CN107629125B - A kind of anti-CD147 nano antibody, its production method and application - Google Patents
A kind of anti-CD147 nano antibody, its production method and application Download PDFInfo
- Publication number
- CN107629125B CN107629125B CN201710799307.6A CN201710799307A CN107629125B CN 107629125 B CN107629125 B CN 107629125B CN 201710799307 A CN201710799307 A CN 201710799307A CN 107629125 B CN107629125 B CN 107629125B
- Authority
- CN
- China
- Prior art keywords
- antibody
- nano antibody
- ser
- seq
- present
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to a kind of nano antibodies of anti-CD147 albumen, while also disclosing the gene order for encoding the nano antibody and the host cell for expressing the nano antibody.Nano antibody gene order announced through the invention and host cell, the nano antibody being capable of high efficient expression, the research and development applied to kinds cancer detection reagent in Escherichia coli.
Description
Technical field
The invention belongs to molecular biology and immunological technique field, and in particular to phage displaying antibody library and nanometer
Antibody recombination and expression techniques are screened more particularly to a kind of bacteriophage nano antibody library for CD147 protein molecular, and nanometer is anti-
Structure, production method and the application of body.
Background technique
CD147 is a kind of wide expression in hematopoiesis and non-hematopoietic cell system, the transmembrane glycoprotein of molecular weight about 43-66kDa,
In 19p13.3, code area encodes 269 amino acid residues for the assignment of genes gene mapping, including 2 C2 type immune globulin areas of extracellular N-terminal,
The intracellular region of 39 amino acid residues of transmembrane region and C-terminal of 24 amino acid residues composition.CD147 wide expression and and other
The interaction of albumen enables to participate in a variety of different pathological processes, and with certain important diseases such as tumour, inflammation
Deng the closely related CD147 of occurrence and development in kinds of tumor cells, the high expression of tissue.Functional study discovery passes through induction week
It surrounds fibrocyte and tumour cell itself generates a variety of matrix metalloproteinases (MMPs, including MMP-1, MMP-2, MMP-9)
Degradation extracellular matrix, promotes the infiltration and transfer of tumour cell.CD147 extracellular region also contains 3 Asn glycosylation sites, table
Bright CD147 molecule N-terminal high glycosylation prompts the N-terminal high glycosylation of CD147 molecule, but glycosylated degree has tissue
Specificity, in different tissues different genera, since albumen distribution is same, glycosylation patterns are also different, different glycosyls
The function that change mode makes protein exhibits different again.Meanwhile the glycosylated degree of CD147 molecule determines that it activates the energy of MMP
Power, the deglycosylated CD147 molecule of purifying not can induce MMP secretion.Largely study carefully the result shows that, in various cancerous tissues, cell,
The universal high expression of CD147 molecule, and with the glycosylation of height.CD147 molecule it is glycosylation modified very rich, sugar chain accounts for about
The half of total molecular weight.It is current studies have shown that in tumour cell CD147 it is glycosylation modified to its inducer substance metalloprotein
The secretion of enzyme may be significant, to influence the occurrence and development of tumour.CD147 is the target molecules on kinds of tumors surface,
The antibody that CD147 is prepared using the relevant technologies is made into diagnostic kit or antibody drug, for cancer diagnosis and control
It treats.
Nano antibody is to research and develop on the basis of conventional antibodies with Protocols in Molecular Biology, and molecular weight is about
It is the antibody molecule for being currently known the smallest combinable antigen for 15KD.Initially there is Belgian scientist Hamers.R in camel
It is found in blood, common antibody protein is made of two heavy chains and two light chains, and is found from camel blood novel anti-
Body only has two heavy chains, and without light chain, these novel antibodies can combine closely as normal antibody in antigen, but unlike single-stranded
The such stick to each other aggregation of antibody is blocking.Compared with conventional antibodies, nano antibody have relative molecular mass is small, affinity is high,
The advantages such as stability is high, dissolubility is good, immunogenicity is low, penetration power is strong, humanization is simple.Therefore, therefore nano antibody is applied
Technical research CD147.
Summary of the invention
To solve the above problems, the present invention provides a kind of anti-CD147 antibody, its coded sequence, preparation method, Yi Jiying
With.Specifically,
On the one hand, the present invention provides a kind of anti-CD147 antibody, contains the CDR1 as shown in SEQ ID NO:3, by SEQ
The CDR2 and the CDR3 as shown in SEQ ID NO:5 that ID NO:4 shows.
Anti- CD147 antibody of the present invention is monoclonal antibody, Fab, F (ab) 2, Fv, scFv, single domain antibody, nanometer
Antibody etc..
Anti- CD147 antibody of the present invention, amino acid sequence is as shown in SEQ ID NO:1.
Anti- CD147 antibody of the present invention, further has label, and the label includes that enzyme marks (such as horseradish peroxide
Compound enzyme label, alkali phosphatase enzyme mark), optical markings (chemiluminescent labeling), fluorescent marker, isotope labelling etc..
Second aspect, the present invention provide a kind of gene, encode antibody of the present invention.
Gene of the present invention contains nucleotide sequence shown in SEQ ID NO:4.
Gene of the present invention can be DNA, cDNA, mRNA, further include noncoding region sequence other than coded sequence
Column, such as 5 ' UTR and 3 ' UTR etc..
The third aspect, the present invention provide a kind of nucleic acid construct, and the nucleic acid construct includes gene of the present invention.
Nucleic acid construct of the present invention can be reading frame, expression cassette, operon, plasmid, clay, viral vectors, bite
Bacteriophage vectors etc..
Fourth aspect, the present invention provide a kind of host, and the host includes gene of the present invention or nucleic acid construct.
Host of the present invention can be the common host of genetic engineering field, including prokaryotic hosts, eucaryon host.Institute
Stating prokaryotic hosts includes but is not limited to Escherichia coli, and the eucaryon host includes but is not limited to yeast (Pichia pastoris, wine brewing ferment
It is female), insect cell, mammalian cell, plant cell etc..
5th aspect, the present invention provide a kind of method for producing anti-CD147 antibody comprising: (1) cultivate place of the invention
Chief cell, (2) make the host expresses antibody, (3) separation and antibody purification from component antibody-containing.
The method of the anti-CD147 antibody of production of the present invention keeps e. coli host cell expression recombination anti-by induction
CD147 antibody separates and purifies CD147 from the supernatant of bacterial cell disruption.
6th aspect, the present invention provide a kind of method for preparing anti-CD147 antibody comprising: (1) it with CD147 albumen is sieved
Select phage displaying antibody library;(2) identification and sequencing of positive colony;(3) expression and purifying of CD147 recombinant antibodies;(4) resist
The identification of CD147 recombinant antibodies function.
The method of the present invention for preparing anti-CD147 antibody is coated with elisa plate/item with the CD147 of purifying, screens phagocytosis
The single domain antibody library of body surface display, process three to four-wheel wash in a pan sieve, and the picking phage clone from the bacteriophage under elution is and auxiliary
Helper phage M13K07 coinfection host strain obtains phasmid, identifies positive colony with PHAGE-ELISA and is sequenced.
7th aspect, the present invention provide the purposes of the anti-CD147 antibody.
The anti-CD147 antibody is used to prepare the detection kit or diagnostic kit of CD147 in test sample;It is described anti-
CD147 antibody can also be used in treatment and express related disease, such as tumour with CD147 high.
Compared with prior art, technical solution of the present invention has the advantage that
(1) molecule is smaller, drug delivery.Anti- CD147 nano antibody of the present invention is relative to common antibody or antibody piece
Section, molecule are smaller.When as targeted molecular, anti-CD147 nano antibody of the present invention is to active site (effector molecule)
Conformation influences and steric hindrance is all smaller, and effector molecule activity is higher, and using the anti-CD147 nano antibody of the present invention as target
With the molal quantity of higher effector molecule in the drug of Unit Weight when to molecule.
(2) immunogenicity is low, and metabolic characteristics is good.Antibody of the present invention is to remain the sequence to form VVH antibody
Ability, and be avoided as much as possible the introducing of inhuman foreign protein has taken into account effective function in conjunction with CD147 and alap different
Source property.In addition, the reduction of heterologous can also extend its half-life period in vivo other than reducing hypersensitivity, improve metabolism
Feature.
(3) affinity is high, and targeting is strong.The small molecule antigens such as segment of scFv or antibody binding protein is due to lacking antibody
The complete structure of molecule usually the affinity of antigen is much not achieved the level of complete antibody.The present invention passes through long term test,
Using the phage display single domain antibody library of high storage capacity, by condition optimizing and repeated screening, the CD147 finally obtained is received
Meter Kang Ti is 7.46 × 10 to the equilibrium dissociation constant of CD147-9M has met or exceeded the affinity magnitude of complete antibody.And
And CD147 nano antibody specific binding CD147 of the present invention, without combining its C-terminal domain C 87, specificity is good.
(4) performance is stablized, and plasticity is good.Relative to common antibody or antibody fragment, anti-CD147 single domain of the present invention is anti-
Therefore body, which only has a chain, can more easily be coupled with effector molecule, and keep the stable binding ability to CD147.This
Two ways is specifically demonstrated in description of the invention, covalently (peptide bond) is coupled by amalgamation and expression and 6 × His tag, is passed through quotient
Productization link reagent and enzyme label is coupled, and the antibody connection product of two ways production all has the ability in conjunction with CD147.
Detailed description of the invention
By reading the following detailed description of the preferred embodiment, various other advantages and benefits are common for this field
Technical staff will become clear.The drawings are only for the purpose of illustrating a preferred embodiment, and is not considered as to the present invention
Limitation.In the accompanying drawings:
Fig. 1: the amino acid sequence of nano antibody of the present invention.
FR1, FR2, FR3, FR4 are four framework regions;CDR1, CDR2, CDR3 are three complementary determining regions.
Fig. 2: PHAGE-ELISA experimental result picture.
Abscissa 2 is negative control group, coating CD147, uses random phage grain in conjunction with step;
Abscissa 3 is blank control group, coating PBS, uses empty phasmid in conjunction with step;
Abscissa 4 is unrelated control, and coating His label uses 12-3 phasmid in conjunction with step;
Abscissa 5 is experimental group, coating CD147, uses 12-3 phasmid in conjunction with step.
Ordinate is 450nm absorbance value.
Fig. 3: nano antibody purifying figure.
Swimming lane 4 is thallus ultrasonication supernatant;
Fig. 4: western blot Western-blot map
Negative control: SDS-PAGE electrophoresis, transferring film are carried out with Ferritin loading, the recombination nano antibody marked with HRP
12-3 combines, develops the color;
Positive control: two swimming lanes carry out SDS-PAGE electrophoresis, transferring film respectively with CD147 the and C87 loading of recombinant expression,
It is combined with commercially available anti-CD147 antibody HAb18, colour developing;Wherein C87, that is, CD147C terminal domains albumen;
Experimental group: two swimming lanes carry out SDS-PAGE electrophoresis, transferring film respectively with CD147 the and C87 loading of recombinant expression, with
The recombination nano antibody 12-3 of HRP label combines, develops the color.
The binding force of Fig. 5: nano antibody 12-3 and CD147 measure, equilibrium dissociation constant KD=7.46 × 10-9M。
Specific embodiment
The illustrative embodiments of the disclosure are more fully described below with reference to accompanying drawings.Although showing this public affairs in attached drawing
The illustrative embodiments opened, it being understood, however, that may be realized in various forms the disclosure without the reality that should be illustrated here
The mode of applying is limited.It is to be able to thoroughly understand the disclosure on the contrary, providing these embodiments, and can be by this public affairs
The range opened is fully disclosed to those skilled in the art.
Embodiment one, the elutriation of anti-CD147 protein nano antibody, the identification and sequencing of positive colony
Using the method for solid phase elutriation, CD147 albumen is diluted to 30-100ug/ul with 1xPBS, coating arrives elisa plate
On item, every empty addition 100ul, 4 DEG C are coated with overnight.PBS is washed three times, and the skim milk of 300ul4%, 37 DEG C of closings 2 are added in every hole
Hour.PBS wash three times after be added phage display library (there are about 1x1012CFU), 37 DEG C 1 hour.Unbonded bacteriophage is sucked out,
Be added PBS wash 5-10 all over (by wheel increase washing times), then PBST wash three times after be added 100ul PH=2.2 glycine-salt
Acid solution, 37 DEG C 7 minutes.Gently the bacteriophage of absorption is washed by piping and druming plate hole, and Tris-Hcl solution (PH=is then added
8.8) 15ul takes 10ul to measure titre, is used for next round elutriation after remaining amplification.After three-wheel elutriation, dripped at random from survey
Picked clones are rescued with helper phage M13K07 on the plate of degree, obtain the phasmid of displaying of target proteins, are used
Phage-elisa is verified, and sample-adding table is as follows:
Table 1:PHAGE-ELISA experimental design and step
The experimental result of PHAGE-ELISA is as shown in Figure 2.The positive phagemid clones for numbering as 12-3 are sent into sequencing,
The nano antibody gene base sequence being inserted into is as follows:
5’-CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTG
TGCAGCCTCTGGATACACCTTCAGTAGCAGCAATTGCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGCGAG
GATGTCGCAATTATTTCTGTTCGTGGTGGCATCACATATTATGCCGACTCCGTGAAGGGCCGATTCACCATCTCCC
GTGACAGCGCCAAGAACACGCTGTCGCTGCAAATGAACAGCCTGAAACCTGAGGACACTGCCGTGTACTACTGTGC
GGCAGATAATCCCAAGTATAGGGCATTGTCCGATGCTCACTGCAATTGGAGTTATCGATACTGGGGCCAGGGGACC
CAGGTCACCGTCTCCTCA-3’
According to said gene base sequence, nano antibody in positive phasmid 12-3 is determined according to e. coli codon
Amino acid sequence are as follows:
NH2-QVQLQESGGGSVQAGGSLRLSCAASGYTFSSSNCMGWFRQAPGKEREDVAIISVRGGITYYADS
VKGRFTISRDSAKNTLSLQMNSLKPEDTAVYYCAADNPKYRALSDAHCNWSYRYWGQGTNVTVSS-COOH
By structural analysis and sequence alignment, FR1-4, CDR1-3 of nano antibody 12-3 are determined, as a result as shown in Figure 1.
The expression and purifying of embodiment two, nano antibody
Above-mentioned base sequence is inserted on PET15b plasmid, construction of expression vector.The PET15b plasmid for taking 2ul to build
It is added in the BL21 competence of 50ul, places 30 minutes be then placed in into 42 DEG C of water-baths heat shock 90 seconds on ice, place on ice
10 minutes, 800 μ L non-resistant LB liquid mediums are added into suspension, 37 DEG C after mixing, 150rpm shaking table oscillation incubation 1h,
3500rpm room temperature is centrifuged 4min, and careful inhale abandons 700 μ L supernatants, stay about 200 μ L culture mediums, blow and beat suspension thalline repeatedly, be coated on
On LB-Amp solid plate;Plate is placed in 37 DEG C of constant incubators, is inverted overnight incubation after just putting culture 10min.Second day
It from picking monoclonal on the plate being incubated overnight, is placed in 10mL LB-Amp fluid nutrient medium, 37 DEG C, the concussion of 200rpm shaking table
Be incubated for 8h, by centrifuge tube bacterium solution be added 1L LB-Amp fluid nutrient medium, 37 DEG C, 200rpm shaking table shaken cultivation 4h, until bacterium solution
OD600>=0.6,1mL 0.1M IPTG is added, 16 DEG C, 150rpm/min induces 15h;Use was collected by centrifugation in bacterium solution in second day
80mlPBS carries out ultrasonication after thallus is resuspended, and condition 200w is crushed 3s, interval 3s.Then 4 DEG C, 8000g is collected by centrifugation
Supernatant crosses nickel column, and nano antibody is adsorbed onto nickel column, washes away foreign protein with cleaning solution (10mM imidazole solution), eluent is added
(250mM imidazole solution) elutes nano antibody, and PBS is added in obtained nano antibody solution and carries out ultrafiltration (3600rpm/
Min, 12min are repeated 3 times), obtained nano antibody is dissolved in PBS, and -80 DEG C of glycerol guarantors of addition 20% after concentration are measured
It deposits spare.
Take the nanometer of recombination bacillus coli bacterial protein, thallus ultrasound precipitation, thallus ultrasound supernatant, affinitive layer purification
Antibody carries out SDS-PAGE, and experimental result is as shown in Figure 3.
The Western blot verifying of embodiment three, nano antibody
1. the label of nano antibody HRP
It takes 40ug antibody-solutions (volume is according to concentration calculation), the activation of I A type of 12ul (100 μ L=1mg) REAGENT is added
Horseradish peroxidase enzyme.With the tune pH value of REAGENT II to 9.5 or so, (about 10ul, because of the difference of the buffer of antibody, amount has
Institute is different, but ensures that PH 9.0 or more, can add REAGENT II (10 μ L), and the usage amount of accurate recording REAGENT II,
In case using in next step) 37 DEG C, 30min.Micro sodium borohydride is added.(it is suitble to enzyme mark object to place for a long time, immediately using without adding
Add.The pipette tips of 200ul are taken, sodium borohydride powder is inserted into, has visible white powder at tip, be then transferred to enzyme mark object, blow
It plays dissolution to mix).It is added REAGENT III (REAGENT II of about 3 times of volumes).Previous step spends II 10ul of REAGENT,
III 30 μ L of REAGENT is added in this step.Ensure that enzyme conjugates PH 7.0 or so, can add the adjustment of REAGENT III), it mixes eventually
Only react.Glycerol adding to 50%, -20 DEG C save.
2.Western blot
Collect protein sample: nano antibody 12-3 (1.07mg/mL), C87 (CD147c terminal domains albumen, 0.65mg/
) and CD147 (0.6mg/mL) mL;PAGE gel electrophoresis: matching glue (15% separation gel), race glue (low pressure 80V, 40min,
High pressure 120V, 50min);It plays glue: gel is taken out from glass plate, cut off the separation gel of spacer gel and bromophenol blue lower part, be left
The separation gel containing destination protein be soaked in transferring film buffer, prevent that gelling is solid, deformation;Wetting: prepare 2 filter paper, 1
Pvdf membrane, size is similar (filter paper is big as glue, and film is larger than glue) with gel size, and pvdf membrane, which is first put into methanol, to be impregnated
30s is placed into transferring film buffer 10 minutes;The production of sandwich: transferred buffering in advance is placed in transfer is pressed from both sides in order
The sponge of liquid immersion, 1 layer of filter paper, gel, nitrocellulose filter, 1 layer of filter paper, sponge guarantee do not have bubble between every layer.Assembling
Sequentially: transferring film presss from both sides black side (cathode)-foam-rubber cushion-filter paper-glue-film-filter paper-foam-rubber cushion-red face (anode);Turn
Transfer: being folded up the transferring film buffer that 4 DEG C of pre-coolings are added into transfer groove, gel face is connected with cathode by film (70V, 70min),
Nitrocellulose filter is connected with anode;Closing: the film after transferring film is complete washes 10min with cleaning solution, then sucks cleaning solution, then plus
5% skim milk closes 1h.It is washed 3 times after closing with TBST, each 10min;
The nano antibody 12-3 for adding HRP to mark, 4 DEG C of overnight incubations;It is washed three times with TBST, each 10min;Add luminescent solution (A
Liquid and B liquid are mixed with 1:1) colour developing, it takes pictures.Western blot Western-blot map is as shown in Figure 4.
The measurement of example IV, anti-CD147 nano antibody binding force
CD147 albumen is diluted to 10ug/ml (every 200 μ L of hole), purified nanotubes antibody obtained in embodiment two is dilute
The BSA to 80 μ g/mL, 40 μ g/mL and 20 μ g/mL (every 200 μ L of hole) the dilution Tween-20+0.1% for being PBS+0.1% is released,
According to the form below is loaded to Octet RED/QK:
Table 2: the binding force measurement of nano antibody 12-3 and CD147
Binding force measurement | Experimental group | Control group |
Equilibrium liquid | PBST+BSA | PBST+BSA |
Sensor coating buffer | CD147+PBST+BSA | CD147+PBST+BSA |
Equilibrium liquid | PBST+BSA | PBST+BSA |
Reaction solution | PBST+BSA+ nano antibody | PBST+BSA |
Dissociation solution | PBST+BSA | PBST+BSA |
The setting of detecting step time is as follows
(1) PBST equilibrium stage, SA sensor react 60s in 200 μ L equilibrium liquids;
(2) Loading stage, SA sensor react 200s in 200 μ L diluted protein solutions;
(3) Association stage, SA sensor react 200s in 200 μ L nano antibody gradient dilution liquid;
(4) Dissociation stage, SA sensor react 200s in 200 μ L dissociation solutions.
The above operation carries out under the conditions of 1000rpm at 30 DEG C.
Testing result is as shown in figure 5, wherein
Ka=1.71 × 105Ms-1
Kd=1.28 × 10-3s-1
KD=Kd/Ka=7.46 × 10-9M
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
In the technical scope disclosed by the present invention, any changes or substitutions that can be easily thought of by anyone skilled in the art,
It should be covered by the protection scope of the present invention.Therefore, protection scope of the present invention should be with the protection model of the claim
Subject to enclosing.
Sequence table
<110>applicant's title: Peking University
<120>a kind of anti-CD147 nano antibody, its production method and application
<130>nothing
<160> 9
<170> PatentIn version 3.5
<210> 1
<211> 129
<212> PRT
<213>anti-CD147 nano antibody
<400> 1
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Ser Ser Ser
20 25 30
Asn Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Asp
35 40 45
Val Ala Ile Ile Ser Val Arg Gly Gly Ile Thr Tyr Tyr Ala Asp Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Ser Ala Lys Asn Thr Leu
65 70 75 80
Ser Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Ala Asp Asn Pro Lys Tyr Arg Ala Leu Ser Asp Ala His Cys
100 105 110
Asn Trp Ser Tyr Arg Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser
115 120 125
Ser
<210> 2
<211> 387
<212> DNA
<213>anti-CD147 nano antibody encoding gene
<400> 2
caggtgcagc tgcaggagtc tgggggaggc tcggtgcagg ctggagggtc tctgagactc 60
tcctgtgcag cctctggata caccttcagt agcagcaatt gcatgggctg gttccgccag 120
gctccaggga aggagcgcga ggatgtcgca attatttctg ttcgtggtgg catcacatat 180
tatgccgact ccgtgaaggg ccgattcacc atctcccgtg acagcgccaa gaacacgctg 240
tcgctgcaaa tgaacagcct gaaacctgag gacactgccg tgtactactg tgcggcagat 300
aatcccaagt atagggcatt gtccgatgct cactgcaatt ggagttatcg atactggggc 360
caggggaccc aggtcaccgt ctcctca 387
<210> 3
<211> 10
<212> PRT
<213> CDR1
<400> 3
Gly Tyr Thr Phe Ser Ser Ser Asn Cys Met
1 5 10
<210> 4
<211> 7
<212> PRT
<213> CDR2
<400> 4
Ser Val Arg Gly Gly Ile Thr
1 5
<210> 5
<211> 21
<212> PRT
<213> CDR3
<400> 5
Ala Ala Asp Asn Pro Lys Tyr Arg Ala Leu Ser Asp Ala His Cys Asn
1 5 10 15
Trp Ser Tyr Arg Tyr
20
<210> 6
<211> 25
<212> PRT
<213> FR1
<400> 6
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser
20 25
<210> 7
<211> 17
<212> PRT
<213> FR2
<400> 7
Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Asp Val Ala Ile
1 5 10 15
Ile
<210> 8
<211> 38
<212> PRT
<213> FR3
<400> 8
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Ser
1 5 10 15
Ala Lys Asn Thr Leu Ser Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Val Tyr Tyr Cys
35
<210> 9
<211> 11
<212> PRT
<213> FR4
<400> 9
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
1 5 10
Claims (7)
1. a kind of anti-CD147 antibody contains the CDR1 as shown in SEQ ID NO:3, as shown in SEQ ID NO:4
The CDR2 and CDR3 as shown in SEQ ID NO:5;
The anti-CD147 antibody is nano antibody;
The amino acid sequence of the anti-CD147 antibody is as shown in SEQ ID NO:1.
2. a kind of gene encodes antibody described in claim 1.
3. the gene as described in claim 2, it includes nucleotide sequences shown in SEQ ID NO:2.
4. a kind of nucleic acid construct, it includes the genes of claim 2 or 3.
5. a kind of host it includes the gene of claim 2 or 3 or includes nucleic acid construct described in claim 4.
6. a kind of method for producing anti-CD147 antibody comprising:
(1) host described in claim 5 is cultivated,
(2) make host expresses antibody,
(3) separation and antibody purification from component antibody-containing.
7. the purposes of anti-CD147 antibody described in claim 1 is used to prepare the diagnostic kit of detection CD147, or uses
In the drug of preparation treatment and CD147 high expression related disease.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201710799307.6A CN107629125B (en) | 2017-09-07 | 2017-09-07 | A kind of anti-CD147 nano antibody, its production method and application |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201710799307.6A CN107629125B (en) | 2017-09-07 | 2017-09-07 | A kind of anti-CD147 nano antibody, its production method and application |
Publications (2)
Publication Number | Publication Date |
---|---|
CN107629125A CN107629125A (en) | 2018-01-26 |
CN107629125B true CN107629125B (en) | 2019-10-15 |
Family
ID=61101771
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201710799307.6A Active CN107629125B (en) | 2017-09-07 | 2017-09-07 | A kind of anti-CD147 nano antibody, its production method and application |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN107629125B (en) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110128536A (en) * | 2018-02-02 | 2019-08-16 | 暨南大学 | The single domain antibody of resisting tumour stem cells marker protein CD133 and its application |
CN108586614B (en) * | 2018-04-24 | 2021-07-23 | 南京市妇幼保健院 | EpCAM single domain antibody D7 |
CN108530538B (en) * | 2018-04-24 | 2021-07-23 | 南京市妇幼保健院 | EpCAM single domain antibody E6 |
CN112266926A (en) * | 2020-10-19 | 2021-01-26 | 上海亚联抗体医药有限公司 | Synthetic antibody library and construction method and application thereof |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102316898A (en) * | 2008-09-29 | 2012-01-11 | 森托科尔奥索生物科技公司 | Anti-CD147 antibodies, methods, and uses |
-
2017
- 2017-09-07 CN CN201710799307.6A patent/CN107629125B/en active Active
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102316898A (en) * | 2008-09-29 | 2012-01-11 | 森托科尔奥索生物科技公司 | Anti-CD147 antibodies, methods, and uses |
Non-Patent Citations (2)
Title |
---|
抗CD133纳米抗体的分离与鉴定;李玲霞等;《生物技术》;20160229;第26卷(第1期);摘要,第87页左栏第1段-第88页左栏第2段 * |
纳米抗体及其应用;杨珂等;《细胞与分子免疫学杂志》;20081231;第24卷(第4期);第425-427页 * |
Also Published As
Publication number | Publication date |
---|---|
CN107629125A (en) | 2018-01-26 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN107629125B (en) | A kind of anti-CD147 nano antibody, its production method and application | |
WO2021244089A1 (en) | Sars-cov-2 spike protein binding molecule and application thereof | |
CN114409773B (en) | Nanobody for novel coronavirus and application thereof | |
CN108341870A (en) | A kind of anti-BSA nano antibodies, its production method and application | |
CN107573417A (en) | Mycoplasma pneumoniae chimeric antigen, the antigen detecting agent and both preparation methods | |
CN114075279B (en) | pH-independent long-acting type antiserum albumin nano antibody and application thereof | |
CN108383907A (en) | Nano antibody for Procalcitonin and its application | |
CN114276445B (en) | Rotavirus recombinant protein specific antibody, plasmid vector and method | |
CN106699884B (en) | Anti-human C-reactive protein antibody and application thereof | |
CN113480659B (en) | Human source anti-mycobacterium tuberculosis complex LAM monoclonal antibody and preparation and application thereof | |
CN116396381A (en) | Preparation and application of human adeno-associated virus (AAV) single domain antibody | |
CN106928363A (en) | A kind of FAP nano antibodies Nb12 | |
WO2022011717A1 (en) | Nanobody against novel coronavirus, and use thereof | |
CN108101993B (en) | A kind of anti-CD147 nano antibody, its production method and application | |
CN107663237B (en) | A kind of anti-CD147 antibody and its production method and application | |
CN105968205A (en) | Nano antibody for anti-prostate specific membrane antigen | |
KR20180050261A (en) | Novel fusion proteins comprising single chain fragment variable and viral coat protein, and uses thereof | |
CN106749659B (en) | Anti-human CRP antibody and application thereof | |
CN113683684B (en) | Anti-hepatitis B virus surface antigen antibody, antibody pair, detection reagent containing same and kit | |
CN106928358B (en) | CD105 nano antibody Nb168 | |
CN106928355B (en) | CD105 nano antibody Nb184 | |
CN113621079A (en) | Fusion protein of Fab antibody and calf intestinal alkaline phosphatase and preparation method thereof | |
CN106928368A (en) | A kind of FAP nano antibodies Nb57 | |
CN106928360B (en) | CD105 nano antibody Nb68 | |
CN106928359B (en) | CD105 nano antibody Nb59 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |