CN107629125B

CN107629125B - A kind of anti-CD147 nano antibody, its production method and application

Info

Publication number: CN107629125B
Application number: CN201710799307.6A
Authority: CN
Inventors: 林坚; 李日飞; 夏斌; 周鹏
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2017-09-07
Filing date: 2017-09-07
Publication date: 2019-10-15
Anticipated expiration: 2037-09-07
Also published as: CN107629125A

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

A kind of anti-CD147 nano antibody, its production method and application

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 1 is positive controls, direct coated phage；

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 1 is molecular weight of albumen MARKER；

Swimming lane 2 is bacterial protein；

Swimming lane 3 is thallus ultrasonication precipitating；

Swimming lane 4 is thallus ultrasonication supernatant；

Swimming lane 5 is the 12-3 nano antibody after His affinity chromatography.

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.

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Claims

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.