ± 0.81) g, compared with negative control group, the tumor suppression of anti-bFGF nano antibodies group again
Rate is 72.24%.Illustrate that anti-bFGF nano antibodies can significantly inhibit the proliferation of melanoma in Mice Body.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, it is other it is any without departing from the spirit and principles of the present invention made by changes, modifications, substitutions, combinations, simplifications,
Equivalent substitute mode is should be, is included within the scope of the present invention.
Sequence table
<110>Ji'nan University
<120>Anti-human bFGF nano antibodies are preparing the application in treating melanoma drug
<160> 42
<170> SIPOSequenceListing 1.0
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<212> PRT
<213>Artificial sequence (Artificial Sequence)
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Gly Ser Asn Phe Ser Asn Asn Asp Met Gly
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Gly Ser Asn Phe Ser Ile Asn Asp Met Gly
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Arg Asn Ile Phe Ser Val Asn His Met Gly
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Gly Arg Thr Phe Ser Ser Gly Ala Met Gly
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Ile Ser Ser Val Gly Arg Thr
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Ile Ser Ser Val Gly Arg Ala
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Ile Thr Pro Gly Gly Thr Arg
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Ile Thr Trp Asp Gly Gly Thr Thr
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His Leu Tyr Gly Asp Tyr Arg Gly Thr Gly Phe
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Tyr Leu Tyr Gly Asp Tyr Arg Gly Thr Gly Phe
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Asn Thr Trp Pro Tyr Glu Ser Ala Tyr
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Ala Ala Arg Ser Tyr Ser Glu Ala Tyr Tyr Leu Ile Gly Ser Ser Asp
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Tyr Asn Tyr
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Leu Gln Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Gly Ser Leu Arg
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Leu Ser Cys Glu Val Ser
20
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Leu Gln Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg
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Leu Ser Cys Lys Ala Ser
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Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg
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Leu Ser Cys Ala Ala Ser
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Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Val Val Ala Gly
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Trp Tyr Arg Gln Ala Pro Gly Lys Arg Arg Glu Val Val Ala Gly
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Tyr Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ala Leu
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Trp Phe Arg Gln Ala Pro Gly Lys Glu Pro Glu Phe Val Ala Thr
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Met Tyr Gly Asp Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
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Ala Lys Asn Met Val Tyr Leu Gln Met Asn Arg Leu Lys Ala Lys Asp
20 25 30
Thr Ala Val Tyr Tyr Cys
35
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Met Tyr Ala Asp Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
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Ala Lys Asn Met Val Tyr Leu Gln Met Asn Arg Leu Lys Pro Glu Asp
20 25 30
Thr Ala Val Tyr Tyr Cys
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Asn Tyr Ala Asn Ser Val Lys Gly Arg Phe Thr Ile Ser Lys Asp Asn
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Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Gln Pro Glu Asp
20 25 30
Thr Ala Val Tyr Tyr Cys
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Tyr Tyr Ala Asp Ser Val Lys Gly Arg Tyr Thr Ile Ser Arg Asp Asn
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Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Asp Leu Lys Pro Glu Asp
20 25 30
Thr Ala Val Tyr Ser Cys
35
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Met Tyr Gly Asp Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
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Ala Lys Asn Met Val Tyr Leu Gln Met Asn Arg Leu Lys Pro Glu Asp
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Thr Ala Val Tyr Tyr Cys
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Trp Gly Lys Gly Thr Gln Val Thr Val Ser Ser
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Ser Gly Gln Gly Thr Gln Val Thr Val Ser Thr
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Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
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Leu Gln Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Gly Ser Leu Arg
1 5 10 15
Leu Ser Cys Glu Val Ser Gly Ser Asn Phe Ser Asn Asn Asp Met Gly
20 25 30
Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Val Val Ala Gly Ile
35 40 45
Ser Ser Val Gly Arg Thr Met Tyr Gly Asp Pro Val Lys Gly Arg Phe
50 55 60
Thr Ile Ser Arg Asp Asn Ala Lys Asn Met Val Tyr Leu Gln Met Asn
65 70 75 80
Arg Leu Lys Ala Lys Asp Thr Ala Val Tyr Tyr Cys His Leu Tyr Gly
85 90 95
Asp Tyr Arg Gly Thr Gly Phe Trp Gly Lys Gly Thr Gln Val Thr Val
100 105 110
Ser Ser
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Leu Gln Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Gly Ser Leu Arg
1 5 10 15
Leu Ser Cys Glu Val Ser Gly Ser Asn Phe Ser Ile Asn Asp Met Gly
20 25 30
Trp Tyr Arg Gln Ala Pro Gly Lys Arg Arg Glu Val Val Ala Gly Ile
35 40 45
Ser Ser Val Gly Arg Ala Met Tyr Ala Asp Pro Val Lys Gly Arg Phe
50 55 60
Thr Ile Ser Arg Asp Asn Ala Lys Asn Met Val Tyr Leu Gln Met Asn
65 70 75 80
Arg Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr Leu Tyr Gly
85 90 95
Asp Tyr Arg Gly Thr Gly Phe Trp Gly Lys Gly Thr Gln Val Thr Val
100 105 110
Ser Ser
<210> 30
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Leu Gln Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Gly Ser Leu Arg
1 5 10 15
Leu Ser Cys Glu Val Ser Gly Ser Asn Phe Ser Ile Asn Asp Met Gly
20 25 30
Trp Tyr Arg Gln Ala Pro Gly Lys Arg Arg Glu Val Val Ala Gly Ile
35 40 45
Ser Ser Val Gly Arg Thr Met Tyr Ala Asp Pro Val Lys Gly Arg Phe
50 55 60
Thr Ile Ser Arg Asp Asn Ala Lys Asn Met Val Tyr Leu Gln Met Asn
65 70 75 80
Arg Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys His Leu Tyr Gly
85 90 95
Asp Tyr Arg Gly Thr Gly Phe Trp Gly Lys Gly Thr Gln Val Thr Val
100 105 110
Ser Ser
<210> 31
<211> 112
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<213>Artificial sequence (Artificial Sequence)
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Leu Gln Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg
1 5 10 15
Leu Ser Cys Lys Ala Ser Arg Asn Ile Phe Ser Val Asn His Met Gly
20 25 30
Tyr Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ala Leu Ile
35 40 45
Thr Pro Gly Gly Thr Arg Asn Tyr Ala Asn Ser Val Lys Gly Arg Phe
50 55 60
Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn
65 70 75 80
Ser Leu Gln Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Thr Trp Pro
85 90 95
Tyr Glu Ser Ala Tyr Ser Gly Gln Gly Thr Gln Val Thr Val Ser Thr
100 105 110
<210> 32
<211> 123
<212> PRT
<213>Artificial sequence (Artificial Sequence)
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Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg
1 5 10 15
Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Gly Ala Met Gly
20 25 30
Trp Phe Arg Gln Ala Pro Gly Lys Glu Pro Glu Phe Val Ala Thr Ile
35 40 45
Thr Trp Asp Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg
50 55 60
Tyr Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met
65 70 75 80
Asn Asp Leu Lys Pro Glu Asp Thr Ala Val Tyr Ser Cys Ala Ala Arg
85 90 95
Ser Tyr Ser Glu Ala Tyr Tyr Leu Ile Gly Ser Ser Asp Tyr Asn Tyr
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 33
<211> 114
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 33
Leu Gln Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Gly Ser Leu Arg
1 5 10 15
Leu Ser Cys Glu Val Ser Gly Ser Asn Phe Ser Ile Asn Asp Met Gly
20 25 30
Trp Tyr Arg Gln Ala Pro Gly Lys Arg Arg Glu Val Val Ala Gly Ile
35 40 45
Ser Ser Val Gly Arg Thr Met Tyr Gly Asp Pro Val Lys Gly Arg Phe
50 55 60
Thr Ile Ser Arg Asp Asn Ala Lys Asn Met Val Tyr Leu Gln Met Asn
65 70 75 80
Arg Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys His Leu Tyr Gly
85 90 95
Asp Tyr Arg Gly Thr Gly Phe Trp Gly Lys Gly Thr Gln Val Thr Val
100 105 110
Ser Ser
<210> 34
<211> 342
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 34
ctgcaggagt ctgggggaga cttggtgcag cctggggggt ctctgagact ctcctgtgaa 60
gtttctggaa gcaacttcag taacaatgac atgggctggt accgccaggc tccagggaag 120
cagcgcgagg tggtcgcagg tattagtagt gttggacgta caatgtatgg agaccccgtg 180
aagggccgat tcaccatctc cagagacaac gccaagaaca tggtgtatct gcaaatgaac 240
agactgaaag ctaaagacac ggccgtctat tactgtcacc tttatggtga ctataggggg 300
actggtttct ggggcaaggg gacccaggtc accgtctcgt cg 342
<210> 35
<211> 342
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 35
ctgcaggagt ctgggggaga cttggtgcag cctggggggt ctctgagact ctcctgtgaa 60
gtttctggaa gcaacttcag tatcaatgac atgggctggt accgccaggc tccggggaag 120
cggcgcgagg tggtcgcagg tattagtagt gttggacgcg caatgtatgc agaccccgtg 180
aagggccgat tcaccatctc cagagacaac gccaagaaca tggtgtactt gcaaatgaac 240
agactgaaac ctgaggacac ggccgtttat tactgttacc tttatggtga ttataggggg 300
actggtttct ggggcaaggg gacccaggtc accgtctcgt cg 342
<210> 36
<211> 342
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 36
ctgcaggagt ctgggggaga cttggtgcag cctggggggt ctctgagact ctcctgtgaa 60
gtttccggaa gcaacttcag tatcaatgac atgggctggt accgccaggc tccagggaag 120
cggcgcgagg tggtcgcagg tattagtagt gttggacgca caatgtatgc agaccccgtg 180
aagggccgat tcaccatctc cagagacaac gccaagaaca tggtgtatct gcaaatgaac 240
agactgaaac ctgaggacac ggccgtctat tactgtcacc tttatggtga ctataggggg 300
actggtttct ggggcaaggg gacccaggtc accgtctcgt cg 342
<210> 37
<211> 336
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 37
ctgcagcagt ctgggggagg cttggtgcag cctggggggt ctctgagact ctcctgtaaa 60
gcctctagaa acatcttcag tgtcaatcac atgggctatt accgccaggc tccagggaag 120
gagcgcgagc tggtcgcgct tattactccc ggtggtacca gaaactatgc aaactccgtg 180
aagggccgat tcaccatctc caaagacaac gccaagaaca cggtgtatct gcagatgaac 240
agcctgcaac ctgaggacac ggccgtctat tactgtaata cctggccata tgagtctgcc 300
tattcgggcc aggggaccca ggtcaccgtc tccaca 336
<210> 38
<211> 369
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 38
ctgcaggagt ctgggggagg attggtgcag gctgggggct ctctgagact ctcctgtgca 60
gcctctggac gcaccttcag tagcggtgcc atgggctggt tccgccaggc tccagggaag 120
gagcctgagt ttgtggcaac tattacgtgg gatgggggta cgacatacta tgcagactcc 180
gtgaagggcc gatacaccat ctccagagac aacgccaaga atacggtata tctgcaaatg 240
aacgacctga aacctgagga cacggccgtt tattcctgtg cagcgagatc ttatagtgag 300
gcttactact taatcggctc gtccgattat aactactggg gtcaggggac ccaggtcacc 360
gtctcctca 369
<210> 39
<211> 342
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 39
ctgcaggagt ctgggggaga cttggtgcag cctggggggt ctctgagact ctcctgtgaa 60
gtttctggaa gcaacttcag tatcaatgac atgggctggt accgccaggc tccagggaag 120
cgacgcgagg tggtcgcagg tattagtagt gttggacgca caatgtatgg agaccccgtg 180
aagggccgat tcaccatctc cagagacaac gccaagaaca tggtgtatct gcaaatgaac 240
agactgaaac ctgaggacac ggccgtctat tactgtcacc tttatggtga ctataggggg 300
actggtttct ggggcaaggg gacccaggtc accgtctcgt cg 342
<210> 40
<211> 30
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 40
ataaaacata tgctgcagga gtctggggga 30
<210> 41
<211> 30
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 41
ataaaacata tgctgcagca gtctggggga 30
<210> 42
<211> 33
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 42
ataaaaaagc ttcttatcgt cgtcatcttt ata 33
Claims (10)
1. a kind of nano antibody of anti-recombinant human bfgf is preparing the application in treating melanoma drug, the anti-recombined human
The nano antibody of bFGF includes the Variable domain being made of framework region FR and complementary determining region CDR, it is characterised in that：
The Variable domain includes CDR1, CDR2 and CDR3 selected from the following：
(1) CDR1 shown in any amino acid sequence in No.1~4 SEQ ID,
(2) CDR2 shown in any amino acid sequence in No.5~8 SEQ ID；
(3) CDR3 shown in any amino acid sequence in No.9~12 SEQ ID.
2. the nano antibody of anti-recombinant human bfgf according to claim 1 is preparing answering in treating melanoma drug
With, which is characterized in that the Variable domain includes selected from any one of following CDR1, CDR2 and CDR3：
(1) CDR3 shown in CDR2 and SEQ ID No.9 shown in CDR1 shown in SEQ ID No.1, SEQ ID No.5；
(2) CDR2 and SEQ ID No.9 shown in CDR1 shown in SEQ ID No.2, SEQ ID No.5 or SEQ ID No.6
Or CDR3 shown in SEQ ID No.10；
(3) CDR3 shown in CDR2 and SEQ ID No.11 shown in CDR1 shown in SEQ ID No.3, SEQ ID No.7；
(4) CDR3 shown in CDR2 and SEQ ID No.12 shown in CDR1 shown in SEQ ID No.4, SEQ ID No.8.
3. the nano antibody of anti-recombinant human bfgf according to claim 1 is preparing answering in treating melanoma drug
With, which is characterized in that described framework region FR FR1, FR2, FR3 and the FR4 selected from the group below：
(1) FR1 shown in any amino acid sequence in No.13~15 SEQ ID；
(2) FR2 shown in any amino acid sequence in No.16~19 SEQ ID；
(3) FR3 shown in any amino acid sequence in No.20~24 SEQ ID；
(4) FR4 shown in any amino acid sequence in No.25~27 SEQ ID.
4. the nano antibody of anti-recombinant human bfgf according to claim 1 is preparing answering in treating melanoma drug
With, it is characterised in that：
The amino acid sequence of the nano antibody of the anti-recombinant human bfgf is any ammonia as shown in NO.28~33 SEQ ID
Base acid sequence.
5. the nano antibody of anti-recombinant human bfgf according to claim 4 is preparing answering in treating melanoma drug
With, it is characterised in that：
The nano antibody of the anti-recombinant human bfgf includes to have with any amino acid sequence shown in NO.28~33 SEQ ID
There is the amino acid sequence of at least 90% sequence identity.
6. the nano antibody of anti-recombinant human bfgf according to claim 5 is preparing answering in treating melanoma drug
With, it is characterised in that：
The nano antibody of the anti-recombinant human bfgf includes to have with any amino acid sequence shown in NO.28~33 SEQ ID
There is the amino acid sequence of at least 95% sequence identity.
7. the nano antibody of anti-recombinant human bfgf according to claim 4 is preparing answering in treating melanoma drug
With, it is characterised in that：
The nucleotides sequence of the amino acid sequence of the nano antibody of the coding anti-recombinant human bfgf is classified as such as SEQ ID NO.34
Any nucleotide sequence shown in~39.
8. the nano antibody of anti-recombinant human bfgf according to claim 4 is preparing answering in treating melanoma drug
With, it is characterised in that：
The amino acid sequence of the nano antibody of the anti-recombinant human bfgf is any ammonia as shown in NO.28~33 SEQ ID
The amino acid sequence that base acid sequence passes through substitution, lacks or add one or several amino acid.
9. the nano antibody of anti-recombinant human bfgf according to claim 1 is preparing answering in treating melanoma drug
With, it is characterised in that：
The drug can be pharmaceutical composition, including pharmaceutically acceptable carrier.
10. the nano antibody of anti-recombinant human bfgf according to claim 1 is preparing answering in treating melanoma drug
With, it is characterised in that：
The drug can be applied in combination therapy.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810574112.6A CN108635579B (en) | 2018-06-06 | 2018-06-06 | Application of anti-human bFGF nano antibody in preparation of drugs for treating melanoma |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810574112.6A CN108635579B (en) | 2018-06-06 | 2018-06-06 | Application of anti-human bFGF nano antibody in preparation of drugs for treating melanoma |
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| Publication Number | Publication Date |
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| CN108635579A true CN108635579A (en) | 2018-10-12 |
| CN108635579B CN108635579B (en) | 2020-08-07 |
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| CN201810574112.6A Active CN108635579B (en) | 2018-06-06 | 2018-06-06 | Application of anti-human bFGF nano antibody in preparation of drugs for treating melanoma |
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Cited By (3)
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| CN109722716A (en) * | 2019-01-22 | 2019-05-07 | 山西农业大学 | A kind of construction method in melanoma nano antibody library |
| CN111410693A (en) * | 2020-04-15 | 2020-07-14 | 山西农业大学 | CDK5 resistant nano antibody and application thereof |
| CN111440238A (en) * | 2020-03-27 | 2020-07-24 | 国典(北京)医药科技有限公司 | Nano antibody of anti-human transforming growth factor β 1 and preparation method and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109722716A (en) * | 2019-01-22 | 2019-05-07 | 山西农业大学 | A kind of construction method in melanoma nano antibody library |
| CN111440238A (en) * | 2020-03-27 | 2020-07-24 | 国典(北京)医药科技有限公司 | Nano antibody of anti-human transforming growth factor β 1 and preparation method and application thereof |
| CN111440238B (en) * | 2020-03-27 | 2022-09-20 | 国典(北京)医药科技有限公司 | Nano antibody of anti-human transforming growth factor beta 1 and preparation method and application thereof |
| CN111410693A (en) * | 2020-04-15 | 2020-07-14 | 山西农业大学 | CDK5 resistant nano antibody and application thereof |
| CN111410693B (en) * | 2020-04-15 | 2020-12-22 | 山西农业大学 | CDK5 resistant nano antibody and application thereof |
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| Publication number | Publication date |
|---|---|
| CN108635579B (en) | 2020-08-07 |
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