CN109265548A - Anti- PD-L1 nano antibody and its coded sequence, preparation method and application - Google Patents
Anti- PD-L1 nano antibody and its coded sequence, preparation method and application Download PDFInfo
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- CN109265548A CN109265548A CN201811065587.9A CN201811065587A CN109265548A CN 109265548 A CN109265548 A CN 109265548A CN 201811065587 A CN201811065587 A CN 201811065587A CN 109265548 A CN109265548 A CN 109265548A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- 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
- C07K16/2827—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 against B7 molecules, e.g. CD80, CD86
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
- G01N2333/70532—B7 molecules, e.g. CD80, CD86
Abstract
The invention discloses anti-PD-L1 nano antibody and its coded sequence, preparation method and application, the amino acid sequence of the antibody VHH chain is as shown in SEQ ID NO:2, SEQ ID NO:11 or SEQ ID NO:20.Compared with prior art, the invention has the following advantages that (1) anti-PD-L1 nano antibody of the present invention has high degree of specificity, detection sensitivity is high；(2) antibody can provide basis in E. coli, thus for the mass production of subsequent immunoreagent and tumors pharmaceutical combination production.
Description
Technical field
The invention belongs to field of biomedicine technology, are related to a kind of new PD-L1 antibody or its functional fragment, specially
Anti- PD-L1 nano antibody and its coded sequence, preparation method and application.
Background technique
1 ligand 1 of the programmed death factor (programmed death 1ligand 1, PD-L1) is also known as CD274, B7-
H1 is B7 family member, is the ligand of PD-1, belongs to I type transmembrane protein.Under normal circumstances, the PD-1 on T cell surface can
Inhibiting the function of T lymphocyte prevents the generation of autoimmune disease to inhibit autoimmune response.But in tumour,
After the PD-L1 of tumor cells expression is in conjunction with the PD-1 on T cell surface, T can be led to by the inhibiting effect to T lymphocyte
Cell Proliferation is lowered, or even induction of T cell apoptosis, promotes the immunologic escape of tumour.And PD-1/PD-L1 negative regulation access is blocked,
Can activating immune system function, killing tumor cell.There are many target PD-L1 monoclonal antibody, such as Roche on the market at present
PD-L1 monoclonal antibody atezolizumab (Tecentriq), Astrazeneca AB research and development Durvalumab (trade name:
Imfinzi), Pfizer/Merck avelumab (trade name: Bavencio), these PD-L1 monoclonal antibodies have been achieved with into clinic
Function application.But monoclonal antibody has the development cycle long, high production cost, molecular weight is big, it is difficult to the disadvantages of penetrating tissue.This
A little factors limit its in clinical application universal, and nano antibody is current the smallest antibody molecule, has stability
The advantages that getting well, at low cost, good water solubility, may pass through blood-brain barrier.And select wasabi as the fusion of expression nano antibody
Segment has expression quantity height, at low cost, the advantages that application convenient for detection.
Summary of the invention
The technical issues of solution: for overcome the deficiencies in the prior art, obtaining a kind of detection sensitivity height, and stability is good,
It is at low cost, it is easy to the nano antibody of practical application, the present invention provides anti-PD-L1 nano antibody and its coded sequences, preparation side
Method and application.
Technical solution: anti-PD-L1 nano antibody, the amino acid sequence of the antibody VHH chain such as SEQ ID NO:2, SEQ
Shown in ID NO:11 or SEQ ID NO:20.
Anti- PD-L1 nano antibody, the antibody VHH chain includes framework region and complementary determining region；Wherein:
The framework region includes following 4 sections of amino acid sequences: FR1, SEQ ID NO:3, FR2, SEQ ID NO:5, FR3,
SEQ ID NO:7, FR4, SEQ ID NO:9；The complementary determining region includes following 3 sections of amino acid sequences: CDR1, SEQ ID
NO:4, CDR2, SEQ ID NO:6, CDR3, SEQ ID NO:8；
Alternatively, the framework region includes following 4 sections of amino acid sequences: FR1, SEQ ID NO:12, FR2, SEQ ID NO:
14, FR3, SEQ ID NO:16, FR4, SEQ ID NO:18；The complementary determining region includes following 3 sections of amino acid sequences:
CDR1, SEQ ID NO:13, CDR2, SEQ ID NO:15, CDR3, SEQ ID NO:17；
Alternatively, the framework region includes following 4 sections of amino acid sequences: FR1, SEQ ID NO:21, FR2, SEQ ID NO:
23, FR3, SEQ ID NO:25, FR4, SEQ ID NO:27；The complementary determining region includes following 3 sections of amino acid sequences:
CDR1, SEQ ID NO:22, CDR2, SEQ ID NO:24, CDR3, SEQ ID NO:26.
The gene of anti-PD-L1 nano antibody is encoded, nucleotides sequence is classified as SEQ ID NO:1, SEQ ID NO:10 or SEQ
ID NO:19。
Recombinant vector includes SEQ ID NO:1, SEQ ID NO:10 or SEQ ID NO:19 sequence in the recombinant vector
Column.
Host cell includes SEQ ID NO:1, SEQ ID NO:10 or SEQ ID NO:19 sequence in the host cell
Column.
The preparation method of anti-PD-L1 nano antibody described above, comprising the following steps:
(1) anti-PD-L1 nano antibody phage library is constructed；
(2) the nano antibody bacteriophage that there is specific bond with PD-L1 is selected；
(3) host of carrier of the culture comprising encoding anti-PD-L1 nano antibody nucleic acid；
(4) purifying obtains anti-PD-L1 nano antibody from host cell.
Preferably, the library of anti-PD-L1 nano antibody is constructed using nested PCR method, wherein first round amplified reaction draws
Object sequence is SEQ ID NO:28-29, and the second wheel amplification primer sequence is SEQ ID NO:30-31.
Immune conjugate, the immune conjugate contain the VHH chain or anti-PD-L1 nano antibody of anti-PD-L1 nano antibody；With
Wasabi albumen.
Above-described anti-PD-L1 nano antibody prepares the kit of detection PD-L1 at (a)；(b) preparation treatment tumour medicine
Application in object.
Above-described immune conjugate prepares the kit of detection PD-L1 at (a)；(b) it prepares in tumor
Application.
The experimental principle of anti-PD-L1 nano antibody of the present invention is: using technique for gene engineering, PD-L1 is immunized
Antibody heavy chain variable region gene is showed in T7 phage surface in camel serum afterwards, recycles PD-L1 from phage antibody library
In filter out specific recognition PD-L1 phage surface nano antibody, by corresponding to the nano antibody that is shown and its
Gene is present in the same recombinant phage, after the bacteriophage nano antibody for obtaining specific recognition PD-L1, by the phagocytosis
The DNA of body carries out PCR amplification, and the nano antibody that being sequenced can be obtained in the recombinant phage corresponds to gene.
The utility model has the advantages that (1) anti-PD-L1 nano antibody of the present invention has high degree of specificity, detection sensitivity is high；(2)
The antibody can be in E. coli, thus is the mass production of subsequent immunoreagent and tumors pharmaceutical combination
Production provides basis.
Detailed description of the invention
Figure 1A is nest-PCR1 product agarose gel electrophoresis figure, and wherein M is DNA marker；1 produces for nest-PCR1
Object；
Figure 1B is nest-PCR2 product agarose gel electrophoresis figure, and wherein M is DNA marker；1 produces for nest-PCR2
Object；
Fig. 1 C is anti-PD-L1 nano antibody library diversity qualification figure, and wherein M is DNA molecular standard；1-10 is anti-PD-
The monoclonal phage PCR product selected at random in L1 nano antibody library；
Fig. 2 is the SDS- of immune conjugate wasabi-antiPD-L1Nb28, wasabi and 3 kinds of anti-PD-L1 nano antibodies
PAGE figure, wherein M is molecular weight standard, and 1 is wasabi-antiPD-L1Nb28 albumen, and 2 be wasabi albumen, and 3-5 is
antiPD-L1Nb15,antiPD-L1Nb22,antiPD-L1Nb28；
Fig. 3 is the specific outcome figure that enzyme-linked immunization identifies nano antibody；
Fig. 4 is that nano antibody detects HEK293T/PD-L1 fluorogram.
Specific embodiment
Following embodiment further illustrates the contents of the present invention, but should not be construed as limiting the invention.Without departing substantially from
In the case where spirit of that invention and essence, to modification made by the method for the present invention, step or condition and replaces, belong to the present invention
Range.Unless otherwise specified, the conventional means that technological means used in embodiment is well known to those skilled in the art.
Embodiment 1
1 anti-PD-L1 nano antibody library construction
(1) 1mg is mixed with PD-L1 extracellular fragment (19-239) antigen of His label with Freund's adjuvant in equal volume, is immunized
One Xinjiang two-humped camel is immunized 5 times altogether once a week, and stimulation B cell expresses the nano antibody of antigentic specificity；
(2) it after being immunized, extracts 100mL camel peripheral blood lymphocytes and extracts total serum IgE；
(3) reverse transcription PCR
I. RT-PCR kit is used, by peripheral blood lymphocytes total serum IgE reverse transcription at cDNA,
II. cDNA is expanded by nest-PCR, obtains the VHH segment of camel heavy chain antibody.
Using above-mentioned resulting cDNA as template, nest-PCR1up (SEQ ID NO:28) and nest-PCR1down (SEQ
ID NO:29) it is primer, carry out first round PCR amplification.After reaction, agarose gel electrophoresis detects PCR product to PCR, coagulates
Gel electrophoresis is the results show that amplification gene segment has specific band at 700bp.Gel extraction purpose band (see Figure 1A).
Using recovery product as template, nest-PCR2up (SEQ ID NO:30) and nest-PCR2down (SEQ ID NO:
31) it is primer, carries out the second wheel PCR amplification.After reaction, agarose gel electrophoresis detects PCR product, amplification gene to PCR
Segment has specific band at 500bp.Gel extraction purpose band, as VHH segment (see Figure 1B).The primer is as follows:
| Primer | Primer sequence |
| nest-PCR1up | 5'>GTCCTGGCTGCTCTTCTACAAGG<3' |
| nest-PCR1down | 5'>GGTACGTGCTGTTGAACTGTTCC<3' |
| nest-PCR2up | 5'>CCGGAATTCTCAGGTGCAGCTGGTGGAGTCTGG<3' |
| nest-PCR2down | 5'>GCCCAAGCTTTGAGGAGACGGTGACCTGGGT<3' |
(4) double digestion of VHH segment
Double digestion is carried out 3 hours with EcoR I and Hind III, after agarose gel electrophoresis, gel extraction.
(5) building of T7 phage antibody library
Nano antibody library is constructed with the T7Select10 kit of Novagen company.VHH segment is connected with T7 carrier,
Carry out the packaging of T7 bacteriophage.By plaque titer determination, the nano antibody library titre of building is about 1 × 107pfu/ml。
(6) amplification of T7 phage antibody library
T7 phage antibody library is expanded with liquid cracking process.
(7) recombination fraction of PD-L1 protein nano antibody library is detected
The plaque chosen in (5) at random extracts phage DNA；Using it as template, according to T7Select10 kit explanation
Book carries out PCR reaction, and agarose gel electrophoresis detects PCR product, and electrophoresis result is shown, the PD-L1 protein nano antibody of building
The recombination positive rate in library is 100% (see Fig. 1 C).
The 2 anti-PD-L1 nano antibodies of nickel ion metal chelate affinity chromatography medium Ni-NTA elutriation
(1) cleaning of Ni-NTA medium:
It takes 100ulNi-NTA medium in 1.5mlEP pipe, adds 1ml sterilizing washing 5 times, use 0.05%TBST for the last time
Instead of aqua sterilisa.
(2) it closes:
1ml BSA confining liquid is added in washed Ni-NTA medium, closes 1h；After closing, TBST is washed 4 times.
(3) bacteriophage weeded out except non-specific binding is born:
Phage library is diluted to 1ml with TBST, in the Ni-NTA medium after being added to closing, is placed on overturning well distributing rocker,
Room temperature combination 30min；Centrifugation, supernatant are the T7 phage library after being negative sieve.
(4) with the screening of the bacteriophage of PDL1 albumen specific bond:
20ug PD-L1 albumen, room temperature combination 30min is added in phage library after negative sieve.Then envelope is added mixture to
In the Ni-NTA medium closed, room temperature combination 30min；Supernatant is abandoned in centrifugation.TBST washes the precipitating of acquisition, washes altogether 5 times；Centrifugation is abandoned
Supernatant；400ul TB culture medium is added to mix, is equally divided into two parts, portion is used to measure the titre of the bacteriophage after screening, and one
Part is used to expand the bacteriophage after screening.
(5) amplification of the bacteriophage after screening:
Bacteriophage after screening is added in 50ml host strain, 37 DEG C of shake cultures, culture is to there is white flock precipitate
When appearance, stop culture；Centrifugation, supernatant are the bacteriophage after the first round screening expanded, the screening for next round；By phase
Same screening step, screening 3-4 wheel.
The bacteriophage of the anti-PD-L1 of 3ELISA identification specificity
Last above-mentioned wheel screens resulting bacteriophage, cultivates on the TB solid medium of 150mm, and select 70 lists
Clone's plaque carries out liquid cracking process amplification in BLT5403 host strain, is centrifuged, supernatant is monoclonal phage；
1ug PD-L1 (extracellular fragment) albumen coating is added in the every hole of elisa plate, and 4 DEG C overnight, and BSA room temperature closes 1h within second day；
Monoclonal phage is added in the every hole of experimental group, and the wild type T7 bacteriophage of equivalent is added in control group, is incubated at room temperature 1h；TBST is washed away
Then rabbit-anti T7 bacteriophage 10A antibody is added in unbonded bacteriophage, be incubated at room temperature 1h；TBST is washed 3 times, and HRP mark is then added
The goat anti-rabbit antibody of note is incubated at room temperature 1h；TBST is washed 3 times, and developing solution is then added, and elisa plate is placed in microplate reader, is read and is inhaled
Light value determines that it is positive colony when experimental port is greater than 2 with control wells light absorption value ratio；Extract positive colony bacteriophage
DNA carries out sequencing analysis.
Finally obtain 3 kinds of nucleotide sequences；Its amino acid sequence is analyzed, these three sequences all have typical nanometer
Antibody structure is made of framework region (FR1, FR2, FR4, FR4) and complementary determining region (CDR1, CDR2 and CDR3).This three plants are bitten
The nucleotide sequence of thallus, amino acid sequence are as follows
Anti- PD-L1-VHH15 nucleotide sequence (SEQ ID NO:1):
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGGCTCTGAGACTCTCCTGTGCA
GCCTCTGGATACACCTCCACTAGCAACTGCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGCGAGGGGGTCGC
AGCTATTTATACTGGTGGTGGTAGCACATACTATGCCGACTCCGTGAAGGGCCGATTCACCATCTCCCAAGACAACG
CCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACTGCCATGTACTACTGTGCGGCAGGGGAT
TTGGACCCCGCCGAATATAGCGACTATGACCCTACCGTCTTTAACTACTGGGGCCAGGGGACCCTGGTCACCGTCTC
CTCA
Anti- PD-L1-VHH15 amino acid sequence (SEQ ID NO:2):
QVQLVESGGGSVQAGGALRLSCAASGYTSTSNCMGWFRQAPGKEREGVAAIYTGGGSTYYADSVKGRFT
ISQDNAKNTVYLQMNSLKPEDTAMYYCAAGDLDPAEYSDYDPTVFNYWGQGTLVTVSS
Framework region (FR1-FR4) and complementary determining region (CDR1-CDR3) amino acid sequence:
FR1 (SEQ ID NO:3): QVQLVESGGGSVQAGGALRLS
CDR1 (SEQ ID NO:4): CAASGYTSTSNCMG
FR2 (SEQ ID NO:5): WFRQAPGK
CDR2 (SEQ ID NO:6): EREGVAAIYTGGGST
FR3 (SEQ ID NO:7): YYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYC
CDR3 (SEQ ID NO:8): AAGDLDPAEYSDYDPTVFNY
FR4 (SEQ ID NO:9): WGQGTLVTVSS
Anti- PD-L1-VHH22 nucleotide sequence (SEQ ID NO:10):
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGACACTCTCCTGTGTA
GCCTCTGGGTTCACTTTTGATGGTTCTGACATGGGCTGGTACCGCCAGGCTCCAGGGACTGAGTGCGAGTTGGTGTC
AACTATTAGTAGTGATGGAAGCACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCCAAGACAACGCCA
AGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGCGCGACTTCACTGC
ACGGGTAGCTGGGCCTTGATAATGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
Anti- PD-L1-VHH22 amino acid sequence (SEQ ID NO:11):
QVQLVESGGGSVQAGGSLTLSCVASGFTFDGSDMGWYRQAPGTECELVSTISSDGSTYYADSVKGRFT
ISQDNAKNTVYLQMNSLKPEDTAVYYCARLHCTGSWALIMGQGTQVTVSS framework region (FR1-FR4) and complementary determining region
(CDR1-CDR3) amino acid sequence:
FR1 (SEQ ID NO:12): QVQLVESGGGSVQAGGSLTLS
CDR1 (SEQ ID NO:13): CVASGFTFDGSDMG
FR2 (SEQ ID NO:14): WYRQAPGT
CDR2 (SEQ ID NO:15): ECELVSTISSDGST
FR3 (SEQ ID NO:16): YYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAVYYC
CDR3 (SEQ ID NO:17): ARLHCTGSWALI
FR4 (SEQ ID NO:18): MGQGTQVTVSS
Anti- PD-L1-VHH28 nucleotide sequence (SEQ ID NO:19):
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTACA
GCCTCTGGATTCACTTTTGATGGTTCTGACATGGGCTGGTACCGCCAGGCTCCAGGGACTGAGTGCGAGTTGGTGTC
AACTATTAGTAGTGATGGTAGCACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCA
AGAACACACTATATCTGCAATTGAACAGCCTGAAACCTGAGGACACTGCCGTGTATTACTGTGCGGCGAGGCTGCCC
CATATTGATGTGGTCGCTACTGCTAAGGGATGTAAGGCGAACAGCTACTTGGGCCAGGGGACCCAGGTCACCGTCTC
CTCA
Anti- PD-L1-VHH28 amino acid sequence (SEQ ID NO:20):
QVQLVESGGGSVQAGGSLRLSCTASGFTFDGSDMGWYRQAPGTECELVSTISSDGSTYYADSVKGRFTI
SRDNAKNTLYLQLNSLKPEDTAVYYCAARLPHIDVVATAKGCKANSYLGQGTQVTVSS
Framework region (FR1-FR4) and complementary determining region (CDR1-CDR3) amino acid sequence:
FR1 (SEQ ID NO:21): QVQLVESGGGSVQAGGSLRLS
CDR1 (SEQ ID NO:22): CTASGFTFDGSDMG
FR2 (SEQ ID NO:23): WYRQAPGT
CDR2 (SEQ ID NO:24): ECELVSTISSDGST
FR3 (SEQ ID NO:25): YYADSVKGRFTISRDNAKNTLYLQLNSLKPEDTAVYYC
CDR3 (SEQ ID NO:26): AARLPHIDVVATAKGCKANSY
FR4 (SEQ ID NO:27): LGQGTQVTVSS
Embodiment 2
1 anti-PD-L1 nano antibody albumen, the vector construction of anti-PD-L1 nano antibody and wasabi fusion protein and expression
Purifying
(1) vector construction of anti-PD-L1-VHH nano antibody (anti-PD-L1Nb15, anti-PD-L1Nb22, anti-PD-L1Nb28)
With expression and purification.
The nucleotide sequence of anti-PD-L1 nano antibody is subjected to double digestion, agarose gel electrophoresis with EcoR I and Hind III
Gel extraction digestion products, insert it into PET32a, construct the expression vector of anti-PD-L1 nano antibody；It is transformed into BL21
(DE3) in, IPTG inducing expression.It is centrifuged bacterium solution and obtains bacterial sediment, be resuspended with lysis buffer, ultrasonication is collected by centrifugation
Supernatant.By the method for Ni-IDA affinity chromatography, the anti-PD-L1 nano antibody of purifying is obtained.Then it is examined with SDS-PAGE electrophoresis
It surveys (see Fig. 2).
(2) fusion protein expression vector for constructing anti-PD-L1Nb28 and wasabi, prepares wasabi-anti-PD-
L1Nb28 albumen.
The amino acid sequence of the wasabi-anti-PD-L1Nb28 fusion protein is as follows.
MVSKGEETTMGVIKPDMKIKLKMEGNVNGHAFVIEGEGEGKPYDGTNTINLEVKEGAPLPFSYDILTT AFSYGNRAFTKYPDDIPNYFKQSFPEGYSWERTMTFEDKGIVKVKSDISMEEDSFIYEIHLKGENFPPNGPVMQKE TTGWDASTERMYVRDGVLKGDVKMKLLLEGGGHHRVDFKTIYRAKKAVKLPDYHFVDHRIEILNHDKDYNKVTVYE IAVARNSTDGMDELYKQVQLVESGGGSVQAGGSLRLSCTASGFTFDGSDMGWYRQAPGTECELVSTISSDGSTYYA
DSVKGRFTISRDNAKNTLYLQLNSLKPEDTAVYYCAARLPHIDVVATAKGCKANSY LGQGTQVTVSS (band underscore
It is wasabi amino acid sequence).
The nucleotide sequence of wasabi-anti-PD-L1Nb28 is as follows:
ATGGTGAGCAAGGGCGAGGAGACCACAATGGGCGTAATCAAGCCCGACATGAAGATCAAGCTGAAGAT GGAGGGCAACGTGAATGGCCACGCCTTCGTGATCGAGGGCGAGGGCGAGGGCAAGCCCTACGACGGCACCAACACC ATCAACCTGGAGGTGAAGGAGGGAGCCCCCCTGCCCTTCTCCTACGACATTCTGACCACCGCGTTCAGTTACGGCA ACAGGGCCTTCACCAAGTACCCCGACGACATCCCCAACTACTTCAAGCAGTCCTTCCCCGAGGGCTACTCTTGGGA GCGCACCATGACCTTCGAGGACAAGGGCATCGTGAAGGTGAAGTCCGACATCTCCATGGAGGAGGACTCCTTCATC TACGAGATACACCTCAAGGGCGAGAACTTCCCCCCCAACGGCCCCGTGATGCAGAAGGAGACCACCGGCTGGGACG CCTCCACCGAGAGGATGTACGTGCGCGACGGCGTGCTGAAGGGCGACGTCAAGATGAAGCTGCTGCTGGAGGGCGG CGGCCACCACCGCGTTGACTTCAAGACCATCTACAGGGCCAAGAAGGCGGTGAAGCTGCCCGACTATCACTTTGTG GACCACCGCATCGAGATCCTGAACCACGACAAGGACTACAACAAGGTGACCGTTTACGAGATCGCCGTGGCCCGCA ACTCCACCGACGGCATGGACGAGCTGTACAAGCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTCGGTGCAGGCTGG
AGGGTCTCTGAGACTCTCCTGTACAGCCTCTGGATTCACTTTTGATGGTTCTGACATGGGCTGGTACCGCCAGGCT
CCAGGGACTGAGTGCGAGTTGGTGTCAACTATTAGTAGTGATGGTAGCACATACTATGCAGACTCCGTGAAGGGCC
GATTCACCATCTCCAGAGACAACGCCAAGAACACACTATATCTGCAATTGAACAGCCTGAAACCTGAGGACACTGC
CGTGTATTACTGTGCGGCGAGGCTGCCCCATATTGATGTGGTCGCTACTGCTAAGGGATGTAAGGCGAACAGCTAC
TTGGGCCAGGGGACCCAGGTCACCGTCTCCTCA (being wasabi nucleotide sequence with underscore)
Coding wasabi-antiPD-L1Nb28 nucleotide sequence is cloned into PET28a carrier, BL21 (DE3) is transformed into
In, IPTG inducing expression.It is centrifuged bacterium solution and obtains bacterial sediment, be resuspended with lysis buffer, supernatant is collected by centrifugation in ultrasonication.
By the method for Ni-IDA affinity chromatography, the wasabi-anti-PD-L1Nb28 of purifying is obtained.(see Fig. 2)
The specificity of the nano antibody of 2 enzyme-linked immunization (ELISA) purification Identification
Envelope antigen albumen PD-L1 (people), PD-L1 (mouse), PD-L1 (monkey), PD-L2 (people), every 0.5 μ g of hole, coating
IgG1 is control, and 4 DEG C overnight；It is washed 3 times with PBST within second day, 1%BSA room temperature is added and closes 2 hours；Each nano antibody is dilute
It releases to 10 μ g/mL, takes 100 μ L and each hole to be incubated for respectively, react at room temperature 1 hour；Unbonded antibody is washed away with PBST, is added 1:
1000 diluted rabbit-anti HA monoclonal antibodies are incubated at room temperature one hour.TBST board-washing 3 times, the diluted HRP label of 1:2000 is added
Goat-anti rabbit secondary antibody is incubated at room temperature one hour.TBST board-washing 3 times, developing solution is added, elisa plate, which is placed in read in microplate reader, to be absorbed
Value.Judge that the specificity of nano antibody, testing result show that three nano antibodies can be with source of people and monkey source according to absorption value
PD-L1 interaction, without with source of mouse PD-L1 and people PD-L2 interaction, nano antibody there is preferable specificity.
Fig. 3 shows the specificity of representative antiPD-L1Nb28.
Wasabi-antiPD-L1Nb28 identifies HEK 293T/PD-L1 cell PD-L1 expression under 3 fluorescence microscopes
PD-L1 plasmid is transfected to HEK293T cell, PBS is cleaned 2 times after 24 hours, and 4% paraformaldehyde is added and fixes
15min.PBS is cleaned 2 times, and 1%BSA is added in 37 DEG C of closing 1h.Wasabi-antiPD- is added in PBS cleaning, experimental group
Wasabi control, PBST cleaning is added in L1Nb28 albumen, control group, and fluorescence microscope detects PD-L1 table on HEK293T cell membrane
Up to situation.It can be seen that there is obvious fluorescence (Fig. 4) in cell film location in HEK293T/PD-L1 experimental group.
Sequence table
<110>Southeast China University
<120>anti-PD-L1 nano antibody and its coded sequence, preparation method and application
<160> 31
<170> SIPOSequenceListing 1.0
<210> 1
<211> 381
<212> DNA
<213>camel (Camel)
<400> 1
caggtgcagc tggtggagtc tgggggaggc tcggtgcagg ctggaggggc tctgagactc 60
tcctgtgcag cctctggata cacctccact agcaactgca tgggctggtt ccgccaggct 120
ccagggaagg agcgcgaggg ggtcgcagct atttatactg gtggtggtag cacatactat 180
gccgactccg tgaagggccg attcaccatc tcccaagaca acgccaagaa cacggtgtat 240
ctgcaaatga acagcctgaa acctgaggac actgccatgt actactgtgc ggcaggggat 300
ttggaccccg ccgaatatag cgactatgac cctaccgtct ttaactactg gggccagggg 360
accctggtca ccgtctcctc a 381
<210> 2
<211> 127
<212> PRT
<213>camel (Camel)
<400> 2
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ala Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Ser Thr Ser Asn
20 25 30
Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Tyr Thr Gly Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Gly Asp Leu Asp Pro Ala Glu Tyr Ser Asp Tyr Asp Pro Thr
100 105 110
Val Phe Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 3
<211> 21
<212> PRT
<213>camel (Camel)
<400> 3
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ala Leu Arg Leu Ser
20
<210> 4
<211> 14
<212> PRT
<213>camel (Camel)
<400> 4
Cys Ala Ala Ser Gly Tyr Thr Ser Thr Ser Asn Cys Met Gly
1 5 10
<210> 5
<211> 8
<212> PRT
<213>camel (Camel)
<400> 5
Trp Phe Arg Gln Ala Pro Gly Lys
1 5
<210> 6
<211> 15
<212> PRT
<213>camel (Camel)
<400> 6
Glu Arg Glu Gly Val Ala Ala Ile Tyr Thr Gly Gly Gly Ser Thr
1 5 10 15
<210> 7
<211> 38
<212> PRT
<213>camel (Camel)
<400> 7
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn
1 5 10 15
Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 8
<211> 20
<212> PRT
<213>camel (Camel)
<400> 8
Ala Ala Gly Asp Leu Asp Pro Ala Glu Tyr Ser Asp Tyr Asp Pro Thr
1 5 10 15
Val Phe Asn Tyr
20
<210> 9
<211> 11
<212> PRT
<213>camel (Camel)
<400> 9
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
1 5 10
<210> 10
<211> 354
<212> DNA
<213>camel (Camel)
<400> 10
caggtgcagc tggtggagtc tgggggaggc tcggtgcagg ctggagggtc tctgacactc 60
tcctgtgtag cctctgggtt cacttttgat ggttctgaca tgggctggta ccgccaggct 120
ccagggactg agtgcgagtt ggtgtcaact attagtagtg atggaagcac atactatgca 180
gactccgtga agggccgatt caccatctcc caagacaacg ccaagaacac ggtgtatctg 240
caaatgaaca gcctgaaacc tgaggacacg gccgtgtatt actgtgcgcg acttcactgc 300
acgggtagct gggccttgat aatgggccag gggacccagg tcaccgtctc ctca 354
<210> 11
<211> 118
<212> PRT
<213>camel (Camel)
<400> 11
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Asp Gly Ser
20 25 30
Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Thr Glu Cys Glu Leu Val
35 40 45
Ser Thr Ile Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Leu His Cys Thr Gly Ser Trp Ala Leu Ile Met Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser
115
<210> 12
<211> 21
<212> PRT
<213>camel (Camel)
<400> 12
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser
20
<210> 13
<211> 14
<212> PRT
<213>camel (Camel)
<400> 13
Cys Val Ala Ser Gly Phe Thr Phe Asp Gly Ser Asp Met Gly
1 5 10
<210> 14
<211> 8
<212> PRT
<213>camel (Camel)
<400> 14
Trp Tyr Arg Gln Ala Pro Gly Thr
1 5
<210> 15
<211> 14
<212> PRT
<213>camel (Camel)
<400> 15
Glu Cys Glu Leu Val Ser Thr Ile Ser Ser Asp Gly Ser Thr
1 5 10
<210> 16
<211> 38
<212> PRT
<213>camel (Camel)
<400> 16
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn
1 5 10 15
Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Val Tyr Tyr Cys
35
<210> 17
<211> 12
<212> PRT
<213>camel (Camel)
<400> 17
Ala Arg Leu His Cys Thr Gly Ser Trp Ala Leu Ile
1 5 10
<210> 18
<211> 11
<212> PRT
<213>camel (Camel)
<400> 18
Met Gly Gln Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210> 19
<211> 381
<212> DNA
<213>camel (Camel)
<400> 19
caggtgcagc tggtggagtc tgggggaggc tcggtgcagg ctggagggtc tctgagactc 60
tcctgtacag cctctggatt cacttttgat ggttctgaca tgggctggta ccgccaggct 120
ccagggactg agtgcgagtt ggtgtcaact attagtagtg atggtagcac atactatgca 180
gactccgtga agggccgatt caccatctcc agagacaacg ccaagaacac actatatctg 240
caattgaaca gcctgaaacc tgaggacact gccgtgtatt actgtgcggc gaggctgccc 300
catattgatg tggtcgctac tgctaaggga tgtaaggcga acagctactt gggccagggg 360
acccaggtca ccgtctcctc a 381
<210> 20
<211> 127
<212> PRT
<213>camel (Camel)
<400> 20
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Asp Gly Ser
20 25 30
Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Thr Glu Cys Glu Leu Val
35 40 45
Ser Thr Ile Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Leu Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Arg Leu Pro His Ile Asp Val Val Ala Thr Ala Lys Gly Cys Lys
100 105 110
Ala Asn Ser Tyr Leu Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 21
<211> 21
<212> PRT
<213>camel (Camel)
<400> 21
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser
20
<210> 22
<211> 14
<212> PRT
<213>camel (Camel)
<400> 22
Cys Thr Ala Ser Gly Phe Thr Phe Asp Gly Ser Asp Met Gly
1 5 10
<210> 23
<211> 8
<212> PRT
<213>camel (Camel)
<400> 23
Trp Tyr Arg Gln Ala Pro Gly Thr
1 5
<210> 24
<211> 14
<212> PRT
<213>camel (Camel)
<400> 24
Glu Cys Glu Leu Val Ser Thr Ile Ser Ser Asp Gly Ser Thr
1 5 10
<210> 25
<211> 38
<212> PRT
<213>camel (Camel)
<400> 25
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
1 5 10 15
Ala Lys Asn Thr Leu Tyr Leu Gln Leu Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Val Tyr Tyr Cys
35
<210> 26
<211> 21
<212> PRT
<213>camel (Camel)
<400> 26
Ala Ala Arg Leu Pro His Ile Asp Val Val Ala Thr Ala Lys Gly Cys
1 5 10 15
Lys Ala Asn Ser Tyr
20
<210> 27
<211> 11
<212> PRT
<213>camel (Camel)
<400> 27
Leu Gly Gln Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210> 28
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 28
gtcctggctg ctcttctaca agg 23
<210> 29
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 29
ggtacgtgct gttgaactgt tcc 23
<210> 30
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 30
ccggaattct caggtgcagc tggtggagtc tgg 33
<210> 31
<211> 31
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 31
gcccaagctt tgaggagacg gtgacctggg t 31
Claims (10)
1. anti-PD-L1 nano antibody, which is characterized in that the amino acid sequence of the antibody VHH chain such as SEQ ID NO:2, SEQ
Shown in ID NO:11 or SEQ ID NO:20.
2. anti-PD-L1 nano antibody, which is characterized in that the antibody VHH chain includes framework region and complementary determining region；Wherein:
The framework region includes following 4 sections of amino acid sequences: FR1, SEQ ID NO:3, FR2, SEQ ID NO:5, FR3, SEQ
ID NO:7, FR4, SEQ ID NO:9；The complementary determining region includes following 3 sections of amino acid sequences: CDR1, SEQ ID NO:4,
CDR2, SEQ ID NO:6, CDR3, SEQ ID NO:8；
Alternatively, the framework region includes following 4 sections of amino acid sequences: FR1, SEQ ID NO:12, FR2, SEQ ID NO:14,
FR3, SEQ ID NO:16, FR4, SEQ ID NO:18；The complementary determining region includes following 3 sections of amino acid sequences: CDR1,
SEQ ID NO:13, CDR2, SEQ ID NO:15, CDR3, SEQ ID NO:17；
Alternatively, the framework region includes following 4 sections of amino acid sequences: FR1, SEQ ID NO:21, FR2, SEQ ID NO:23,
FR3, SEQ ID NO:25, FR4, SEQ ID NO:27；The complementary determining region includes following 3 sections of amino acid sequences: CDR1,
SEQ ID NO:22, CDR2, SEQ ID NO:24, CDR3, SEQ ID NO:26.
3. encoding the gene of anti-PD-L1 nano antibody, which is characterized in that its nucleotides sequence is classified as SEQ ID NO:1, SEQ ID
NO:10 or SEQ ID NO:19.
4. recombinant vector, which is characterized in that include SEQ ID NO:1, SEQ ID NO:10 or SEQ ID in the recombinant vector
NO:19 sequence.
5. host cell, which is characterized in that include SEQ ID NO:1, SEQ ID NO:10 or SEQ ID in the host cell
NO:19 sequence.
6. the preparation method of anti-PD-L1 nano antibody as claimed in claim 1 or 2, which comprises the following steps:
(1) anti-PD-L1 nano antibody phage library is constructed；
(2) the nano antibody bacteriophage that there is specific bond with PD-L1 is selected；
(3) host of carrier of the culture comprising encoding anti-PD-L1 nano antibody nucleic acid；
(4) purifying obtains anti-PD-L1 nano antibody from host cell.
7. the preparation method of anti-PD-L1 nano antibody according to claim 6, which is characterized in that use nested PCR method
The library of anti-PD-L1 nano antibody is constructed, wherein first round amplification primer sequence is SEQ ID NO:28-29, the second wheel
Amplification primer sequence is SEQ ID NO:30-31.
8. immune conjugate, which is characterized in that the VHH chain or anti-PD-L1 that the immune conjugate contains anti-PD-L1 nano antibody are received
Meter Kang Ti；With wasabi albumen.
9. anti-PD-L1 nano antibody of any of claims 1 or 2 prepares the kit of detection PD-L1 at (a)；(b) preparation treatment
Application in tumour medicine.
10. the kit that immune conjugate according to any one of claims 8 prepares detection PD-L1 at (a)；(b) preparation treatment tumour medicine
Application in object.
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| CN110256562A (en) * | 2019-07-05 | 2019-09-20 | 石河子大学 | PD-1 nano antibody, preparation method and applications |
| WO2020156507A1 (en) * | 2019-02-01 | 2020-08-06 | 信达生物制药(苏州)有限公司 | Novel anti-pd-l1 antibodies and use thereof |
| CN111909272A (en) * | 2020-08-12 | 2020-11-10 | 华东理工大学 | anti-PD-L1 nano antibody and application thereof |
| WO2020259566A1 (en) * | 2019-06-27 | 2020-12-30 | 启愈生物技术(上海)有限公司 | Anti-pd-l1 nanobody and fc fusion protein and application thereof |
| CN112210009A (en) * | 2020-09-10 | 2021-01-12 | 哈尔滨博易诚生物科技有限公司 | Single-domain antibody aiming at PD1 and application thereof |
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| WO2023232036A1 (en) * | 2022-05-31 | 2023-12-07 | 明济生物制药（北京）有限公司 | Anti-cd40 antibody, anti-pd-l1×cd40 bispecific antibody, and use thereof |
| CN117186223A (en) * | 2022-05-31 | 2023-12-08 | 明济生物制药（北京）有限公司 | anti-PD-L1 antibody, nucleic acid encoding same, preparation method and application |
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