CN110218254B

CN110218254B - CD 3-resistant nano antibody CD3/Nb25 and preparation method and application thereof

Info

Publication number: CN110218254B
Application number: CN201810175293.5A
Authority: CN
Inventors: 卢小玲; 杨晓梅; 段斯亮
Original assignee: Guangxi Medical University
Current assignee: Guangxi Medical University
Priority date: 2018-03-02
Filing date: 2018-03-02
Publication date: 2020-12-04
Anticipated expiration: 2038-03-02
Also published as: CN110218254A

Abstract

The invention discloses a CD 3-resistant nano antibody CD3/Nb25 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 nucleotide sequence and the host cell of the nano antibody disclosed by the invention, the nano antibody can be efficiently expressed in escherichia coli, is applied to the research and development of a CD3 molecular detection reagent, and is used for preparing a tumor inhibitor or a tumor cell inhibitor and preparing a medicine for inhibiting the activity of CD3 and promoting the proliferation of T cells.

Description

CD 3-resistant nano antibody CD3/Nb25 and preparation method and application thereof

Technical Field

The invention relates to a nano antibody CD3/Nb25 for resisting CD3 in the field of biomedicine and a preparation method and application thereof.

Background

The CD3 molecule is an important differentiation antigen on the T cell membrane and is a characteristic marker of mature T cells. It forms a complex with the T cell surface receptor TCR and plays an important role in the intracellular signal transduction process. The CD3 antibody can specifically recognize T cell surface CD3 molecule, causing cross-linking of T cell TCR-CD3 complex, directly generating activation signal, resulting in T cell activation and proliferation. The current research shows that the CD3 antibody plays an important role in tumor treatment.

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 heavy chain 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 CD3/Nb25, 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 23 rd to 32 th amino acid of SEQ ID No.8 in the sequence table; the amino acid sequence of the CDR2 is amino acid 49-57 of SEQ ID No.8 in the sequence table; the amino acid sequence of the CDR3 is the 95 th to 112 th amino acid 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 amino acid at the 1 st to 22 th positions of SEQ ID No.8 in the sequence table; the amino acid sequence of FR2 is the 33 rd-48 th amino acid of SEQ ID No.8 in the sequence table; the amino acid sequence of FR3 is the 58 th-94 th amino acid of SEQ ID No.8 in the sequence table; the amino acid sequence of the FR4 is the 113 th and 123 th amino acids 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 CD3, which comprises the amino acid sequences of the framework region FR and the complementarity determining region CDR, wherein the framework region FR is selected from the following group of FRs: 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 CD3 has the amino acid sequence of SEQ ID NO: 8.

In order to facilitate the purification of the nano antibody CD3/Nb25, the amino terminal or the carboxyl terminal of the protein shown by the amino acids 1-123 of the 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 (in general)Is 5 pieces)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL
HA	9	YPYDVPDYA

In another aspect of the invention, the nano antibody CD3/Nb25 can be synthesized artificially, or can be obtained by synthesizing the coding gene and then performing biological expression. The coding gene of the nano antibody CD3/Nb25 can be obtained by deleting one or more codons of amino acid residues in a DNA sequence shown by 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 CD3/Nb 25.

The biomaterial related to the nano antibody CD3/Nb25 provided by the invention is any one of B1) to B12):

B1) nucleic acid molecules encoding the nanobody CD3/Nb 25;

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 CD3/Nb25 described in B2), also called CD3/Nb25 gene expression cassette, refers to DNA capable of expressing the nanobody CD3/Nb25 in host cells, and the DNA may include not only a promoter for initiating transcription of the nanobody CD3/Nb25 gene, but also a terminator for terminating transcription of the nanobody CD3/Nb25 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 CD3/Nb25 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) is a recombinant vector pComb3-CD3/Nb25 obtained by introducing a coding gene (nucleotide sequence is nucleotides 1-369 of SEQ ID No.9 in a sequence table) of the nano antibody CD3/Nb25 into pComb3, and the recombinant vector pComb3-CD3/Nb25 expresses the nano antibody CD3/Nb25 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 CD3/Nb25 of the present 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 to the nucleotide sequence of the nanobody CD3/Nb25 of the present invention B1), as long as encoding the nanobody CD3/Nb25 and having the nanobody CD3/Nb25 activity, are derived from the nucleotide sequence of the present invention and are identical to the sequence of the present 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 CD3/Nb 25;

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 CD3/Nb 25.

In order to solve the technical problems, the invention also provides a derivative antibody of the nano antibody CD3/Nb 25.

The derivative antibody of the nano antibody CD3/Nb25 provided by the invention is a) or b) or c) or d) or e):

a) a single-chain antibody containing the nano-antibody CD3/Nb 25;

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

c) a fusion antibody containing the nanobody CD3/Nb 25;

d) fab containing the nanobody CD3/Nb 25;

e) an intact antibody comprising the Nanobody CD3/Nb 25.

In order to solve the technical problems, the invention also provides a preparation method of the nano antibody CD3/Nb 25.

The preparation method of the nano antibody CD3/Nb25 provided by the invention comprises the steps of introducing a nucleic acid molecule for coding the nano antibody CD3/Nb25 into a receptor cell to obtain a transgenic cell for expressing the nano antibody CD3/Nb25, and culturing the transgenic cell to obtain the nano antibody CD3/Nb 25.

In the preparation method of the nano antibody CD3/Nb25, the nucleotide sequence of the nucleic acid molecule for coding the nano antibody CD3/Nb25 is shown as SEQ ID No.9 in the sequence table.

In the preparation method of the nano antibody CD3/Nb25, 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 the application of the nano antibody CD3/Nb25 in preparing 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 CD3/Nb25 in the preparation of tumor inhibitors or tumor cell inhibitors;

a5, and application of the nano antibody CD3/Nb25 in preparing products for inhibiting CD3 activity or promoting T cell proliferation;

a6, the application of the biomaterial in preparing products for inhibiting CD3 activity or promoting T cell proliferation;

a7, the use of the derivative antibody in the preparation of products for inhibiting CD3 activity or promoting T cell proliferation;

a8, and application of the preparation method of the nano antibody CD3/Nb25 in preparation of products for inhibiting CD3 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 a nano antibody for resisting CD3, 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 CD3 molecular detection reagents, and is used for preparing tumor inhibitors or tumor cell inhibitors and preparing medicines for inhibiting CD3 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 channel is a molecular marker of 2000bp, the second channel is a DNA electrophoresis band of a CD3 nano antibody, and a PCR product band is about 450 bp.

FIG. 2 is an electrophoresis diagram of SDS-PAGE of CD3 nanobody CD3/Nb25 after 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 CD3/Nb25 with B cells; FIG. 3B shows the result of the binding experiment of the Nanobody CD3/Nb25 to 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 CD3/Nb25, and the amino acid sequence of the nano antibody CD3/Nb25 is shown as SEQ ID No.8 in a sequence table and is coded by the nucleotide sequence of SEQ ID No. 9.

The nucleotide electrophoresis diagram of the nano antibody CD3/Nb25 is shown in figure 1, wherein the first channel is a 2000bp molecular marker, the second channel is a CD3 nano antibody DNA electrophoresis band, and the PCR product band is about 450 bp.

The DNA fragment between the PstI and NotI recognition sequences of the vector pComb3(Biovector product) 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-CD3/Nb25, pComb3-CD3/Nb25 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-CD3/Nb25 expresses a nano antibody CD3/Nb25 shown in SEQ ID No. 8. The recombinant strain WK6-pComb3-CD3/Nb25 is obtained by introducing pComb3-CD3/Nb25 into Escherichia coli WK 6.

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

(1) coating WK6-pComb3-CD3/Nb25 on LB plate containing ampicillin and glucose (in the LB plate, the concentrations of ampicillin and glucose were 100. mu.g/mL and 20mg/mL, respectively), and culturing 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 reaches 0.6-1, adding IPTG (isopropyl-beta-D-beta-D-3/Nb-25) to obtain a culture solution WK6-pComb3-CD 3/Nb-25, enabling the concentration of IPTG in the culture solution WK6-pComb3-CD 3/Nb-25 to be 1mM, and carrying out shake culture on the culture solution WK6-pComb3-CD3/Nb25 at 28 ℃ on a shaker (the rotation speed of the shaker is 220rpm) overnight (12 hours) to obtain an induction solution WK6-pComb3-CD3/Nb 25;

(4) centrifuging the induction liquid WK6-pComb3-CD3/Nb25 of 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 bioactive nanoparticles for rapid and sensitive detection of nonfluenza H3N2.nanoscale Res Lett 2014,9: 528.); the method comprises the following specific steps: collecting the bacterial liquid containing the thalli in the step (4), centrifuging at 6000rpm for 20min, removing supernatant after thalli precipitation, adding 4-5ml of lysis solution (20 ml of cell lysis solution formula: 40 microliter 0.5M EDTA (PH8.0), 4ml 10% SDS, 1ml 1M Tris-cl (PH6.8), 14.96ml distilled water) into the thalli precipitation, and carrying out lysis at 250rpm at 4 ℃ for 2h to obtain lysis mixed solution. Then adding lysis solution with the volume ratio of 4:1 to the lysis mixed solution, and carrying out lysis for 2h at 4 ℃. Then, the mixture was centrifuged at 4000rpm for 30min, and the supernatant lysate was collected.

(6) Nanobody CD3/Nb25 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.). Specifically, the supernatant lysate collected after the bacteria lysis in the step (5) is added with 600 microliter of magnesium chloride per tube and mixed evenly, then 1.5ml of nickel magnetic beads are added and mixed evenly, and the mixture is vibrated and combined for 1h at 4 ℃. Poured onto the column, washed with PBS until the nickel beads turn blue, then washed with 20mM imidazole for 2 column volumes, which can be increased to 3 column volumes if the nickel column is dirty. Washing with 2ml 50mM imidazole once, washing with 1ml 100mM imidazole once, adding 1ml 500mM imidazole, standing for 10min, and collecting protein; adding 2ml of 500mM imidazole, standing for 10min, and collecting protein; then, 1ml of 500mM imidazole was added thereto, and the mixture was left for 10min to collect the protein. Mixing the three collected proteins to obtain crude protein extract. And (3) putting the crude protein extracting solution into an ultrafiltration centrifugal tube, and repeatedly washing the crude protein extracting solution with PBS until the concentration of imidazole is 1 mM. The protein on the ultrafiltration tube is the prepared nano antibody CD3/Nb 25.

SDA-PAGE electrophoretograms of the nano antibody CD3/Nb25 are respectively shown in FIG. 2. The result shows that the purity of the nano antibody CD3/Nb25 obtained by the method reaches over 90 percent.

Example 2 determination of the binding Rate of Nanobodies to CD3

Determination of binding rate of nano antibody CD3/Nb25 and CD3 (direct method)

T cells and B cells were isolated from peripheral blood cells of healthy volunteers, and the Nanobody CD3/Nb25 (1. mu.g) of example 1 was added to 1X 10 cells, respectively⁶The above T cells and B cells were incubated at 4 ℃ for 30min in the absence of light, washed 2 times with PBS, and then incubated at 4 ℃ for 30min with 5. mu.l of PE anti-HA tag antibody (abcam, Clone:20B12), and after washing 2 times with PBS, the samples were subjected to BACKMAN flow cytometer, and the results are shown in FIGS. 3A and 3B. FIG. 3A is the percentage of binding of the blank control and CD3 nanobody CD3/Nb25, respectively, to B cells; FIG. 3B is the percentage of binding of the blank control and CD3 nanobody CD3/Nb25, respectively, to T cells; in FIGS. 3A and 3B, the horizontal axis represents fluorescence intensity (PE) and the vertical axis represents percentage by number (% of Max), S2 represents a blank, and S1 represents CD3 nanobody CD3/Nb 25. The result shows that the CD3 nano antibody CD3/Nb25 can be well combined with T cells, and the combination rate is as high as 35.8%.

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> CD 3-resistant nano antibody CD3/Nb25 and preparation method and application thereof

<130> GY18100128

<160> 9

<170> PatentIn version 3.5

<210> 1

<211> 22

<212> PRT

<213> Artificial sequence

<220>

<221> CONFLICT

<222> (1)..(22)

<223> FR1

<400> 1

Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg

1 5 10 15

Leu Ser Cys Ala Ala Ser

20

<210> 2

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> CONFLICT

<222> (1)..(16)

<223> FR2

<400> 2

Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val Ala 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

Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys

1 5 10 15

Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala

20 25 30

Met Tyr Tyr Cys Ser

35

<210> 4

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> CONFLICT

<222> (1)..(11)

<223> FR4

<400> 4

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

1 5 10

<210> 5

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> CONFLICT

<222> (1)..(10)

<223> CDR1

<400> 5

Gly Asp Thr Tyr Ser Arg Tyr Tyr Met Gly

1 5 10

<210> 6

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> CONFLICT

<222> (1)..(9)

<223> CDR2

<400> 6

Ser Pro Asp Gly Gly Ser Thr Asn Tyr

1 5

<210> 7

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<221> CONFLICT

<222> (1)..(18)

<223> CDR3

<400> 7

Ala Asp Leu Asn Gly Thr Trp Tyr Arg Arg Leu Ser Gly Arg Ala His

1 5 10 15

Asn Tyr

<210> 8

<211> 123

<212> PRT

<213> Artificial sequence

<400> 8

Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg

1 5 10 15

Leu Ser Cys Ala Ala Ser Gly Asp Thr Tyr Ser Arg Tyr Tyr Met Gly

20 25 30

Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val Ala Ser Ile

35 40 45

Ser Pro Asp Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly Arg

50 55 60

Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met

65 70 75 80

Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ser Ala Asp

85 90 95

Leu Asn Gly Thr Trp Tyr Arg Arg Leu Ser Gly Arg Ala His 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 ctgggggagg ctcggtgcag gctggagggt ctctgagact ctcctgtgca 60

gcctctggag acacctacag taggtactac atgggctggt tccgccaggt tccagggaag 120

gagcgtgagg gggtcgcaag tatttcacct gatggtggaa gcacaaacta tgccgactcc 180

gtgaagggcc gattcaccat ctcccgagac aacgccaaga acacgctgta tctacaaatg 240

aacagcctga aacctgagga cactgccatg tactactgtt cggcagatct caacggtact 300

tggtaccgac gcctgtccgg gcgggcgcat aactactggg gccaggggac ccaggtcacc 360

gtctcctca 369

Claims

1. A nanobody comprising an epitope-complementing region and a framework region; the method is characterized in that:

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

the amino acid sequence of the CDR1 is the 23 rd to 32 th amino acid of SEQ ID No.8 in the sequence table;

the amino acid sequence of the CDR2 is amino acid 49-57 of SEQ ID No.8 in the sequence table;

the amino acid sequence of the CDR3 is the 95 th to 112 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 containing the recombinant vector of B4);

the nucleic acid molecule is a cDNA molecule or DNA molecule with a nucleotide sequence of SEQ ID No.9 in a sequence table.

3. The method for preparing the nanobody of claim 1, comprising introducing a nucleic acid molecule encoding the nanobody of claim 1 into recipient cells to obtain transgenic cells expressing the nanobody, and culturing the transgenic cells to obtain the nanobody, wherein the nucleotide sequence of the nucleic acid molecule encoding the nanobody of claim 1 is shown as SEQ ID No.9 in the sequence listing.

4. The method of claim 3, wherein: the recipient cell is a microbial cell.