CN107474135A - Anti- PD 1 nano antibody PD 1/Nb20 and preparation method and application - Google Patents
Anti- PD 1 nano antibody PD 1/Nb20 and preparation method and application Download PDFInfo
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- CN107474135A CN107474135A CN201710085196.2A CN201710085196A CN107474135A CN 107474135 A CN107474135 A CN 107474135A CN 201710085196 A CN201710085196 A CN 201710085196A CN 107474135 A CN107474135 A CN 107474135A
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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
- C07K16/2818—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 CD28 or CD152
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/46—Hybrid immunoglobulins
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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
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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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
- C07K2317/565—Complementarity determining region [CDR]
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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®
Abstract
The invention discloses anti-PD 1 nano antibody PD 1/Nb20 and preparation method and application.Nano antibody provided by the present invention, include determinant complementary region and framework region；The determinant complementary region of the nano antibody is made up of CDR1, CDR2 and CDR3；The nano antibody nucleotide sequence and host cell announced by the present invention, the nano antibody can in Escherichia coli high efficient expression, applied to the research and development of the Molecular Detection reagents of PD 1, prepare tumor inhibitor or inhibiting tumour cells agent and prepare the medicine for suppressing the activity of PD 1 and promoting T cell propagation.
Description
Technical field
The present invention relates to biomedical sector moderate resistance PD-1 nano antibody PD-1/Nb20 and preparation method and application.
Background technology
Programmed death acceptor 1 (programmed death 1, PD-1) is a kind of cross-film of activating T cell surface expression
Glycoprotein molecule, belong to CD28 superfamily members, bred negativity adjustment effect to T cell, important regulation is played in immune response
Effect.PD-1 is mainly expressed in the T cell and B cell of activation, is a kind of surface receptor of activated form T cell, and PD-1 has two
Individual part, be respectively apoptosis-ligand 1 (PD-L1, also known as B7-H1) and apoptosis-ligand 1 (PD-L2, again
Name B7-DC).Tumor microenvironment in body can induce the high expression PD-1 molecules of the T cell of infiltration, and tumour cell can high expression
PD-1 part PD-L1 and PD-L2, cause after PD-1 paths sustained activation PD-L1 couples with PD-1 in tumor microenvironment, T is thin
Born of the same parents' function is suppressed, it is impossible to the signal of attack tumour is sent to immune system.PD-1 inhibitor can block PD-1's and PD-L1
With reference to, block negative regulation signal, make T cell activity recovery, so as to strengthen immune response.High specific, hypoergia it is anti-
PD-1 monoclonal antibodies are affirmed in multinomial clinical test, such as the entitled Keytruda of MSD Corp.'s production
(Pembrolizumab) medicine, and entitled Opdivo (Nivolumab) nivolumab of Shi Guibao companies production
Medicine.
But antibody drug is applied and many problems be present, for example R＆D cycle length, production cost are too high；It is difficult to extensive
Production；Stability difference is degradable, and storage cost is high；It is easily contaminated, maintenance cost high cost；And there is immunogenicity etc.,
Limit its application clinically.
Nano antibody technology, be biomedical science man on the basis of conventional antibodies, with Protocols in Molecular Biology knot
The antibody engineering revolution that the concept of nano-particle science is carried out is closed, so as to the newest and minimum antibody molecule developed.1993
The reports such as year Hamers, camel body is interior, and there is natural deletions light chain and the heavy chain antibody of heavy chain constant region 1 (CH1), Ke Longqi
Variable region obtains the single domain antibody being only made up of a weight chain variable district, referred to as VHH (variable domain of heavy
Chain of heavy-chain antibody), renamed as " nano antibody " (nanobody, Nb).Nanometer is anti-
Body has the minimum antigen-binding fragment of complete function, and its crystal structure is oval, diameter 2.5nm, long 4nm.Nb has
Many unique properties, it is well suited for carrying out genetic modification, is presented in the Precise Diagnosis of disease and targeted therapy etc. wide
Application prospect.Nano antibody many simpler than antibody in chemical composition and in shape, without chemical drains, its heat resistance
It is stronger with anti acid alkali performance, it is easier to be combined with each other or combined with other compounds, energy coverlet gene code, easily closed with microorganism
Into.Nano antibody has good tolerance to environment, possesses the conformational stability of height, and molecular mass is smaller, clinical
Therapeutic effect it is more preferable, while these little albumen molecules are easier to synthesize, and price is also lower.Unique property of nano antibody,
It is set to present more wide application prospect in the Precise Diagnosis of disease and immune targeted therapy etc..
The content of the invention
The technical problems to be solved by the invention are how to prepare the medicine of effectively treatment tumour.
In order to solve the above technical problems, present invention firstly provides nano antibody.
The first aspect of the present invention, there is provided a kind of nano antibody, referred to as PD-1/Nb20, the nano antibody, which includes, to be determined
Cluster complementary region CDR and framework region FR；The determinant complementary region CDR of the nano antibody is made up of CDR1, CDR2 and CDR3；It is described
CDR1 amino acid sequence is the 26-35 amino acids of SEQ ID No.8 in sequence table；The amino acid sequence of the CDR2 is
SEQ ID No.8 52-60 amino acids in sequence table；The amino acid sequence of the CDR3 is SEQ ID in sequence table
No.8 98-114 amino acids.
Preferably, the framework region FR of the nano antibody is made up of FR1, FR2, FR3 and FR4；Wherein described FR1 amino acid
Sequence is the 1-25 amino acids of SEQ ID No.8 in sequence table；The amino acid sequence of the FR2 is SEQ ID in sequence table
No.8 36-51 amino acids；The amino acid sequence of the FR3 is the 61-97 bit aminos of SEQ ID No.8 in sequence table
Acid；The amino acid sequence of the FR4 is the 115-127 amino acids of SEQ ID No.8 in sequence table.
Preferably, the amino acid sequence of the nano antibody is as shown in SEQ ID No.8 in sequence table.
The present invention also accordingly provides a kind of VHH chains of PD-1 nano antibody, including framework region FR and complementary determining region
CDR, the amino acid sequence for the FR that the framework region FR is selected from the group：SEQ ID NO：FR1 shown in 1, SEQ ID NO：Shown in 2
FR2, SEQ ID NO：FR3 shown in 3, SEQ ID NO：FR4 shown in 4；What the complementary determining region CDR was selected from the group
CDR amino acid sequence：SEQ ID NO：CDR1 shown in 5, SEQ ID NO：CDR2 shown in 6, SEQ ID NO：Shown in 7
CDR3。
Preferably, the VHH chains of described PD-1 nano antibody, it has SEQ ID NO：Amino acid sequence shown in 8.
In order that the nano antibody PD-1/Nb20 is easy to purify, can in sequence table SEQ ID No.8 1-127
The upper label as shown in table 1 of amino terminal or carboxyl terminal connection of protein shown in amino acids.
The sequence of table 1, label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being usually 5) | RRRRR |
| Poly-His | 2-10 (being usually 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
| HA | 9 | YPYDVPDYA |
Still further aspect of the present invention, the nano antibody PD-1/Nb20 can be artificial synthesized, also can first synthesize it and encode base
Cause, then carry out biological expression and obtain.The encoding gene of the nano antibody PD-1/Nb20 can be by by SEQ ID in sequence table
The codon of one or several amino acid residues is lacked in DNA sequence dna shown in No.9, and/or carries out one or several base-pairs
Missense mutation, and/or connect at its 5 ' end and/or 3 ' ends the coded sequence of label shown in table 1 and obtain.
In order to solve the above technical problems, present invention also offers the biological material related to the nano antibody PD-1/Nb20
Material.
The biomaterial related to the nano antibody PD-1/Nb20 provided by the present invention, be B1) to B12) in appoint
It is a kind of：
B1 the nucleic acid molecules of the nano antibody PD-1/Nb20) are encoded；
B2 B1) is contained) expression cassettes of the nucleic acid molecules；
B3 B1) is contained) recombinant vectors of the nucleic acid molecules；
B4 B2) is contained) recombinant vector of the expression cassette；
B5 B1) is contained) recombinant microorganisms of the nucleic acid molecules；
B6 B2) is contained) recombinant microorganism of the expression cassette；
B7 B3) is contained) recombinant microorganism of the recombinant vector；
B8 B4) is contained) recombinant microorganism of the recombinant vector；
B9 B1) is contained) the transgenetic animal cell systems of the nucleic acid molecules；
B10 B2) is contained) the transgenetic animal cell system of the expression cassette；
B11 B3) is contained) the transgenetic animal cell system of the recombinant vector；
B12 B4) is contained) the transgenetic animal cell system of the recombinant vector.
In above-mentioned biomaterial, the nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The core
Acid molecule can also be RNA, such as mRNA or hnRNA.
In above-mentioned biomaterial, B2) described in the expression containing the nucleic acid molecules for encoding the nano antibody PD-1/Nb20
Box, also known as PD-1/Nb20 expression casettes, it is the DNA for referring to express nano antibody PD-1/Nb20 in host cell,
The DNA not only may include the promoter for starting nano antibody PD-1/Nb20 genetic transcriptions, may also include and terminates nano antibody PD-
The terminator of 1/Nb20 genetic transcriptions.Further, the expression cassette may also include enhancer sequence.
The recombinant vector of the nano antibody PD-1/Nb20 expression casettes can be contained with existing expression vector establishment.
In above-mentioned biomaterial, the carrier can be plasmid, sticking grain, bacteriophage or viral vector.
In above-mentioned biomaterial, the recombinant vector can be by B1) nucleic acid molecules imported into pComb3 and obtain
Recombinant vector.In one embodiment of the invention, B3) recombinant vector is by the volume of the nano antibody PD-1/Nb20
Code gene (nucleotides sequence is classified as the 1-381 positions nucleotides of SEQ ID No.9 in sequence table) imports in pComb3 obtained weight
Nano antibody shown in group carrier pComb3-PD-1/Nb20, recombinant vector pComb3-PD-1/Nb20 expression SEQ ID No.8
PD-1/Nb20。
In above-mentioned biomaterial, the microorganism can be yeast, bacterium, algae or fungi.
In above-mentioned biomaterial, the transgenetic animal cell system does not include propagating materials；The recombinant microorganism can be
By B1) nucleic acid molecules imported into Escherichia coli WK6 obtained recombinant microorganism.
Those of ordinary skill in the art can be easily using known method, such as the side of orthogenesis and point mutation
Method, to the B1 of the present invention) nucleotide sequence of the nano antibody PD-1/Nb20 is mutated.Those by manually modified,
With the B1 with the present invention) nucleosides of more than 75% or 75% homogeneity of nucleotide sequence of the nano antibody PD-1/Nb20
Acid, it is derived from the present invention as long as encoding the nano antibody PD-1/Nb20 and with nano antibody PD-1/Nb20 activity
Nucleotide sequence and be equal to the present invention sequence.
Term " homogeneity " used herein refers to the sequence similarity with native sequence nucleic acid." homogeneity " includes and this hair
The nucleotide sequence of protein shown in bright coding SEQ ID No.8 has 75% or higher, or 85% or higher, or 90%
Or it is higher, or the nucleotide sequence of 95% or higher homogeneity.Homogeneity can with the naked eye or computer software is evaluated.Make
With computer software, the homogeneity between two or more sequences can use percentage (%) to represent, it can be used for evaluating phase
Close the homogeneity between sequence.
Above-mentioned 75% or more than 75% homogeneity, can be 75%, 80%, 85%, 90% or more than 95% homogeneity.
In above-mentioned biomaterial, B1) nucleic acid molecules for it is following 1) or 2) or 3)：
1) nucleotide sequence is the cDNA molecule or DNA molecular of SEQ ID No.9 in sequence table；
2) nucleotide sequence with 1) limiting has 75% or more than 75% homogeneity, and encodes the nano antibody PD-
1/Nb20 cDNA molecules or genomic DNA molecule；
3) nucleotide sequence hybridization with 1) restriction, and encode the nano antibody PD-1/Nb20's under strict conditions
CDNA molecules or genomic DNA molecule.
In order to solve the above technical problems, present invention also offers the derivative antibody of the nano antibody PD-1/Nb20.
The nano antibody PD-1/Nb20 provided by the present invention derivative antibody, for it is following a) or b) or c) or d) or
E) or f)：
A) single-chain antibody of the nano antibody PD-1/Nb20 is contained；
B) fusion antibody of a) single-chain antibody is contained；
C) fusion antibody of the nano antibody PD-1/Nb20 is contained；
D) Fab of the nano antibody PD-1/Nb20 is contained；
E) complete antibody of the nano antibody PD-1/Nb20 is contained；
F) compound containing 1 or 2 or 3 nano antibody of claim；The compound refers to toxin, medicine, cell
The protein such as the factor, or all nano materials such as liposome, nano-micelle, chitosan, silicon, nanogold.
In order to solve the above technical problems, present invention also offers the preparation method of the nano antibody PD-1/Nb20.
The nano antibody PD-1/Nb20 provided by the present invention preparation method, including the nano antibody will be encoded
PD-1/Nb20 nucleic acid molecules import the transgenic cell that recipient cell obtains expressing the nano antibody PD-1/Nb20, culture
The transgenic cell, obtain the nano antibody PD-1/Nb20.
In above-mentioned nano antibody PD-1/Nb20 preparation method, the nucleic acid of the coding nano antibody PD-1/Nb20
The nucleotide sequence of molecule is as shown in SEQ ID No.9 in sequence table.
In above-mentioned nano antibody PD-1/Nb20 preparation method, the recipient cell can be microbial cell, such as large intestine bar
Bacterium, concretely Escherichia coli WK6.
In order to solve the above technical problems, present invention also offers any of following A 1-A8 purposes：
The application of A1, the nano antibody PD-1/Nb20 in tumor inhibitor or inhibiting tumour cells agent is prepared；
The application of A2, the biomaterial in tumor inhibitor or inhibiting tumour cells agent is prepared；
The application of A3, the derivative antibody of the nano antibody in tumor inhibitor or inhibiting tumour cells agent is prepared；
A4, the preparation method of the nano antibody PD-1/Nb20 are in tumor inhibitor or inhibiting tumour cells agent is prepared
Application；
A5, the nano antibody PD-1/Nb20 answering in preparation suppresses PD-1 activity or promotion T cell propagation product
With；
A6, the biomaterial are preparing the application in suppressing PD-1 activity or promoting T cell propagation product；
A7, the derivative antibody are preparing the application in suppressing PD-1 activity or promoting T cell propagation product；
A8, the preparation method of the nano antibody PD-1/Nb20 are preparing suppression PD-1 activity or are promoting T cell propagation production
Application in product.
The said goods can be medicine.
The core of amino acid sequence or its any fragment amino acid sequence in amplification coding sequence table shown in SEQ ID No.8
The primer pair of acid molecule, falls within protection scope of the present invention.
The invention provides a kind of anti-PD-1 nano antibody, the nucleotide sequence for encoding the nano antibody and host are thin
Born of the same parents, with and its preparation method and application.The nano antibody can in Escherichia coli high efficient expression, applied to PD-1 Molecular Detections
The research and development of reagent, prepare tumor inhibitor or inhibiting tumour cells agent and prepare suppression PD-1 activity and promote T cell propagation
Medicine.
Brief description of the drawings
Fig. 1 is the DNA electrophoretograms of nano antibody, and from left to right the DNA bands of gel pore are respectively：First is 2000bp
Molecular labeling, remaining duct is PCR primer, and PCR primer band is about 500bp.
Fig. 2 is SDS-PAGEs of the PD-1 nano antibodies PD-1/Nb20 after nickel post resin gel affinitive layer purification electricity
Swimming figure；Swimming lane M represents molecular weight of albumen Marker.
Fig. 3 A are nano antibody PD-1/Nb20 and the Binding experiment result of unactivated T cell；Fig. 3 B are nano antibody PD-
1/Nb20 and activating T cell Binding experiment result.
Embodiment
The present invention is further described in detail with reference to embodiment, the embodiment provided is only for explaining
The bright present invention, the scope being not intended to be limiting of the invention.
Experimental method in following embodiments, it is conventional method unless otherwise specified.
Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
After Escherichia coli WK6 in following embodiments agrees to through the sub- female laboratory of life science institute of Southeast China University ten thousand,
The public can obtain the biomaterial from Guangxi Medical University, and the biomaterial is only attached most importance to used in the related experiment of duplicate invention, no
It can be used as other purposes.
The preparation of embodiment 1, nano antibody
The invention provides a kind of nano antibody from camel, its entitled PD-1/Nb20, nano antibody PD-1/
Nb20 amino acid sequence is as shown in SEQ ID No.8 in sequence table, by the nucleotide sequence coded of SEQ ID No.9.
Nano antibody PD-1/Nb20 nucleotides electrophoretogram is as shown in figure 1, be for wherein first 2000bp molecule mark
Note, remaining duct is PCR primer, and PCR primer band is about 500bp.
DNA fragmentation between PstI the and NotI recognition sequences of carrier pComb3 (Biovector products) is replaced with into SEQ
DNA molecular shown in ID No.9, other sequences are constant, obtain recombinant vector pComb3-PD-1/Nb20, pComb3-PD-1/
The DNA fragmentation that Nb20 and pComb3 difference is only that between PstI the and NotI recognition sequences by pComb3 replaces with SEQ ID
DNA molecular shown in No.9.Nano antibody PD-1/ shown in recombinant vector pComb3-PD-1/Nb20 expression SEQ ID No.8
Nb20.PComb3-PD-1/Nb20 is imported in Escherichia coli WK6, obtains recombinant bacterium WK6-pComb3-PD-1/Nb20.
The specific preparation process of nano antibody is as follows：
(1) WK6-pComb3-PD-1/Nb20 is coated on to LB flat boards (the LB flat boards containing ampicillin and glucose
In, the concentration of ampicillin and glucose is respectively 100 μ g/mL and 20mg/mL) on, 37 DEG C of overnight incubations (12 hours)；
(2) select single bacterium colony be seeded in LB nutrient solutions that 5mL contains ampicillin (in LB nutrient solutions, ammonia benzyl mould
The concentration of element is 100 μ g/mL) in, 37 DEG C of shaking table cultures are overnight (12 hours)；
(3) nutrient solution of 1mL steps (2) overnight incubation is taken to be seeded in 330mL TB nutrient solutions, 37 DEG C of shaking table cultures are extremely
When OD values reach 0.6-1, IPTG is added, WK6-pComb3-PD-1/Nb20 nutrient solutions is obtained, makes WK6-pComb3-PD-1/
IPTG concentration is 1mM in Nb20 nutrient solutions, by WK6-pComb3-PD-1/Nb20 nutrient solutions at 28 DEG C in shaking table (shaking table
Rotating speed is 220rpm) on overnight incubation (12 hours), obtain WK6-pComb3-PD-1/Nb20 induction liquid；
(4) the WK6-pComb3-PD-1/Nb20 induction liquid of step (3) is centrifuged at 4 DEG C, collects thalline；
(5) document (Zhu M, Hu Y, Li G, Ou W, Mao P, Xin S, Wan Y is utilized:Combining magnetic
nanoparticle with biotinylated nanobodies for rapid and sensitive detection
of influenza H3N2.Nanoscale Res Lett 2014,9:528.) osmosis in, antibody crude extract is obtained；
(6) document (Zhu M, Hu Y, Li G, Ou W, Mao P, Xin S, Wan Y is utilized:Combining magnetic
nanoparticle with biotinylated nanobodies for rapid and sensitive detection
of influenza H3N2.Nanoscale Res Lett 2014,9:528.) prepared by the nickel post ion affinity chromatography method in receives
Meter Kang Ti PD-1/Nb20.Nano antibody PD-1/Nb20 SDA-PAGE electrophoretograms are respectively such as Fig. 2.As a result show, the above method
Obtained nano antibody PD-1/Nb20 purity reaches more than 90%.
The measure of embodiment 2, nano antibody and PD-1 Percentage bounds
Nano antibody PD-1/Nb20 and PD-1 Percentage bounds measure (direct method)
T cell is separated from healthy volunteer human peripheral blood cell, is incubated in 96 orifice plates, by 10 μ g/ml PHA
(sigma, L9017) is added in T cell co-culture 72h after, the nano antibody PD-1/Nb20 (1 μ g) of embodiment 1 is added 1 ×
1064 DEG C of lucifuges are incubated 30min in individual above-mentioned T cell, after PBS is washed 2 times, add 5 μ l PE anti-HA tag antibody
(abcam, Clone:20B12) 4 DEG C of incubation 30min, after PBS is washed 2 times, by BACKMAN flow cytometers on sample, as a result such as
Shown in Fig. 3 B, non-activated T cell is used as control as shown in Figure 3A.Fig. 3 A are blank control and PD-1 nano antibodies PD-1/
Nb20 respectively and unactivated T cell combination percentage；Fig. 3 B are blank control and PD-1 nano antibodies PD-1/Nb20 difference
And the combination percentage of the T cell of activation；Transverse axis is fluorescence intensity (PE) in Fig. 3 A and 3B, and the longitudinal axis is number percent (%of
Max), S2 represents blank control, and S1 represents PD-1 nano antibodies PD-1/Nb20.Graphical results can be seen that PD-1 nano antibodies
PD-1/Nb20 can be good at being combined with the T cell activated.
Finally it should be noted that：The above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although
The present invention is described in detail with reference to the foregoing embodiments, it will be understood by those within the art that：It still may be used
To be modified to the technical scheme described in foregoing embodiments, or equivalent substitution is carried out to which part technical characteristic；
And these modification or replace, do not make appropriate technical solution essence depart from various embodiments of the present invention technical scheme spirit and
Scope.
SEQUENCE LISTING
<110>Guangxi Medical University
<120>Anti- PD-1 nano antibody PD-1/Nb20 and preparation method and application
<130> GY17100059
<160> 9
<170> PatentIn version 3.5
<210> 1
<211> 25
<212> PRT
<213>Artificial sequence
<220>
<221> CONFLICT
<222> (1)..(25)
<223> FR1
<400> 1
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser
20 25
<210> 2
<211> 16
<212> PRT
<213>Artificial sequence
<220>
<221> CONFLICT
<222> (1)..(16)
<223> FR2
<400> 2
Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val Ser Ser Ile
1 5 10 15
<210> 3
<211> 37
<212> PRT
<213>Artificial sequence
<220>
<221> CONFLICT
<222> (1)..(37)
<223> FR3
<400> 3
Glu Tyr Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
1 5 10 15
Asn Thr Leu Tyr Leu Gln Leu Asn Ser Leu Lys Thr Glu Asp Thr Ala
20 25 30
Met Tyr Tyr Cys Thr
35
<210> 4
<211> 13
<212> PRT
<213>Artificial sequence
<220>
<221> CONFLICT
<222> (1)..(13)
<223> FR4
<400> 4
Leu Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala
1 5 10
<210> 5
<211> 10
<212> PRT
<213>Artificial sequence
<220>
<221> CONFLICT
<222> (1)..(10)
<223> CDR1
<400> 5
Gly Phe Thr Ser Arg Asn Tyr Ala Met Thr
1 5 10
<210> 6
<211> 9
<212> PRT
<213>Artificial sequence
<220>
<221> CONFLICT
<222> (1)..(9)
<223> CDR2
<400> 6
Ser Ser Asp Asp Asp Ser Thr Tyr Tyr
1 5
<210> 7
<211> 17
<212> PRT
<213>Artificial sequence
<220>
<221> CONFLICT
<222> (1)..(17)
<223> CDR3
<400> 7
Lys Glu Phe Val Ala Val Val Pro Val Leu Lys Leu Gly Arg Pro Arg
1 5 10 15
Asp
<210> 8
<211> 127
<212> PRT
<213>Artificial sequence
<400> 8
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ser Arg Asn Tyr
20 25 30
Ala Met Thr Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Ser Ser Asp Asp Asp Ser Thr Tyr Tyr Glu Tyr Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Leu Asn Ser Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Thr Lys Glu Phe Val Ala Val Val Pro Val Leu Lys Leu Gly Arg Pro
100 105 110
Arg Asp Leu Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala
115 120 125
<210> 9
<211> 381
<212> DNA
<213>Artificial sequence
<400> 9
caggtgcagc tgcaggagtc tggaggaggc ttggtgcagc ctggggggtc tctgagactc 60
tcctgtgcag cctctggatt cacctccagg aattatgcca tgacatgggt ccgccaggct 120
ccagagaagg gactcgagtg ggtctcaagt atttctagtg atgatgatag tacatactat 180
gaatactccg tgaagggacg attcaccatc tccagagaca acgccaagaa cacgctgtat 240
ctgcaattga acagcctgaa aactgaggac acggccatgt attactgtac aaaagagttc 300
gttgcggtag tacctgtgct aaagctaggc cgcccccggg acctcggcca ggggacccag 360
gtcaccgtct cctcagcggc c 381
Claims (10)
1. nano antibody, include determinant complementary region；It is characterized in that：
The determinant complementary region of the nano antibody is made up of CDR1, CDR2 and CDR3；
The amino acid sequence of the CDR1 is the 26-35 amino acids of SEQ ID No.8 in sequence table；
The amino acid sequence of the CDR2 is the 52-60 amino acids of SEQ ID No.8 in sequence table；
The amino acid sequence of the CDR3 is the 98-114 amino acids of SEQ ID No.8 in sequence table.
2. nano antibody according to claim 1, it is characterised in that：The nano antibody by the determinant complementary region and
The framework region composition.
3. nano antibody according to claim 1 or 2, it is characterised in that：The amino acid sequence of the nano antibody such as sequence
In list shown in SEQ ID No.8.
Any of 4. the biomaterial related to 1 or 2 or 3 nano antibody of claim, be B1) to B12)：
B1 the nucleic acid molecules of 1 or 2 or 3 nano antibody of claim) are encoded；
B2 B1) is contained) expression cassettes of the nucleic acid molecules；
B3 B1) is contained) recombinant vectors of the nucleic acid molecules；
B4 B2) is contained) recombinant vector of the expression cassette；
B5 B1) is contained) recombinant microorganisms of the nucleic acid molecules；
B6 B2) is contained) recombinant microorganism of the expression cassette；
B7 B3) is contained) recombinant microorganism of the recombinant vector；
B8 B4) is contained) recombinant microorganism of the recombinant vector；
B9 B1) is contained) the transgenetic animal cell systems of the nucleic acid molecules；
B10 B2) is contained) the transgenetic animal cell system of the expression cassette；
B11 B3) is contained) the transgenetic animal cell system of the recombinant vector；
B12 B4) is contained) the transgenetic animal cell system of the recombinant vector.
5. biomaterial according to claim 4, it is characterised in that：B1) nucleic acid molecules for it is following 1) or 2) or 3)：
1) nucleotide sequence is the cDNA molecule or DNA molecular of SEQ ID No.9 in sequence table；
2) nucleotide sequence with 1) limiting has 75% or more than 75% homogeneity, and encodes described in claim 1 or 2 or 3
The cDNA molecules or genomic DNA molecule of nano antibody；
3) nucleotide sequence hybridization with 1) restriction, and encode 1 or 2 or 3 nano antibody of claim under strict conditions
CDNA molecules or genomic DNA molecule.
6. the derivative antibody of 1 or 2 or 3 nano antibody of claim, for it is following a) or b) or c) or d) or e) or f)：
A) single-chain antibody containing 1 or 2 or 3 nano antibody of claim；
B) fusion antibody of a) single-chain antibody is contained；
C) fusion antibody containing 1 or 2 or 3 nano antibody of claim；
D) Fab containing 1 or 2 or 3 nano antibody of claim；
E) complete antibody containing 1 or 2 or 3 nano antibody of claim；
F) compound containing 1 or 2 or 3 nano antibody of claim.
7. the preparation method of 1 or 2 or 3 nano antibody of claim, including 1 or 2 or 3 nanometer of claim will be encoded
The nucleic acid molecules of antibody import the transgenic cell that recipient cell obtains expressing the nano antibody, and it is thin to cultivate the transgenosis
Born of the same parents, obtain the nano antibody.
8. according to the method for claim 7, it is characterised in that：Coding 1 or 2 or 3 nano antibody of claim
Nucleic acid molecules nucleotide sequence as shown in SEQ ID No.9 in sequence table.
9. according to the method for claim 7, it is characterised in that：The recipient cell is microbial cell.
10. any of following A 1-A8 purposes：
The application of A1,1 or 2 or 3 nano antibody of claim in tumor inhibitor or inhibiting tumour cells agent is prepared；
Application of any biomaterial in tumor inhibitor or inhibiting tumour cells agent is prepared in A2, claim 4-5；
Application of the derivative antibody in tumor inhibitor or inhibiting tumour cells agent is prepared described in A3, claim 6；
The application of A4, the methods described of claim 7 or 8 in tumor inhibitor or inhibiting tumour cells agent is prepared；
A5,1 or 2 or 3 nano antibody of claim answering in preparation suppresses PD-1 activity or promotion T cell propagation product
With；
Any biomaterial is in preparing suppression PD-1 activity or promoting T cell propagation product in A6, claim 4-5
Using；
Derivative antibody described in A7, claim 6 is preparing the application in suppressing PD-1 activity or promoting T cell propagation product；
A8, claim 7 or 8 or 9 methods describeds are preparing the application in suppressing PD-1 activity or promoting T cell propagation product.
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