CN108101993B - 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 PDF

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CN108101993B
CN108101993B CN201810002537.XA CN201810002537A CN108101993B CN 108101993 B CN108101993 B CN 108101993B CN 201810002537 A CN201810002537 A CN 201810002537A CN 108101993 B CN108101993 B CN 108101993B
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antibody
nano antibody
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present
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CN108101993A (en
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林坚
李日飞
夏斌
周鹏
周斌
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JIANGSU JICUI MOLECULE ENGINEERING RESEARCH INSTITUTE Co.,Ltd.
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Peking University
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)

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Abstract

The present invention relates to the nano antibodies and its VHH chain of a kind 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 and some diseases associated with inflammation (such as rheumatism and psoriasis) antibody drug 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 has broad application prospects.
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 has met or exceeded the affinity magnitude of complete antibody.
(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 13-1 phasmid in conjunction with step；
Abscissa 5 is experimental group, coating CD147, uses 13-1 phasmid in conjunction with step.
Ordinate is 450nm absorbance value.
Fig. 3: nano antibody purifying figure, from left to right
Swimming lane 1 is bacterial protein；
Swimming lane 2 is thallus ultrasonication protein precipitation；
Swimming lane 3 is thallus ultrasonication supernatant protein；
Swimming lane 4 is the 13-1 nano antibody VHH chain of His affinitive layer purification；
Swimming lane 5 is molecular weight of albumen MARKER.
Fig. 4: western blot Western-blot map
With CD147 progress the SDS-PAGE electrophoresis, transferring film of recombinant expression, the recombination nano antibody 13-1 marked with HRP, The nano antibody MG53 of HRP label respectively in connection with two swimming lanes and develops the color.
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, 100ul is added in every hole, and 4 DEG C are coated with overnight.PBS is washed three times, and the skim milk of 300ul 4%, 37 DEG C of closings are added in every hole 2 hours.Phage display nano antibody library (about 1x10 is added after washing three times in PBS12CFU), 37 DEG C 1 hour.It is sucked out unbonded Bacteriophage, PBS is added and washes 5-10 all over (increasing washing times by wheel), then the PH=2.2 of 100ul is added after washing three times with PBST Glycine-HCI solution, 37 DEG C 7 minutes.Gently the bacteriophage of absorption is washed by piping and druming plate hole, and Tris-HCl is then added Solution (PH=8.8) 15ul takes 10ul to measure titre, is used for next round elutriation after remaining amplification.
After three-wheel elutriation, rescued at random from picked clones on the plate for surveying titre with helper phage M13K07 It helps, obtains the phasmid of displaying of target proteins, verified with phage-elisa, 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 13-1 are sent into sequencing, The nano antibody gene base sequence being inserted into is as follows:
5’-GCCATGGCCCAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAG ACTCTCCTGTACAGCCTCTAGTGGCACCAACTACATGGCCTGGTTCCGCCGGGCTCCAGGGAAGGAGCGCGAGGGG GTCGCAGGTATTTCAGCTGGTGGTAGTATCACATACTATACCGACTCCGTGAAGGGCCGATTCACCATCTCCCGAG ACAACGCCCAGAACACGGTGTATCTGCAAATGAACGGCCTGAAACCTGAGGACACTGCCATGTACTACTGTGCGGC CCGAGGGACATTTGGGCGGACCTCTTGGGGCGGTGAAAACTGGCATCGCCCTTCTCAGTATGACACCTGGGGCCAG GGGACCCAGGTCACCGTCTCCTCAG-3’
According to said gene base sequence, nano antibody in positive phasmid 13-1 is determined according to e. coli codon Amino acid sequence are as follows:
NH2-AMAQVQLQESGGGSVQAGGSLRLSCTASSGTNYMAWFRRAPGKEREGVAGISAGGSITYYTDSV KGRFTISRDNAQNTVYLQMNGLKPEDTAMYYCAARGTFGRTSWGGENWHRPSQYDTWGQGTQVTVSS-COOH
By structural analysis and sequence alignment, FR1-4, CDR1-3 of 13-1 in nano antibody are determined, as a result such as Fig. 1 institute Show.
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；Bacterium solution was collected by centrifugation in second day and uses 80ml PBS carries out ultrasonication after thallus is resuspended, and ultrasound 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) elute nano antibody, in obtained nano antibody solution be added PBS carry out ultrafiltration (3600rpm, 12min is repeated 3 times), obtained nano antibody is dissolved in PBS, and -80 DEG C of glycerol preservations of addition 20% are standby after measurement concentration With.
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 (according to concentration calculation volume), 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, and the usage amount of accurate recording REAGENT II, in case Use 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 addition. The pipette tips of 200ul are taken, sodium borohydride powder is inserted into, there is visible white powder at tip, are then transferred to enzyme mark object, are blown and beaten molten Solution mixes).It is added REAGENT III (3 times of II volume of about REAGENT).Previous step spends II 10ul of REAGENT, III 30 μ L of REAGENT is added in this step.Ensure enzyme conjugates PH 7.0 or so (additional amount of visual REAGENT II, accordingly Add the adjustment of REAGENT III), it mixes and terminates reaction.Glycerol adding to 50%, -20 DEG C save.
2.Western blot
Take protein sample: nano antibody 13-1 (3.98mg/mL), MG53 nano antibody (0.94mg/mL) and CD147 (0.6mg/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, it is remaining to contain mesh The separation gel of albumen be soaked in transferring film buffer, prevent that gelling is solid, deformation；Wetting: preparing 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, impregnates 30s, then It is put into transferring film buffer 10 minutes；The production of sandwich: transferred buffer in advance is placed in transfer is pressed from both sides in order and is impregnated Sponge, 1 layer of filter paper, gel, nitrocellulose filter, 1 layer of filter paper, sponge, guarantee every layer between there is no bubble.Assembling sequence: Transferring film presss from both sides black side (cathode)-foam-rubber cushion-filter paper-glue-film-filter paper-foam-rubber cushion-red face (anode)；Transferring film 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 (70V, 70min), nitre Acid cellulose film 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 13-1 or nano antibody MG53 of HRP label, 4 DEG C of overnight incubations are added；It is washed three times with TBST, often Secondary 10min；Add luminescent solution (A liquid and B liquid are mixed with 1:1) to develop the color, takes pictures.Western blot Western-blot map such as Fig. 4 institute Show, nano antibody 13-1 can specifically bind CD147, and nano antibody MG53 cannot then combine CD147.
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 (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.
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