CN108218988B - Nano antibody PD-1/Nb52 of anti-PD-1 and the preparation method and application thereof - Google Patents
Nano antibody PD-1/Nb52 of anti-PD-1 and the preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses nano antibody PD-1/Nb52 of anti-PD-1 and the preparation method and application thereof.Nano antibody provided by the present invention, antigenic determinant complementary region and skeleton area comprising nano antibody PD-1/Nb52；The antigenic determinant complementary region of the nano antibody PD-1/Nb52 is made of CDR1, CDR2 and CDR3；The nucleotide sequence and host cell of nano antibody PD-1/Nb52 announced through the invention, nano antibody PD-1/Nb52 can in Escherichia coli high efficient expression, applied to the research and development of PD-1 Molecular Detection reagent, preparation inhibits PD-1 activity and prepares tumor inhibitor or inhibiting tumour cells agent and promote the drug of T cell proliferation.
Description
Technical field
The present invention relates to nano antibody PD-1/Nb52 of PD-1 anti-in field of biomedicine and the preparation method and application thereof.
Background technique
Programmed death receptor 1 (programmed death 1, PD-1) is a kind of cross-film of activating T cell surface expression
Glycoprotein molecule belongs to CD28 superfamily member, has been proliferated negativity adjustment effect to T cell, important adjusting is played in immune response
Effect.PD-1 is mainly expressed in the T cell of activation and B cell, is a kind of surface receptor of activated form T cell, and PD-1 has two
A ligand, be respectively apoptosis-ligand 1 (PD-L1 also known as B7-H1) and apoptosis-ligand 1 (PD-L2, again
Name B7-DC).The intracorporal tumor microenvironment of machine can induce the T cell height of infiltration to express PD-1 molecule, and tumour cell can high expression
The ligand PD-L1 and PD-L2 of PD-1 causes after PD-1 access sustained activation PD-L1 couples with PD-1 in tumor microenvironment, and T is thin
Born of the same parents' function is suppressed, and the signal of attack tumour cannot be issued to immune system.PD-1 inhibitor can block PD-1's and PD-L1
In conjunction with blocking negative regulation signal makes T cell activity recovery, to enhance immune response.High specific, hypoergia it is anti-
PD-1 monoclonal antibody is affirmed in multinomial clinical test, such as the entitled Keytruda of MSD Corp.'s production
(Pembrolizumab) entitled Opdivo (Nivolumab) nivolumab's of drug and the production of Shi Guibao company
Drug.
But antibody drug is applied there are many problems, such as R&D cycle are long, production cost is excessively high；It is difficult to extensive
Production；Stability difference is degradable, stores at high cost；It is easy to be contaminated, maintenance cost expense is high；And there is immunogenicity etc.,
Limit its application range clinically.
Nano antibody technology is biomedical science man on the basis of conventional antibodies, with Protocols in Molecular Biology knot
The antibody engineering revolution that the concept of nanoparticle science carries out is closed, thus the newest and the smallest antibody molecule developed.1993
The reports such as year Hamers, camel body is interior, and there is the heavy chain antibody of natural deletions light chain and heavy chain constant region 1 (CH1), Ke Longqi
Variable region obtains the single domain antibody being only made of a heavy chain variable region, referred to as VHH (variable domain of heavy
Chain of heavy-chain antibody), it is renamed as " nano antibody " (nanobody, Nb).Nanometer is anti-
Body has the smallest antigen-binding fragment of complete function, and crystal structure is oval, diameter 2.5nm, long 4nm.Nb has
Many unique properties, be well suited for carry out genetic modification, disease Precise Diagnosis and in terms of present it is wide
Application prospect.Nano antibody many simpler than antibody in chemical composition and in shape does not have chemical drains, heat resistance
It is stronger with anti acid alkali performance, it is easier to be combined with each other or in conjunction with other compounds, can be encoded by single-gene, be easy to be closed with microorganism
At.Nano antibody has good tolerance to environment, has 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.The unique property of nano antibody,
Make its disease Precise Diagnosis and it is immune in terms of present more extensive application prospect.
Summary of the invention
How the technical problem to be solved by the present invention is to prepare the drug of effectively treatment tumour.
In order to solve the above technical problems, present invention firstly provides nano antibodies.
The first aspect of the present invention provides a kind of nano antibody, referred to as PD-1/Nb52, which includes antigen
Determinant complementary region CDR and skeleton area FR；The antigenic determinant complementary region CDR of the nano antibody is by CDR1, CDR2 and CDR3
Composition；The amino acid sequence of the CDR1 is the 23-32 amino acids of SEQ ID No.8 in sequence table；The ammonia of the CDR2
Base acid sequence is the 49-56 amino acids of SEQ ID No.8 in sequence table；The amino acid sequence of the CDR3 is in sequence table
The 94-112 amino acids of SEQ ID No.8.
Preferably, the skeleton area FR of the nano antibody is made of FR1, FR2, FR3 and FR4；The wherein amino acid of the FR1
Sequence is the 1-22 amino acids of SEQ ID No.8 in sequence table；The amino acid sequence of the FR2 is SEQ ID in sequence table
The 33-48 amino acids of No.8；The amino acid sequence of the FR3 is the 57-93 bit amino of SEQ ID No.8 in sequence table
Acid；The amino acid sequence of the FR4 is the 113-123 amino acids of SEQ ID No.8 in sequence table.
Preferably, the amino acid sequence of the nano antibody PD-1/Nb52 is as shown in SEQ ID No.8 in sequence table.
The present invention also accordingly provides the VHH chain of PD-1 nano antibody PD-1/Nb52 a kind of, including skeleton area FR and antigen
Determinant complementary region CDR, the amino acid sequence of the skeleton area FR FR selected from the group below: FR1, SEQ shown in SEQ ID NO:1
FR4 shown in FR3 shown in FR2 shown in ID NO:2, SEQ ID NO:3, SEQ ID NO:4；The antigenic determinant is complementary
The amino acid sequence of area CDR CDR selected from the group below: CDR2 shown in CDR1 shown in SEQ ID NO:5, SEQ ID NO:6,
CDR3 shown in SEQ ID NO:7.
Preferably, the VHH chain of the PD-1 nano antibody PD-1/Nb52, it has amino shown in SEQ ID NO:8
Acid sequence.
In order to make the nano antibody PD-1/Nb52 convenient for purifying, can in sequence table SEQ ID No.8 1-123
The amino terminal or carboxyl terminal of protein shown in amino acids connect upper label as shown in Table 1.
The sequence of table 1, label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (usually 5) | RRRRR |
| Poly-His | 2-10 (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/Nb52 can be artificial synthesized, can also first synthesize it and encode base
Cause, then carry out biological expression and obtain.The encoding gene of the nano antibody PD-1/Nb52 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 the coded sequence of label shown in table 1 at its 5 ' end and/or 3 ' ends and obtain.
In order to solve the above technical problems, the present invention also provides biological materials relevant to the nano antibody PD-1/Nb52
Material.
Biomaterial relevant to the nano antibody PD-1/Nb52 provided by the present invention is B1) appointing into B12)
It is a kind of:
B1 the nucleic acid molecules of the nano antibody PD-1/Nb52) are encoded；
B2) contain B1) expression cassettes of the nucleic acid molecules；
B3) contain B1) recombinant vectors of the nucleic acid molecules；
B4) contain B2) recombinant vector of the expression cassette；
B5) contain B1) recombinant microorganisms of the nucleic acid molecules；
B6) contain B2) recombinant microorganism of the expression cassette；
B7) contain B3) recombinant microorganism of the recombinant vector；
B8) contain B4) recombinant microorganism of the recombinant vector；
B9) contain B1) the transgenetic animal cell systems of the nucleic acid molecules；
B10) contain B2) the transgenetic animal cell system of the expression cassette；
B11) contain B3) the transgenetic animal cell system of the recombinant vector；
B12) contain B4) 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 is also possible to 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/Nb52
Box, also known as PD-1/Nb52 expression casette are the DNA for referring to express nano antibody PD-1/Nb52 in host cell,
The DNA not only may include the promoter for starting nano antibody PD-1/Nb52 genetic transcription, may also include and terminates nano antibody PD-
The terminator of 1/Nb52 genetic transcription.Further, the expression cassette may also include enhancer sequence.
The recombinant vector of the nano antibody PD-1/Nb52 expression casette can be contained with existing expression vector establishment.
In above-mentioned biomaterial, the carrier can be plasmid, sticking grain, bacteriophage or viral vectors.
In above-mentioned biomaterial, the recombinant vector can be that the B1) nucleic acid molecules are imported into obtained in pComb3
Recombinant vector.In one embodiment of the invention, B3) recombinant vector is by the volume of the nano antibody PD-1/Nb52
Code gene (the 1-369 nucleotide that nucleotides sequence is classified as SEQ ID No.9 in sequence table) imports weight obtained in pComb3
Group carrier pComb3-PD-1/Nb52, recombinant vector pComb3-PD-1/Nb52 express nano antibody shown in SEQ ID No.8
PD-1/Nb52。
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 propagation material；The recombinant microorganism can be
B1) the nucleic acid molecules are imported into recombinant microorganism obtained in Escherichia coli WK6.
Those of ordinary skill in the art can easily adopt by known method, such as the side of directed evolution and point mutation
Method, to B1 of the invention) nucleotide sequence of the nano antibody PD-1/Nb52 is mutated.Those by manually modified,
Nucleosides with 75% or 75% or more the identity of nucleotide sequence with B1 of the invention) the nano antibody PD-1/Nb52
Acid is derived from the present invention as long as encoding the nano antibody PD-1/Nb52 and having nano antibody PD-1/Nb52 activity
Nucleotide sequence and be equal to sequence of the invention.
Term " identity " used herein refers to the sequence similarity with native sequence nucleic acid." identity " 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 higher or 95% or higher identity nucleotide sequence.Identity can with the naked eye or computer software is evaluated.Make
With computer software, the identity between two or more sequences can be indicated with percentage (%), can be used to evaluate phase
Close the identity between sequence.
Above-mentioned 75% or 75% or more identity can be 75%, 80%, 85%, 90% or 95% or more identity.
In above-mentioned biomaterial, B1) nucleic acid molecules be 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) there is 75% or 75% or more identity with the nucleotide sequence 1) limited, and encodes the nano antibody PD-
The cDNA molecule or genomic DNA molecule of 1/Nb52；
3) and encode the nano antibody PD-1/Nb52's with the nucleotide sequence hybridization that 1) limits under strict conditions
CDNA molecule or genomic DNA molecule.
In order to solve the above technical problems, the present invention also provides the derivative antibody of the nano antibody PD-1/Nb52.
The derivative antibody of the nano antibody PD-1/Nb52 provided by the present invention, for it is following a) or b) or c) or d) or
E):
A) contain the single-chain antibody of the nano antibody PD-1/Nb52；
B) contain the fusion antibody of a) single-chain antibody；
C) contain the fusion antibody of the nano antibody PD-1/Nb52；
D) contain the Fab of the nano antibody PD-1/Nb52；
E) contain the complete antibody of the nano antibody PD-1/Nb52.
In order to solve the above technical problems, the present invention also provides the preparation methods of the nano antibody PD-1/Nb52.
The preparation method of the nano antibody PD-1/Nb52 provided by the present invention, including the nano antibody will be encoded
The nucleic acid molecules of PD-1/Nb52 import the transgenic cell that recipient cell obtains expressing the nano antibody PD-1/Nb52, culture
The transgenic cell obtains the nano antibody PD-1/Nb52.
In the preparation method of above-mentioned nano antibody PD-1/Nb52, the nucleic acid of the coding nano antibody PD-1/Nb52
The nucleotide sequence of molecule is as shown in SEQ ID No.9 in sequence table.
In the preparation method of above-mentioned nano antibody PD-1/Nb52, 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, the present invention also provides any purposes in following A 1-A8:
A1, the nano antibody PD-1/Nb52 are preparing the application in tumor inhibitor or inhibiting tumour cells agent；
A2, the biomaterial are preparing the application in tumor inhibitor or inhibiting tumour cells agent；
A3, the nano antibody PD-1/Nb52 derivative antibody in preparing tumor inhibitor or inhibiting tumour cells agent
Application；
A4, the nano antibody PD-1/Nb52 preparation method in preparing tumor inhibitor or inhibiting tumour cells agent
Application；
A5, the nano antibody PD-1/Nb52 inhibit PD-1 activity in preparation or promote answering in T cell proliferation product
With；
A6, the biomaterial inhibit PD-1 activity in preparation or promote the application in T cell proliferation product；
A7, the derivative antibody inhibit PD-1 activity in preparation or promote the application in T cell proliferation product；
A8, the nano antibody PD-1/Nb52 preparation method preparation inhibit PD-1 activity or promote T cell proliferation produce
Application in product.
The said goods can be drug.
The core of amino acid sequence shown in SEQ ID No.8 or its any fragment amino acid sequence in amplification coding sequence table
The primer pair of acid molecule, also belongs to protection scope of the present invention.
The present invention provides the nano antibody PD-1/Nb52 of anti-PD-1 a kind of, encode the core of nano antibody PD-1/Nb52
Nucleotide sequence and host cell, with and its preparation method and application.Nano antibody PD-1/Nb52 can be high in Escherichia coli
Effect expression prepares tumor inhibitor or inhibiting tumour cells agent and preparation suppression applied to the research and development of PD-1 Molecular Detection reagent
PD-1 activity processed and the drug for promoting T cell proliferation.
Detailed description of the invention
Fig. 1 is the DNA electrophoretogram of nano antibody, and from left to right the DNA band of gel pore is respectively: first is 2000bp
Molecular labeling, second is PCR product, and PCR product band is about 400bp；
Fig. 2 is the electricity of SDS-PAGE of the PD-1 nano antibody PD-1/Nb52 after nickel column resin gel affinitive layer purification
Swimming figure；Swimming lane M indicates molecular weight of albumen Marker, unit KDa；
Fig. 3 A is the Binding experiment result of the 293T cell of nano antibody PD-1/Nb52 and untransfected PD-1；Fig. 3 B is to receive
The Binding experiment result of the 293T cell of meter Kang Ti PD-1/Nb52 and transfection PD-1.
Specific embodiment
The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.
Experimental method in following embodiments is unless otherwise specified conventional method.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Escherichia coli WK6 in following embodiments after the Southeast China University laboratory life science institute Wan Yakun is agreed to,
The public can obtain the biomaterial from Guangxi Medical University, which only attaches most importance to used in the related experiment of duplicate invention, no
It can be used as other purposes to use.
The preparation of embodiment 1, nano antibody
The present invention provides a kind of nano antibody for deriving from camel, entitled PD-1/Nb52, nano antibody PD-1/
The amino acid sequence of Nb52 is as shown in SEQ ID No.8 in sequence table, by the nucleotide sequence coded of SEQ ID No.9.
The nucleotide electrophoretogram of nano antibody PD-1/Nb52 as shown in Figure 1, wherein first be 2000bp molecule mark
Note, remaining duct are PCR product, and PCR product band is about 400bp.
The PstI and NotI of carrier pComb3 (Biovector product) are identified that the DNA fragmentation between sequence replaces with SEQ
DNA molecular shown in ID No.9, other sequences are constant, obtain recombinant vector pComb3-PD-1/Nb52, pComb3-PD-1/
The difference of Nb52 and pComb3, which is only that, replaces with SEQ ID for the DNA fragmentation between the PstI of pComb3 and NotI identification sequence
DNA molecular shown in No.9.Recombinant vector pComb3-PD-1/Nb52 expresses nano antibody PD-1/ shown in SEQ ID No.8
Nb52.PComb3-PD-1/Nb52 is imported in Escherichia coli WK6, recombinant bacterium WK6-pComb3-PD-1/Nb52 is obtained.
Specific preparation process is as follows for nano antibody:
(1) WK6-pComb3-PD-1/Nb52 is coated on to LB plate (the LB plate containing ampicillin and glucose
In, the concentration of ampicillin and glucose is respectively 100 μ g/mL and 20mg/mL) on, (10-14 is small for 25-37 DEG C of overnight incubation
When)；
(2) select single bacterium colony be seeded in LB culture solution that 5mL contains ampicillin (in LB culture solution, ammonia benzyl mould
The concentration of element is 100 μ g/mL) in, 25-37 DEG C of shaking table culture is overnight (10-14 hours)；
(3) culture solution of 1mL step (2) overnight incubation is taken to be seeded in 300-350mL TB culture solution, 25-37 DEG C of shaking table
When culture reaches 0.6-1 to OD value, IPTG is added, obtains WK6-pComb3-PD-1/Nb52 culture solution, makes WK6-pComb3-
The concentration of IPTG is 1mM in PD-1/Nb52 culture solution, by WK6-pComb3-PD-1/Nb52 culture solution in shaking at 20-30 DEG C
Overnight incubation (10-14 hours) on bed (revolving speed of shaking table is 220-250rpm), obtains WK6-pComb3-PD-1/Nb52 induction
Liquid；
(4) the WK6-pComb3-PD-1/Nb52 induction liquid of step (3) is centrifuged at 4 DEG C, collects thallus；
(5) document (Zhu M, Hu Y, Li G, Ou W, Mao P, Xin S, Wan Y:Combining magnetic is utilized
nanoparticle with biotinylated nanobodies for rapid and sensitive detection
Of influenza H3N2.Nanoscale Res Lett 2014,9:528.) in osmosis, obtain antibody crude extract；
(6) document (Zhu M, Hu Y, Li G, Ou W, Mao P, Xin S, Wan Y:Combining magnetic is utilized
nanoparticle with biotinylated nanobodies for rapid and sensitive detection
Of influenza H3N2.Nanoscale Res Lett 2014,9:528.) in nickel column ion affinity chromatography method preparation receive
Meter Kang Ti PD-1/Nb52.The SDA-PAGE electrophoretogram of nano antibody PD-1/Nb52 is respectively such as Fig. 2, nano antibody PD-1/Nb52
Size be about 15KDa.The results show that the purity of nano antibody PD-1/Nb52 obtained by the above method can achieve 90% with
On.
The measurement of embodiment 2, nano antibody and PD-1 Percentage bound
Nano antibody PD-1/Nb52 and PD-1 Percentage bound measurement (direct method)
With surely turn PD-1 293T cell detection nano antibody PD-1/Nb52 and PD-1 Percentage bound, by the nanometer of embodiment 1
1-6 × 10 are added in antibody PD-1/Nb52 (1 μ g)6Incubation 20-40min is protected from light for 4 DEG C in a above-mentioned 293T cell, and PBS is washed 2 times
Afterwards, 5 μ l PE anti-HA tag antibody (abcam, Clone:20B12), 4 DEG C of incubation 20-40min, PBS washings 2 are added
After secondary, by BACKMAN flow cytometer on sample, as a result as shown in Figure 3B, the 293T cell of untransfected PD-1 is used as control such as
Shown in Fig. 3 A.Fig. 3 A is blank control and PD-1 nano antibody the PD-1/Nb52 knot with the 293T cell of untransfected PD-1 respectively
Close percentage；Fig. 3 B is blank control and PD-1 nano antibody PD-1/Nb52 surely turn respectively PD-1 293T cell combination hundred
Divide rate；Horizontal axis is fluorescence intensity (PE) in Fig. 3 A and 3B, and the longitudinal axis is number percent (%of Max), and S2 represents blank control,
S1 represents PD-1 nano antibody PD-1/Nb52.Graphical results can be seen that PD-1 nano antibody PD-1/Nb52 can be good at
Surely turn the 293T cell combination of PD-1.
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
Present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: it still may be used
To modify the technical solutions described in the foregoing embodiments or equivalent replacement of some of the technical features；
And these are modified or replaceed, technical solution of various embodiments of the present invention that it does not separate the essence of the corresponding technical solution spirit and
Range.
SEQUENCE LISTING
<110>Guangxi Medical University
<120>the nano antibody PD-1/Nb52 and the preparation method and application thereof of anti-PD-1
<130> GY17100594
<160> 9
<170> PatentIn version 3.3
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Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ser Ile
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Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
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Gly Tyr Thr Tyr Ser Asn Tyr Tyr Met Gly
1 5 10
<210> 6
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Asp Thr Leu Gly Tyr Thr Arg Tyr
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Ala Pro Arg Ser Pro Tyr Tyr Arg Gly Gln Thr Phe Trp Glu Gly Ala
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Tyr Asn Tyr
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Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ser Gly Gly Ser Leu Arg
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20 25 30
Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ser Ile
35 40 45
Asp Thr Leu Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe
50 55 60
Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr Leu Tyr Leu Gln Met Ser
65 70 75 80
Ser Leu Gln Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Pro Arg
85 90 95
Ser Pro Tyr Tyr Arg Gly Gln Thr Phe Trp Glu Gly Ala Tyr Asn Tyr
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 9
<211> 369
<212> DNA
<213>artificial sequence
<400> 9
ctgcaggagt ctggaggagg ctcggtgcag tccggagggt ctctgagact cacctgtgca 60
gcctctggat acacctacag taactactac atgggctggt tccgccaggc tccaggaaag 120
gagcgcgagg gggtcgcaag tattgatact cttggttata caagatacgc agactccgtg 180
aaggggcgat tcaccatctc ccgtgacaac gataagaaca cgctgtatct gcaaatgagc 240
agcctgcaac cggaggacac ggccatgtat tactgtgcgg cccctcgttc tccttactac 300
cggggtcaga cgttctggga gggggcgtat aactactggg gccaggggac ccaggtcacc 360
gtctcctca 369
Claims (6)
1. nano antibody includes antigenic determinant complementary region and skeleton area；It is characterized by:
The antigenic determinant complementary region of the nano antibody is made of CDR1, CDR2 and CDR3；
The amino acid sequence of the CDR1 is the 23-32 amino acids of SEQ ID No.8 in sequence table；
The amino acid sequence of the CDR2 is the 49-56 amino acids of SEQ ID No.8 in sequence table；
The amino acid sequence of the CDR3 is the 94-112 amino acids of SEQ ID No.8 in sequence table；
The amino acid sequence of the nano antibody is as shown in SEQ ID No.8 in sequence table.
2. it is B1 biomaterial relevant to nano antibody described in claim 1) any one of to B12):
B1 the nucleic acid molecules of nano antibody described in claim 1) are encoded；
B2) contain B1) expression cassettes of the nucleic acid molecules；
B3) contain B1) recombinant vectors of the nucleic acid molecules；
B4) contain B2) recombinant vector of the expression cassette；
B5) contain B1) recombinant microorganisms of the nucleic acid molecules；
B6) contain B2) recombinant microorganism of the expression cassette；
B7) contain B3) recombinant microorganism of the recombinant vector；
B8) contain B4) recombinant microorganism of the recombinant vector；
B9) contain B1) the transgenetic animal cell systems of the nucleic acid molecules；
B10) contain B2) the transgenetic animal cell system of the expression cassette；
B11) contain B3) the transgenetic animal cell system of the recombinant vector；
B12) contain B4) the transgenetic animal cell system of the recombinant vector.
3. biomaterial according to claim 2, it is characterised in that: B1) nucleic acid molecules be it is following 1) or 2):
1) nucleotide sequence is the cDNA molecule or DNA molecular of SEQ ID No.9 in sequence table；
2) there is 75% or 75% or more identity with the nucleotide sequence 1) limited, and it is anti-to encode nanometer described in claim 1
The cDNA molecule or genomic DNA molecule of body.
4. the preparation method of nano antibody described in claim 1, the nucleic acid point including nano antibody described in claim 1 will be encoded
Son imports the transgenic cell that recipient cell obtains expressing the nano antibody, cultivates the transgenic cell, obtains described receive
Meter Kang Ti.
5. according to the method described in claim 4, it is characterized by: the nucleic acid of nano antibody described in the coding claim 1
The nucleotide sequence of molecule is as shown in SEQ ID No.9 in sequence table.
6. according to the method described in claim 4, it is characterized by: the recipient cell is microbial cell.
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| CN201711223392.8A CN108218988B (en) | 2017-11-29 | 2017-11-29 | Nano antibody PD-1/Nb52 of anti-PD-1 and the preparation method and application thereof |
| PCT/CN2018/074474 WO2019104860A1 (en) | 2017-11-29 | 2018-01-29 | Anti-pd-1 nanobody pd-1/nb52, preparation method therefor and use thereof |
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| CN201711223392.8A CN108218988B (en) | 2017-11-29 | 2017-11-29 | Nano antibody PD-1/Nb52 of anti-PD-1 and the preparation method and application thereof |
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| CN111423510B (en) * | 2019-01-10 | 2024-02-06 | 迈威(上海)生物科技股份有限公司 | Recombinant anti-human PD-1 antibody and application thereof |
| CN110423277B (en) * | 2019-03-21 | 2022-09-06 | 南京东极医药科技有限公司 | Nano antibody of PD-1 and clinical application thereof |
| CN110256562B (en) * | 2019-07-05 | 2022-10-25 | 石河子大学 | PD-1 nano antibody, preparation method and application thereof |
| CN112442122B (en) * | 2019-09-04 | 2021-09-21 | 上海洛启生物医药技术有限公司 | Blocking type PD-1 nano antibody and its coding sequence and use |
| CN114984207B (en) * | 2022-05-09 | 2024-01-26 | 浙江特瑞思药业股份有限公司 | anti-PD-1 nano antibody preparation |
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| CN105061597B (en) * | 2015-06-09 | 2016-04-27 | 北京东方百泰生物科技有限公司 | The monoclonal antibody of a kind of anti-PD-1 and preparation method thereof |
| WO2017024515A1 (en) * | 2015-08-11 | 2017-02-16 | Wuxi Biologics (Cayman) Inc. | Novel anti-pd-1 antibodies |
| CN106432494B9 (en) * | 2015-08-11 | 2022-02-15 | 广州誉衡生物科技有限公司 | Novel anti-PD-1 antibodies |
| CN105175544B (en) * | 2015-10-20 | 2021-04-09 | 安徽瀚海博兴生物技术有限公司 | anti-PD-1 humanized monoclonal antibody and application thereof |
| CN106267191A (en) * | 2016-09-03 | 2017-01-04 | 山西纳安生物科技有限公司 | Nano antibody biological medicament Percutaneously administrable preparation system and preparation method and application |
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| WO2019104860A1 (en) | 2019-06-06 |
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