CN107474135B

CN107474135B - anti-PD-1 nano antibody PD-1/Nb20 and preparation method and application thereof

Info

Publication number: CN107474135B
Application number: CN201710085196.2A
Authority: CN
Inventors: 卢小玲; 段斯亮
Original assignee: Guangxi Medical University
Current assignee: Guangxi Medical University
Priority date: 2017-02-17
Filing date: 2017-02-17
Publication date: 2020-08-18
Anticipated expiration: 2037-02-17
Also published as: CN107474135A

Abstract

The invention discloses an anti-PD-1 nano antibody PD-1/Nb20 and a preparation method and application thereof. The nano antibody provided by the invention comprises a determinant complementary region and a framework region; the determinant complementary region of the nanobody consists of CDR1, CDR2 and CDR 3; through the nano antibody nucleotide sequence and the host cell disclosed by the invention, the nano antibody can be efficiently expressed in escherichia coli, and is applied to the research and development of PD-1 molecular detection reagents, the preparation of tumor inhibitors or tumor cell inhibitors and the preparation of medicines for inhibiting PD-1 activity and promoting T cell proliferation.

Description

anti-PD-1 nano antibody PD-1/Nb20 and preparation method and application thereof

Technical Field

The invention relates to an anti-PD-1 nano antibody PD-1/Nb20 in the field of biomedicine and a preparation method and application thereof.

Background

Programmed death receptor 1 (PD-1) is a transmembrane glycoprotein molecule which activates the surface expression of T cells, belongs to a member of the CD28 superfamily, has negative regulation effect on the proliferation of T cells and plays an important regulation effect in immune response. PD-1 is expressed predominantly in activated T cells and B cells, and is a surface receptor for activated T cells, and PD-1 has two ligands, apoptosis-ligand 1(PD-L1, also known as B7-H1) and apoptosis-ligand 1(PD-L2, also known as B7-DC). The tumor microenvironment in the body can induce infiltrated T cells to highly express PD-1 molecules, and the tumor cells can highly express ligands PD-L1 and PD-L2 of PD-1, so that after PD-1 channels in the tumor microenvironment continuously activate PD-L1 to be linked with PD-1, the function of the T cells is inhibited, and signals for attacking tumors cannot be sent to an immune system. The PD-1 inhibitor can block the combination of PD-1 and PD-L1, block negative regulation signals, and restore the activity of T cells, thereby enhancing the immune response. Highly specific, low-reactivity anti-PD-1 monoclonal antibodies have been confirmed in several clinical trials, such as the drug manufactured by Shimadong under the name Keytruda (Pembrolizumab) and the drug manufactured by Shinobao under the name Opdivo (Nivolumab) nivolumab.

However, there are many problems in the application of antibody drugs, such as long development cycle and high production cost; difficult to mass produce; poor stability, easy degradation and high storage cost; is easy to be polluted, and the maintenance cost is high; and has immunogenicity and the like, thereby limiting the application range of the medicine in clinic.

The nano antibody technology is an antibody engineering revolution which is carried out by biomedical scientists by combining molecular biology technology with the concept of nano particle science on the basis of traditional antibodies, thereby developing the latest and smallest antibody molecules. In 1993, Hamers et al reported that a camelid has a heavy chain antibody with a naturally deleted light chain and heavy chain constant region 1(CH1) in it, and the variable region was cloned to obtain a single domain antibody consisting of only one heavy chain variable region, called VHH (variable domain of heavyain of heavy-chain antibody), which has now been renamed as "Nanobody" (Nb). The nano antibody has the smallest antigen binding fragment with complete function, and the crystal structure of the nano antibody is elliptic, the diameter of the nano antibody is 2.5nm, and the length of the nano antibody is 4 nm. Nb has a plurality of unique properties, is very suitable for genetic modification, and shows wide application prospects in the aspects of accurate diagnosis, targeted treatment and the like of diseases. The nano antibody is much simpler in chemical composition and shape than an antibody, has no chemical hydrophobicity, is stronger in heat resistance and acid and alkali resistance, is easier to combine with each other or other compounds, can be encoded by a single gene, and is easy to synthesize by microorganisms. The nano antibody has good tolerance to the environment, high conformational stability, smaller molecular weight and better clinical treatment effect, and meanwhile, the small protein molecules are easier to synthesize and have lower price. The unique property of the nano antibody enables the nano antibody to show wider application prospects in the aspects of accurate diagnosis of diseases, immune targeted therapy and the like.

Disclosure of Invention

The technical problem to be solved by the invention is how to prepare the medicine for effectively treating the tumor.

In order to solve the technical problems, the invention firstly provides a nano antibody.

In a first aspect of the invention, there is provided a nanobody, designated PD-1/Nb20, comprising a determinant complementary region CDR and a framework region FR; the determinant complementary region CDR of the nanobody consists of CDR1, CDR2 and CDR 3; the amino acid sequence of the CDR1 is the 26 th to 35 th amino acids of SEQ ID No.8 in the sequence table; the amino acid sequence of the CDR2 is amino acid 52-60 of SEQ ID No.8 in the sequence table; the amino acid sequence of the CDR3 is the 98 th to 114 th amino acids of SEQ ID No.8 in the sequence table.

Preferably, the framework region FR of the nanobody consists of FR1, FR2, FR3 and FR 4; wherein the amino acid sequence of FR1 is the 1 st-25 th amino acid of SEQ ID No.8 in the sequence table; the amino acid sequence of the FR2 is the 36 th-51 th amino acid of SEQ ID No.8 in the sequence table; the amino acid sequence of the FR3 is the 61 st-97 th amino acid of SEQ ID No.8 in the sequence table; the amino acid sequence of the FR4 is the amino acid at the 115-th and 127-th positions of SEQ ID No.8 in the sequence table.

Preferably, the amino acid sequence of the nano antibody is shown as SEQ ID No.8 in the sequence table.

The invention also correspondingly provides a VHH chain of the nano antibody of the PD-1, which comprises the framework region FR and the complementarity determining region CDR, wherein the framework region FR is selected from the amino acid sequences of the FRs of the following groups: SEQ ID NO: FR1 as shown in 1, SEQ ID NO: FR2 as shown in 2, SEQ ID NO: FR3 as shown in SEQ ID NO: FR4 shown in FIG. 4; the CDR is selected from the amino acid sequence of CDR of the following group: SEQ ID NO: 5, CDR1 shown in SEQ ID NO: 6, CDR2 shown in SEQ ID NO: CDR3 shown in FIG. 7.

Preferably, the VHH chain of the nanobody of PD-1 has the amino acid sequence of SEQ ID NO: 8.

In order to facilitate the purification of the nano antibody PD-1/Nb20, the amino terminal or the carboxyl terminal of the protein shown by the amino acids 1 to 127 of SEQ ID No.8 in the sequence table can be connected with the label shown in the table 1.

TABLE 1 sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG
	8	DYKDDDDK
			Strep-tag II	8	WSHPQFEK
c-myc	10	EQKLISEEDL
			HA
	9	YPYDVPDYA

On the other hand, the nano antibody PD-1/Nb20 can be synthesized artificially, or can be obtained by synthesizing the coding gene and then carrying out biological expression. The coding gene of the nano antibody PD-1/Nb20 can be obtained by deleting one or more codons of amino acid residues in a DNA sequence shown in SEQ ID No.9 in a sequence table, and/or carrying out missense mutation of one or more base pairs, and/or connecting a coding sequence of a label shown in the table 1 at the 5 'end and/or the 3' end of the coding gene.

In order to solve the technical problems, the invention also provides a biological material related to the nano antibody PD-1/Nb 20.

The biological material related to the nano antibody PD-1/Nb20 provided by the invention is any one of B1) to B12):

B1) nucleic acid molecules encoding the nanobody PD-1/Nb 20;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector comprising the nucleic acid molecule of B1);

B4) a recombinant vector comprising the expression cassette of B2);

B5) a recombinant microorganism comprising the nucleic acid molecule of B1);

B6) a recombinant microorganism comprising the expression cassette of B2);

B7) a recombinant microorganism containing the recombinant vector of B3);

B8) a recombinant microorganism containing the recombinant vector of B4);

B9) a transgenic animal cell line comprising the nucleic acid molecule of B1);

B10) a transgenic animal cell line comprising the expression cassette of B2);

B11) a transgenic animal cell line containing the recombinant vector of B3);

B12) a transgenic animal cell line comprising the recombinant vector of B4).

In the above biological material, the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

In the above biomaterial, the expression cassette containing a nucleic acid molecule encoding the nanobody PD-1/Nb20 described in B2), also called PD-1/Nb20 gene expression cassette, refers to DNA capable of expressing the nanobody PD-1/Nb20 in a host cell, and the DNA may include not only a promoter for initiating transcription of the nanobody PD-1/Nb20 gene, but also a terminator for terminating transcription of the nanobody PD-1/Nb20 gene. Further, the expression cassette may also include an enhancer sequence.

The existing expression vector can be used for constructing a recombinant vector containing the nano antibody PD-1/Nb20 gene expression cassette.

In the above biological material, the vector may be a plasmid, a cosmid, a phage, or a viral vector.

In the above biological material, the recombinant vector may be a recombinant vector obtained by introducing the nucleic acid molecule of B1) into pComb 3. In one embodiment of the invention, B3) the recombinant vector is a recombinant vector pComb3-PD-1/Nb20 obtained by introducing a coding gene (nucleotide sequence is 1-381 th nucleotide of SEQ ID No.9 in a sequence table) of the nano antibody PD-1/Nb20 into pComb3, and the recombinant vector pComb3-PD-1/Nb20 expresses the nano antibody PD-1/Nb20 shown in SEQ ID No. 8.

In the above biological material, the microorganism may be yeast, bacteria, algae or fungi.

In the above biological material, the transgenic animal cell line does not include propagation material; the recombinant microorganism may be a recombinant microorganism obtained by introducing the nucleic acid molecule of B1) into Escherichia coli WK 6.

The nucleotide sequence of the nanobody PD-1/Nb20 of the invention B1) can be easily mutated by a person of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or more than 75% identity with the nucleotide sequence of the nanobody PD-1/Nb20 of the invention B1), as long as the nucleotides encoding the nanobody PD-1/Nb20 and having the nanobody PD-1/Nb20 activity, are derived from the nucleotide sequence of the invention and are identical to the sequence of the invention.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence having 75% or more, or 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence of the present invention encoding the protein represented by SEQ ID No. 8. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

The above-mentioned identity of 75% or more may be 75%, 80%, 85%, 90% or 95% or more.

In the above biological material, the nucleic acid molecule of B1) is 1) or 2) or 3) described below:

1) the nucleotide sequence is a cDNA molecule or a DNA molecule of SEQ ID No.9 in a sequence table;

2) a cDNA molecule or a genome DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by 1) and codes the nano antibody PD-1/Nb 20;

3) hybridizing with the nucleotide sequence defined in 1) under strict conditions, and encoding the cDNA molecule or genome DNA molecule of the nano antibody PD-1/Nb 20.

In order to solve the technical problems, the invention also provides a derivative antibody of the nano antibody PD-1/Nb 20.

The derivative antibody of the nano antibody PD-1/Nb20 provided by the invention is a) or b) or c) or d) or e) or f):

a) a single-chain antibody containing the nano antibody PD-1/Nb 20;

b) a fusion antibody comprising a) said single chain antibody;

c) a fusion antibody containing the nano antibody PD-1/Nb 20;

d) fab containing the nanobody PD-1/Nb 20;

e) a whole antibody containing the nano-antibody PD-1/Nb 20;

f) a complex comprising the nanobody of

claim

1 or 2 or 3; the compound refers to proteins such as toxin, medicine, cytokine and the like, or all nano materials such as liposome, nano micelle, chitosan, silicon, nano gold and the like.

In order to solve the technical problems, the invention also provides a preparation method of the nano antibody PD-1/Nb 20.

The preparation method of the nano antibody PD-1/Nb20 provided by the invention comprises the steps of introducing a nucleic acid molecule for coding the nano antibody PD-1/Nb20 into a receptor cell to obtain a transgenic cell for expressing the nano antibody PD-1/Nb20, and culturing the transgenic cell to obtain the nano antibody PD-1/Nb 20.

In the preparation method of the nano antibody PD-1/Nb20, the nucleotide sequence of the nucleic acid molecule for coding the nano antibody PD-1/Nb20 is shown as SEQ ID No.9 in a sequence table.

In the preparation method of the nano antibody PD-1/Nb20, the receptor cell can be a microbial cell, such as Escherichia coli, and specifically can be Escherichia coli WK 6.

In order to solve the technical problem, the invention also provides any one of the following applications A1-A8:

a1 and application of the nano antibody PD-1/Nb20 in preparation of tumor inhibitors or tumor cell inhibitors;

a2, the application of the biological material in preparing tumor inhibitors or tumor cell inhibitors;

a3, and the application of the derivative antibody of the nano antibody in preparing a tumor inhibitor or a tumor cell inhibitor;

a4 and the application of the preparation method of the nano antibody PD-1/Nb20 in preparing tumor inhibitors or tumor cell inhibitors;

a5 and application of the nano antibody PD-1/Nb20 in preparing products for inhibiting PD-1 activity or promoting T cell proliferation;

a6, the application of the biological material in preparing products for inhibiting PD-1 activity or promoting T cell proliferation;

a7, and application of the derivative antibody in preparing products for inhibiting PD-1 activity or promoting T cell proliferation;

a8 and application of the preparation method of the nano antibody PD-1/Nb20 in preparation of products for inhibiting PD-1 activity or promoting T cell proliferation.

The product may be a medicament.

The primer pair for amplifying the nucleic acid molecule of the amino acid sequence shown in SEQ ID No.8 in the coding sequence list or any fragment of the amino acid sequence also belongs to the protection scope of the invention.

The invention provides an anti-PD-1 nano antibody, a nucleotide sequence and a host cell for encoding the nano antibody, and a preparation method and application thereof. The nano antibody can be efficiently expressed in escherichia coli, is applied to the research and development of PD-1 molecular detection reagents, and is used for preparing tumor inhibitors or tumor cell inhibitors and medicines for inhibiting PD-1 activity and promoting T cell proliferation.

Drawings

FIG. 1 is a DNA electrophoresis diagram of a nanobody, and DNA bands from left to right of a gel hole are respectively: the first path is a molecular marker of 2000bp, the rest pore paths are PCR products, and the PCR product band is about 500 bp.

FIG. 2 is an electrophoresis diagram of SDS-PAGE of PD-1 nanobody PD-1/Nb20 after purification by nickel column resin gel affinity chromatography; lane M shows the protein molecular weight Marker.

FIG. 3A shows the result of the binding experiment of the Nanobody PD-1/Nb20 with unactivated T cells; FIG. 3B shows the result of the binding experiment of the Nanobody PD-1/Nb20 with activated T cells.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention.

The experimental procedures in the following examples are conventional unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The E.coli WK6 in the examples described below was obtained from Guangxi medical university after agreement with the Wanyakun laboratory of the institute of Life sciences of southeast university, and the biomaterial was only used for repeating the experiments related to the present invention, and was not used for other purposes.

Example 1 preparation of Nanobodies

The invention provides 1 camel-derived nano antibody, which is named as PD-1/Nb20, and the amino acid sequence of the nano antibody PD-1/Nb20 is shown as SEQ ID No.8 in a sequence table and is coded by a nucleotide sequence of SEQ ID No. 9.

The nucleotide electrophoresis diagram of the nano antibody PD-1/Nb20 is shown in figure 1, wherein the first path is a molecular marker of 2000bp, the rest pore paths are PCR products, and the PCR product band is about 500 bp.

The DNA fragment between the PstI and NotI recognition sequences of the vector pComb3 (product of Biovector) was replaced with the DNA molecule shown in SEQ ID No.9, and the other sequences were not changed, resulting in a recombinant vector pComb3-PD-1/Nb20, pComb3-PD-1/Nb20 differing from pComb3 only in that the DNA fragment between the PstI and NotI recognition sequences of pComb3 was replaced with the DNA molecule shown in SEQ ID No. 9. The recombinant vector pComb3-PD-1/Nb20 expresses a nano antibody PD-1/Nb20 shown in SEQ ID No. 8. The pComb3-PD-1/Nb20 is introduced into Escherichia coli WK6 to obtain recombinant strain WK6-pComb3-PD-1/Nb 20.

The specific preparation steps of the nano antibody are as follows:

(1) WK6-pComb3-PD-1/Nb20 was spread on LB plates containing ampicillin and glucose (in the LB plates, the concentrations of ampicillin and glucose were 100. mu.g/mL and 20mg/mL, respectively), and cultured overnight (12 hours) at 37 ℃;

(2) a single colony was selected and inoculated into 5mL of LB medium containing ampicillin (ampicillin concentration: 100. mu.g/mL in LB medium), and shake-cultured overnight (12 hours) at 37 ℃;

(3) inoculating 1mL of culture solution cultured overnight in the step (2) into 330mL of TB culture solution, carrying out shake culture at 37 ℃ until OD value reaches 0.6-1, adding IPTG (isopropyl-beta-D-2-PD-1/Nb 20) to obtain a WK6-pComb3-PD-1/Nb20 culture solution, enabling the concentration of IPTG in the WK6-pComb3-PD-1/Nb20 culture solution to be 1mM, and carrying out shake culture on the WK6-pComb3-PD-1/Nb20 culture solution at 28 ℃ on a shaker (the rotation speed of the shaker is 220rpm) overnight (12 hours) to obtain a WK6-pComb3-PD-1/Nb20 induction solution;

(4) centrifuging the induction liquid WK6-pComb3-PD-1/Nb20 obtained in the step (3) at 4 ℃, and collecting thalli;

(5) obtaining a crude antibody extract by an infiltration method in the literature (Zhu M, Hu Y, Li G, Ou W, Mao P, Xin S, Wan Y: Combining magnetic nanoparticles with biologically functionalized nanoparticles for rapid and sensitive detection of antibiotics H3N2.nanoscale Res Lett 2014,9: 528.);

(6) nanobody PD-1/Nb20 was prepared by nickel column ion affinity chromatography in the literature (Zhu M, Hu Y, Li G, Ou W, Mao P, Xin S, Wan Y: Combining magnetic nanoparticles with biologically functionalized nanoparticles for rapid and sensitive detection of antibiotics H3N2.nanoscale Res Lett 2014,9: 528.). SDA-PAGE electrophoretograms of the nano antibody PD-1/Nb20 are respectively shown in FIG. 2. The result shows that the purity of the nano antibody PD-1/Nb20 obtained by the method reaches over 90 percent.

Example 2 determination of binding ratio of Nanobodies to PD-1

Determination of binding rate of nano antibody PD-1/Nb20 and PD-1 (direct method)

T cells were isolated from peripheral blood cells of healthy volunteers, cultured in a 96-well plate, 10. mu.g/ml PHA (sigma, L9017) was added to the T cells for co-culture for 72 hours, and then the Nanobody PD-1/Nb20 (1. mu.g) of example 1 was added to 1 × 10⁶After each of the above T cells was incubated at 4 ℃ for 30min in the absence of light, washed 2 times with PBS, 5. mu.l of PE anti-HA tag antibody (abcam, Clone:20B12) was added and incubated at 4 ℃ for 30min, and after washing 2 times with PBS, the sample was subjected to BACKMAN flow cytometer, and the results are shown in FIG. 3B, and the non-activated T cells were used as controls in FIG. 3A. FIG. 3A is the percent binding of the blank control and PD-1 nanobody PD-1/Nb20, respectively, to unactivated T cells; FIG. 3B is the percent binding of the blank control and PD-1 nanobody PD-1/Nb20, respectively, to activated T cells; in FIGS. 3A and 3B, the horizontal axis represents fluorescence intensity (PE) and the vertical axis represents percentage by number (% of ofMax), S2 represents a blank, and S1 represents PD-1 nanobody PD-1/Nb 20. The results shown in the figure show that the PD-1 nano antibody PD-1/Nb20 can be well combined with activated T cells.

Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

SEQUENCE LISTING

<110> Guangxi university of medical science

<120> anti-PD-1 nano antibody PD-1/Nb20 and preparation method and application thereof

<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

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<213> Artificial sequence

<220>

<221>CONFLICT

<222>(1)..(17)

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<400>7

Lys Glu Phe Val Ala Val Val Pro Val Leu Lys Leu Gly Arg Pro Arg

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Asp

<210>8

<211>127

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<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

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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

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<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

1. A nanobody comprising a determinant complementarity region; the method is characterized in that:

the determinant complementary region of the nanobody consists of CDR1, CDR2 and CDR 3;

the amino acid sequence of the CDR1 is the 26 th to 35 th amino acids of SEQ ID No.8 in the sequence table;

the amino acid sequence of the CDR2 is amino acid 52-60 of SEQ ID No.8 in the sequence table;

the amino acid sequence of the CDR3 is the 98 th-114 th amino acid of SEQ ID No.8 in the sequence table;

the amino acid sequence of the nano antibody is shown as SEQ ID No.8 in a sequence table.

2. The biological material related to the nanobody of claim 1, which is any one of B1) to B12):

B1) a nucleic acid molecule encoding the nanobody of claim 1;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector comprising the nucleic acid molecule of B1);

B4) a recombinant vector comprising the expression cassette of B2);

B5) a recombinant microorganism comprising the nucleic acid molecule of B1);

B6) a recombinant microorganism comprising the expression cassette of B2);

B7) a recombinant microorganism containing the recombinant vector of B3);

B8) a recombinant microorganism containing the recombinant vector of B4);

B9) a transgenic animal cell line comprising the nucleic acid molecule of B1);

B10) a transgenic animal cell line comprising the expression cassette of B2);

B11) a transgenic animal cell line containing the recombinant vector of B3);

B12) a transgenic animal cell line comprising the recombinant vector of B4).

3. The biomaterial of claim 2, wherein: B1) the nucleic acid molecule is 1) or 2) or 3) below:

2) a cDNA molecule or a genome DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by 1) and codes the nanobody of claim 1;

3) a cDNA molecule or a genomic DNA molecule which hybridizes with the nucleotide sequence defined in 1) under stringent conditions and encodes the nanobody of claim 1.

4. The nanobody derivative antibody of claim 1, which is a) or b) or c) or d) or e) or f):

a) a single chain antibody comprising the nanobody of claim 1;

b) a fusion antibody comprising a) said single chain antibody;

c) a fusion antibody comprising the nanobody of claim 1;

d) a Fab comprising the nanobody of claim 1;

e) an intact antibody comprising a nanobody of claim 1;

f) a complex comprising the nanobody of claim 1.

5. The method for preparing the nanobody of claim 1, comprising introducing a nucleic acid molecule encoding the nanobody of claim 1 into a recipient cell to obtain a transgenic cell expressing the nanobody, and culturing the transgenic cell to obtain the nanobody;

the nucleotide sequence of the nucleic acid molecule for coding the nanobody of claim 1 is shown as SEQ ID No.9 in the sequence table.

6. The method of claim 5, wherein: the recipient cell is a microbial cell.

7. Any one of the following uses A1-A4:

a1, the use of the nanobody of claim 1 in the preparation of tumor suppressors or tumor cell suppressors;

use of A2 or the biomaterial of any one of claims 2 to 3 for the preparation of a tumor suppressor or a tumor cell suppressor;

a3, the use of the derivative antibody of claim 4 for preparing tumor suppressor or tumor cell suppressor;

a4, use of the method of claim 5 for preparing tumor suppressor or tumor cell suppressor.