Anti-VIM nano antibody and application thereof

Abstract

The invention relates to an anti-VIM nano antibody and application thereof, belongs to the technical field of biology, and aims to solve the problem of Vimentin VIM detection, and has the key points that a complementary determining region CDR of the nano antibody comprises CDR1, CDR2 and CDR3 sequences: (I): (1) the amino acid sequence of the CDR1 is shown as SEQ ID NO.27, and (2) the amino acid sequence of the CDR2 is shown as SEQ ID NO.33; and (3) the amino acid sequence of the CDR3 is shown as SEQ ID NO.39. The nano antibody and the polypeptide thereof have the effects that the nano antibody and the polypeptide thereof have very high affinity and activity, can specifically recognize and combine with VIM, can be used for capturing and detecting VIM positive cells, and can be applied to immunofluorescence analysis or immunohistochemical analysis and the like through a proper antibody labeling technology.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to an anti-VIM nano antibody, a polypeptide comprising the nano antibody, and preparation and application thereof. Background Art

[0002] Vimentin (VIM) is a type III intermediate filament protein with a molecular weight of approximately 54 kDa, and is mainly found in cells of mesoderm origin. Vimentin plays a key role in many biological processes, including building the cytoskeleton, participating in signal transduction pathways, and significantly upregulating its expression during epithelial-mesenchymal transition (EMT), making it a hallmark indicator of EMT. Therefore, the detection of vimentin is not only of far-reaching significance in the study of VIM-related pathways, but also plays a vital role in the field of oncology.

[0003] Specifically, as a landmark molecule of EMT, vimentin is not only involved in regulating the EMT process, but also involves the formation of mesenchymal phenotype tumor stem cells, and is closely related to the tumor's invasiveness, metastasis ability and drug resistance; when vimentin is used in combination with other tumor markers (such as alpha-fetoprotein), the specificity and sensitivity of diagnosis can be significantly improved; changes in the expression level of vimentin are also regarded as an important indicator for evaluating the efficacy of tumor treatment; vimentin plays a key role in the occurrence, development, invasion and metastasis of tumors, and has become a key target for studying the biological behavior of tumors; the dynamic changes of CTCs are closely related to the EMT process, and the detection of phenotypic changes in CTCs by VIM reflects the heterogeneity of tumors and the differences in EMT status.

[0004] These findings further emphasize the importance of vimentin detection and VIM-positive CTCs detection and capture in oncology research and clinical applications.

[0005] Currently, VIM and its positive CTCs are detected by recognizing the corresponding antigens with monoclonal antibodies (EPR3776, RV202) or polyclonal antibodies (10366-1-AP), which can be used in different experimental applications, including Western Blot (WB), immunohistochemistry (IHC), immunocytochemistry (ICC / IF), flow cytometry (Flow Cyt) and enzyme-linked immunosorbent assay (ELISA), to meet the needs of VIM detection.

[0006] However, traditional antibodies are large in size and expensive to prepare, which limits the practical application of affinity enrichment and detection. Summary of the invention

[0007] In order to solve the problem of vimentin (VIM) detection, in a first aspect, according to the anti-VIM nanobody in some embodiments of the present application, the complementarity determining region CDR of the nanobody includes CDR1, CDR2 and CDR3 sequences:

[0008] (I): (1) the amino acid sequence of the CDR1 is as shown in SEQ ID NO.27, (2) the amino acid sequence of the CDR2 is as shown in SEQ ID NO.33; and (3) the amino acid sequence of the CDR3 is as shown in SEQ ID NO.39; or (II): an amino acid sequence obtained by modifying, replacing, deleting or adding one or more amino acids as the amino acid sequences of (1), (2) and (3) in (I), and having the same function as the amino acid sequence of (I).

[0009] According to the anti-VIM nanoantibodies in some embodiments of the present application, wherein: the amino acid sequence obtained by replacing one or more amino acids in the amino acid sequences of (1), (2) and (3) of (I) in (II) comprises (II-1): the amino acid sequence of CDR1 is as shown in SEQ ID NO.28, the amino acid sequence of CDR2 is as shown in SEQ ID NO.33, and the amino acid sequence of CDR3 is as shown in SEQ ID NO.39; or (II-2): the amino acid sequence of CDR1 is as shown in SEQ ID NO.29, the amino acid sequence of CDR2 is as shown in SEQ ID NO.34, and the amino acid sequence of CDR3 is as shown in SEQ ID NO.39; or (II-3): the amino acid sequence of CDR1 is as shown in SEQ ID NO.30, the amino acid sequence of CDR2 is as shown in SEQ ID NO.35, and the amino acid sequence of CDR3 is as shown in SEQ ID NO.39; or (II-4): the amino acid sequence of CDR1 is as shown in SEQ ID NO.31, the amino acid sequence of CDR2 is as shown in SEQ ID NO.36, and the amino acid sequence of CDR3 is as shown in SEQ ID NO.39; or (II-5): the amino acid sequence of CDR1 is shown in SEQ ID NO.32, the amino acid sequence of CDR2 is shown in SEQ ID NO.37, and the amino acid sequence of CDR3 is shown in SEQ ID NO.39; or (II-6): the amino acid sequence of CDR1 is shown in SEQ ID NO.32, the amino acid sequence of CDR2 is shown in SEQ ID NO.38, and the amino acid sequence of CDR3 is shown in SEQ ID NO.39.

[0010] According to the anti-VIM nanobody in some embodiments of the present application, wherein: (III) the framework region FR of the nanobody includes FR1, FR2, FR3 and FR4 sequences, wherein: (1) the amino acid sequence of the FR1 is as shown in SEQ ID NO.49, (2) the amino acid sequence of the FR2 is as shown in SEQ ID NO.57, (3) the amino acid sequence of the FR3 is as shown in SEQ ID NO.65, (4) the amino acid sequence of the FR4 is as shown in SEQ ID NO.72; or (IV) an amino acid sequence having more than 50% homology with the amino acid sequences described in (1), (2), (3) and (4) of (III).

[0011] According to the anti-VIM nanoantibodies in some embodiments of the present application, (IV) has an amino acid sequence with more than 50% homology with the amino acid sequence described in (1), (2), (3) and (4) of (III), including (IV-1): the amino acid sequence of FR1 is as shown in SEQ ID NO.40, the amino acid sequence of FR2 is as shown in SEQ ID NO.57, the amino acid sequence of FR3 is as shown in SEQ ID NO.65, and the amino acid sequence of FR4 is as shown in SEQ ID NO.72; or (IV-2): the amino acid sequence of FR1 is as shown in SEQ ID NO.40, the amino acid sequence of FR2 is as shown in SEQ ID NO.53, the amino acid sequence of FR3 is as shown in SEQ ID NO.65, and the amino acid sequence of FR4 is as shown in SEQ ID NO.72; or (IV-3): the amino acid sequence of FR1 is as shown in SEQ ID NO.40, the amino acid sequence of FR2 is as shown in SEQ ID NO.59, the amino acid sequence of FR3 is as shown in SEQ ID NO.64, and the amino acid sequence of FR4 is as shown in SEQ ID NO.72; or (IV-4): the amino acid sequence of FR1 is shown in SEQ ID NO.43, the amino acid sequence of FR2 is shown in SEQ ID NO.56, the amino acid sequence of FR3 is shown in SEQ ID NO.64, and the amino acid sequence of FR4 is shown in SEQ ID NO.72; or (IV-5): the amino acid sequence of FR1 is shown in SEQ ID NO.45, the amino acid sequence of FR2 is shown in SEQ ID NO.58, the amino acid sequence of FR3 is shown in SEQ ID NO.68, and the amino acid sequence of FR4 is shown in SEQ ID NO.72; or (IV-6): the amino acid sequence of FR1 is shown in SEQ ID NO.45, the amino acid sequence of FR2 is shown in SEQ ID NO.58, the amino acid sequence of FR3 is shown in SEQ ID NO.63, and the amino acid sequence of FR4 is shown in SEQ ID NO.72; or (IV-7): the amino acid sequence of FR1 is shown in SEQ ID NO.47, the amino acid sequence of FR2 is shown in SEQ ID NO.60, and the amino acid sequence of FR3 is shown in SEQ ID NO. NO.62, the amino acid sequence of FR4 is shown in SEQ ID NO.72; or (IV-8): the amino acid sequence of FR1 is shown in SEQ ID NO.47, the amino acid sequence of FR2 is shown in SEQ ID NO.54, the amino acid sequence of FR3 is shown in SEQ ID NO.62, and the amino acid sequence of FR4 is shown in SEQ ID NO.72; or (IV-9): the amino acid sequence of FR1 is shown in SEQ ID NO.46, the amino acid sequence of FR2 is shown in SEQ ID NO.or (IV-12): the amino acid sequence of FR1 is shown in SEQ ID NO.46, the amino acid sequence of FR2 is shown in SEQ ID NO.54, the amino acid sequence of FR3 is shown in SEQ ID NO.62, and the amino acid sequence of FR4 is shown in SEQ ID NO.72; or (IV-13): the amino acid sequence of FR1 is shown in SEQ ID NO.47, the amino acid sequence of FR2 is shown in SEQ ID NO.55, the amino acid sequence of FR3 is shown in SEQ ID NO.63, and the amino acid sequence of FR4 is shown in SEQ ID NO.72. ID NO.40, the amino acid sequence of FR2 is shown in SEQ ID NO.53, the amino acid sequence of FR3 is shown in SEQ ID NO.70, and the amino acid sequence of FR4 is shown in SEQ ID NO.72; or (IV-14): the amino acid sequence of FR1 is shown in SEQ ID NO.43, the amino acid sequence of FR2 is shown in SEQ ID NO.51, the amino acid sequence of FR3 is shown in SEQ ID NO.64, and the amino acid sequence of FR4 is shown in SEQ ID NO.72; or (IV-15): the amino acid sequence of FR1 is shown in SEQ ID NO.44, the amino acid sequence of FR2 is shown in SEQ ID NO.50, the amino acid sequence of FR3 is shown in SEQ ID NO.63, and the amino acid sequence of FR4 is shown in SEQ ID NO.72; or (IV-16): the amino acid sequence of FR1 is shown in SEQ ID NO.48, the amino acid sequence of FR2 is shown in SEQ ID NO.54, the amino acid sequence of FR3 is shown in SEQ ID NO.62, and the amino acid sequence of FR4 is shown in SEQ ID NO. NO.72; or (IV-17): the amino acid sequence of FR1 is shown in SEQ ID NO.41, the amino acid sequence of FR2 is shown in SEQ ID NO.55, the amino acid sequence of FR3 is shown in SEQ ID NO.61, and the amino acid sequence of FR4 is shown in SEQ ID NO.73; or (IV-18): the amino acid sequence of FR1 is shown in SEQ ID NO.44, the amino acid sequence of FR2 is shown in SEQ ID NO.51, and the amino acid sequence of FR3 is shown in SEQ ID NO.67, the amino acid sequence of FR4 is shown in SEQ ID NO.72; or (IV-19): the amino acid sequence of FR1 is shown in SEQ ID NO.41, the amino acid sequence of FR2 is shown in SEQ ID NO.52, the amino acid sequence of FR3 is shown in SEQ ID NO.61, and the amino acid sequence of FR4 is shown in SEQ ID NO.73; or (IV-20): the amino acid sequence of FR1 is shown in SEQ ID NO.42, the amino acid sequence of FR2 is shown in SEQ ID NO.50, the amino acid sequence of FR3 is shown in SEQ ID NO.66, and the amino acid sequence of FR4 is shown in SEQ ID NO.72.

[0012] According to the anti-VIM nanobody in some embodiments of the present application, the nanobody has (V) an amino acid sequence as shown in SEQ ID NO.1; or (VI) an amino acid sequence obtained by modifying, replacing, deleting or adding one or more amino acids as described in (V), and an amino acid sequence with the same function as the amino acid sequence described in (I).

[0013] According to the anti-VIM nanobody in some embodiments of the present application, the amino acid sequence of the nanobody of (VI) is shown in any one of SEQ ID NO.2 to SEQ ID NO.26.

[0014] In a second aspect, the polypeptide according to some embodiments of the present application comprises any one of the Nanobodies described.

[0015] On the third side, a nucleic acid molecule encoding any one of the Nanobodies described in some embodiments of the present application.

[0016] In a fourth aspect, the expression vector according to some embodiments of the present application comprises the nucleic acid molecule.

[0017] In a fifth aspect, a host cell is transformed or transfected with the expression vector according to some embodiments of the present application.

[0018] In a sixth aspect, the binding substance or conjugate according to some embodiments of the present application comprises any one of the chemically labeled or biologically labeled Nanobodies.

[0019] In the seventh aspect, the adsorbent according to some embodiments of the present application includes any one of the nanoantibodies; or the polypeptide; or the nucleic acid molecule; or the expression vector; or the host cell; or the conjugate; or the conjugate, and a carrier.

[0020] In the eighth aspect, according to some embodiments of the present application, the kit is characterized in that it comprises any one of the nanoantibodies; or the polypeptide; or the nucleic acid molecule; or the expression vector; or the host cell; or the conjugate; or the conjugate; or the adsorbent, and an auxiliary agent acceptable in detection.

[0021] In the ninth aspect, the device according to some embodiments of the present application is used to capture, adsorb and / or detect VIM, including any one of the nanoantibodies; or the polypeptide; or the nucleic acid molecule; or the expression vector; or the host cell; or the conjugate; or the conjugate; or the adsorbent; or the kit.

[0022] In the tenth aspect, the use of the Nanobody; or the polypeptide; or the nucleic acid molecule; or the expression vector; or the host cell; or the conjugate; or the conjugate; or the adsorbent; or the kit according to any one of some embodiments of the present application in the preparation of a preparation for specific capture, adsorption and / or detection of VIM.

[0023] Use of the nanobody according to any one of some embodiments of the present application; or the polypeptide; or the nucleic acid molecule; or the expression vector; or the host cell; or the conjugate; or the conjugate; or the adsorbent; or the kit in the preparation of a tumor detection preparation that specifically captures, adsorbs and / or detects VIM.

[0024] Use of the nanobody according to any one of some embodiments of the present application; or the polypeptide; or the nucleic acid molecule; or the expression vector; or the host cell; or the conjugate; or the conjugate; or the adsorbent; or the kit in the preparation of an enriched and / or purified cell preparation for specific capture, adsorption and / or detection of VIM.

[0025] Use of the nanobody described in any one of some embodiments of the present application; or the polypeptide; or the nucleic acid molecule; or the expression vector; or the host cell; or the conjugate; or the conjugate; or the adsorbent; or the kit in the preparation of immunofluorescence analysis or immunohistochemistry analysis reagents for heterosexual capture, adsorption and / or detection of VIM.

[0026] Beneficial effects: The nanoantibody of the present invention is an anti-VIM nanoantibody with a new amino acid sequence discovered through screening. The nanoantibody and its polypeptide have high affinity and activity, can specifically recognize and bind to VIM, can be used for VIM detection and purification, and capture and detection of VIM-positive cells, and can be applied to immunofluorescence analysis or immunohistochemistry analysis through appropriate antibody labeling technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is the kinetic curve of the Nanobody in Example 5 of the present invention, wherein, V1. the response curve of Nanobody V1, V2. the response curve of Nanobody V2, V3. the response curve of Nanobody V3, V4. the response curve of Nanobody V4, V5. the response curve of Nanobody V5, V6. the response curve of Nanobody V6, V7. the response curve of Nanobody V7, V8. the response curve of Nanobody V8, V9. the response curve of Nanobody V9.

[0028] Figure 2 This is the labeling result after V1-biotin capture in Example 8 of the present invention, (a). Cells captured by magnetic beads, (b). Magnetic beads, (c). Cells.

[0029] Figure 3 These are the results of fluorescence staining of V1-FITC in Example 9 of the present invention, A. human MCF-7 cells, B. African green monkey kidney Vero E6 cells, C. mouse l929 cells.

[0030] Figure 4 It is a flow cytometric graph of the fluorescence staining of V1-FITC in Example 9 of the present invention.

[0031] Figure 5 This is the immunohistochemical staining result of V1-HRP in Example 10 of the present invention. DETAILED DESCRIPTION

[0032] The above and other aspects of the present invention will be further described below, wherein:

[0033] (1) Unless otherwise indicated, the term "sequence" as used herein (e.g., in phrases such as "antibody sequence," "variable region sequence," "V HH The term "sequence" or "protein sequence" should generally be understood to include the relevant amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding the amino acid sequence, unless the context requires a narrower interpretation.

[0034] (2) Unless otherwise indicated, all methods, steps, techniques and operations not specifically described are known and well-known to those skilled in the art. For example, reference is still made to the comprehensive background technology cited above and other references cited therein.

[0035] (3) The term "specificity" refers to the ability of a particular antigen-binding molecule (such as a Nanobody or polypeptide of the invention) to bind to different types of antigens or antigenic determinants. The specificity of an antigen-binding molecule can be determined based on its affinity and / or activity. Affinity is expressed as the dissociation equilibrium constant (K) between the antigen and the antigen-binding molecule. D ), is a measure of the binding strength between antigen and antigen-binding molecule, K D The smaller the value, the stronger the binding strength between the antigen and the antigen-binding molecule. D The larger the value, the weaker the binding strength between the antigen and the antigen-binding molecule. a represents the binding constant, K a The larger the value, the faster the binding. a The smaller the K, the slower the binding; d represents the dissociation constant, K d The larger the K, the faster the dissociation. d The smaller the K, the slower the dissociation; and D =K d / K a .

[0036] (4) The term "homologous" refers to a family of Nanobody sequences that bind to the same antigen, have the same number of amino acids and have an amino acid sequence identity greater than 75%.

[0037] (5) Amino acid substitution can generally be described as a substitution in which an amino acid residue can be substituted with an amino acid having a similar chemical structure or with an amino acid having a dissimilar chemical structure, as long as there is little or no effect on the function, activity or other biological properties of the polypeptide. Preferably, the amino acid residue can be substituted with an amino acid having a similar chemical structure.

[0038] For the above-mentioned substitution modes, the situations disclosed in documents WO04 / 037999, WO 98 / 49185, WO 00 / 46383 and WO 01 / 09300 can be cited, but they are not limited thereto. In addition, the (preferred) types and / or combinations of the substitutions can be selected based on the relevant information of other references cited in WO 04 / 037999 and WO 06 / 122786.

[0039] The amino acid substitutions of the present invention may include, but are not limited to, substitutions in which one amino acid in the following groups (a) to (e) is replaced by another amino acid in the same group: (a) Ala, Ser, Thr, Pro and Gly; (b) Asp, Asn, Glu and Gln; (c) His, Lys and Arg; (d) Met, Leu, Ile, Val and Cys; (e) Phe, Tyr and Trp.

[0040] Preferred amino acid substitutions can be listed but are not limited to the following: Ala is substituted with Gly or Ser; Arg is substituted with Lys; Asn is substituted with Gln or His; Asp is substituted with Glu; Cys is substituted with Ser or Thr; Gln is substituted with Asn; Glu is substituted with Asp; Gly is substituted with Ala or Pro; His is substituted with Asn or Gln; Ile is substituted with Leu or Val; Leu is substituted with Ile or Val; Lys is substituted with Arg, Glu or Gln; Met is substituted with Leu, Tyr or Ile; Phe is substituted with Met, Tyr or Leu; Ser is substituted with Thr; Thr is substituted with Ser; Tyr is substituted with Trp; Trp is substituted with Tyr.

[0041] For the framework region, it is more conservative than the complementary determining region. Those skilled in the art will reasonably screen the sequence structure of the framework region according to the actual use and function of the nanobody. As the amino acid sequence of the framework region, an amino acid sequence with a homology of more than 50% is preferred, and an amino acid sequence with a homology of more than 70% is further preferred, and an amino acid sequence with a homology of more than 95% is further preferred.

[0042] Nanobody sequence number and FR sequence information comparison table-1

[0043] Antibody Name Antibody serial number FR1 sequence SEQ ID FR2 sequence SEQ ID V1 SEQ ID No.1 QLQESGGGLVQPGGSLRLSCVTS No.49 MGWYRQAPGKERELVAHI No.57 V2 SEQ ID No.2 QLQESGGGLVQPGGSLRLSCVAS No.40 MGWYRQAPGKERELVAHI No.57 V3 SEQ ID No.3 QLQESGGGLVQPGGSLRLSCVAS No.40 MGWYRQAPGKERELVATI No.53 V4 SEQ ID No.4 QLQESGGGLVQPGGSLRLSCVAS No.40 MGWYRQAPGKEREFVATI No.59 V5 SEQ ID No.5 QLQESGGGLAQPGGSLRLSCAAS No.43 MGWYRQAPGEQREFVATI No.56 V6 SEQ ID No.6 QLQESGGGLVQAGGSLRLSCATS No.45 MGWYRQAPGKQRELVASI No.58 V7 SEQ ID No.7 QLQESGGGLVQAGGSLRLSCATS No.45 MGWYRQAPGKQRELVASI No.58 V8 SEQ ID No.8 QLQESGGGLVQPGGSLRLSCVVS No.47 MGWYRRAPGKERELVAVI No.60 V9 SEQ ID No.9 QLQESGGGLVQPGGSLRLSCVVS No.47 MGWYRQGPGKERELVAVI No.54 V11 SEQ ID No.10 QLQESGGGLVQPGGSLRLSCAAS No.46 MGWYRQAPGKERELVATI No.53 V12 SEQ ID No.11 QLQESGGGLAQPGGSLRLSCAAS No.43 MGWYRQAPGEQREFVATI No.56 V13 SEQ ID No.12 QLQESGGGLVQAGGSLRLSCATS No.45 MGWFRQAPGQGLEAVAAI No.52 V14 SEQ ID No.13 QLQESGGGLVQPGGSLRLSCAAS No.46 MGWYRQGPGKERELVAVI No.54 V15 SEQ ID No.14 QLQESGGGLVQPGGSLRLSCVAS No.40 MGWYRQAPGKERELVATI No.53 V16 SEQ ID No.15 QLQESGGGLAQPGGSLRLSCAAS No.43 MGWFRQAPGKEREFVAAI No.51 V17 SEQ ID No.16 QLQESGGGLVQAGGSLRLSCAAS No.44 MGWFRQAPGKEREFVSAI No.50 V18 SEQ ID No.17 QLQESGGGFVQPGGSLRLSCAAS No.48 MGWYRQGPGKERELVAVI No.54 V1-ah SEQ ID No.18 QLVESGGGLVQPGGSLRLSCAAS No.41 MGWFRQAPGKGLEAVAAI No.55 V2-ah SEQ ID No.19 QLVESGGGLVQPGGSLRLSCAAS No.41 MGWFRQAPGKGLEAVAAI No.55 V3-com SEQ ID No.20 QLQESGGGLVQAGGSLRLSCAAS No.44 MGWFRQAPGKEREFVAAI No.51 V4-com SEQ ID No.21 QLQESGGGLVQAGGSLRLSCAAS No.44 MGWFRQAPGKEREFVAAI No.51 V5-FGLA SEQ ID No.22 QLVESGGGLVQPGGSLRLSCAAS No.41 MGWFRQAPGQGLEAVAAI No.52 V6-FGLA SEQ ID No.23 QLVESGGGLVQPGGSLRLSCAAS No.41 MGWFRQAPGQGLEAVAAI No.52 V7-hs SEQ ID No.24 QLQASGGGFVQPGGSLRLSCAAS No.42 MGWFRQAPGKEREFVSAI No.50 V8-hs SEQ ID No.25 QLQASGGGFVQPGGSLRLSCAAS No.42 MGWFRQAPGKEREFVSAI No.50 V9-hs SEQ ID No.26 QLQASGGGFVQPGGSLRLSCAAS No.42 MGWFRQAPGKEREFVSAI No.50

[0044] Nanobody sequence number and FR sequence information comparison table-2

[0045]

[0046]

[0047] The contribution of the framework region to affinity is small, so amino acid substitutions in the framework region generally do not affect the affinity of the nanobody, and as long as they can exist in a soluble form, the amino acids in the framework region are also suitable for the above amino acid substitutions. Among them, humanization is a typical example of amino acid substitutions in the framework region. In the present invention, SEQ ID No: 10 to SEQ ID No: 26 are humanized forms of SEQ ID No: 1 to SEQ ID No: 9, and none of them affects the affinity of the original sequence.

[0048] Furthermore, the total number of residues in a Nanobody may be in the interval 110 - 120. However, parts, fragments or analogs of Nanobodies are not particularly limited in their length and / or size, as long as such parts, fragments or analogs meet the further requirements listed below and are also suitable for the purposes described herein.

[0049] The nanobodies of the present invention belong to the same family of nanobodies, whose amino acid sequences have the same total length, and whose framework regions FR and antigen-binding regions CDR have the same length and high sequence identity, similar structures, and substantially equivalent antigen-binding abilities.

[0050] As a method for preparing "nanoantibodies", in its broadest sense, it is not limited to a specific biological resource or a specific preparation method. For example, the nanoantibodies of the present invention can be obtained by: (1) isolating the V chain of a naturally occurring heavy chain antibody; HH domain; (2) by expressing a naturally occurring V HH The nucleotide sequence of the domain; (3) by HH domains are "humanized" (as described below) or by expressing a polypeptide encoding the humanized V HH (4) using synthetic or semi-synthetic techniques to prepare proteins, polypeptides or other amino acid sequences; (5) preparing nucleic acids encoding Nanobodies by applying nucleic acid synthesis techniques and then expressing the nucleic acids thus obtained; and / or (6) by any combination of the foregoing.

[0051] In addition, based on a variation of the Nanobodies of the invention, also includes a V HH The amino acid sequence of the nanobody corresponds to the structural domain but has been humanized. Humanization is to use the V of a conventional 4-chain antibody from humans. H One or more amino acid residues existing at corresponding positions in the structural domain replace the naturally occurring V HH One or more amino acid residues of a domain sequence.

[0052] According to one non-limiting embodiment of the invention, the above-mentioned polypeptide consists essentially of a Nanobody. "Essentially consisting of" means that the amino acid sequence of the polypeptide of the invention is identical or corresponds to the amino acid sequence of a Nanobody, wherein a limited number of amino acid residues, such as 1 to 10 amino acid residues, and preferably 1 to 6 amino acid residues, such as 1, 2, 3, 4, 5 or 6 amino acid residues, are added to the amino terminus (N-terminus) and / or carboxyl terminus (C-terminus) of the Nanobody or polypeptide.

[0053] The above amino acid residues may not change the biological properties of the Nanobody, and may add other functionalities to the Nanobody. For example, the amino acid residues may:

[0054] a is a purification tag, i.e. an amino acid sequence or residue that facilitates the purification of the Nanobody, for example, using an affinity technique for the sequence or residue for purification. Some preferred but non-limiting examples of such residues are groups of His-tags (His6 or His8), GST-tag, MBP-tag, Myc-tag, Strep-tag, Flag-tag, HA-tag, V5-tag, S-tag, E-tag;

[0055] b is a soluble tag, i.e., a tag that helps increase the solubility of the nanobody, such as SUMO;

[0056] c is the N-terminal amino acid residue, for example, Met, Ala, Gln or MetAlaGln, AlaGln, whereby it can be expressed in a heterologous host cell or host organism;

[0057] d is a C-terminal Cys residue, which can react with -SH on the ligand or with the Au surface, for example;

[0058] e is a hinge to provide a link or spacer between the nanobody and other groups, for example, a combination of GlySer, IgG hinge, IgA hinge, or other artificially synthesized hinges;

[0059] f is one or more amino acid residues which may be provided with a functional group and / or have been functionalized in a known manner, for example amino acid residues such as lysine or cysteine ​​allow for the attachment of a PEG group as is known in the art.

[0060] The polypeptide of the present invention may also include two or more of the above-described Nanobodies, also known as multivalent polypeptides.

[0061] A bivalent polypeptide comprises two nanobodies, optionally linked by one hinge sequence, a trivalent polypeptide comprises three nanobodies, optionally linked by two hinge sequences, and a tetravalent polypeptide comprises four nanobodies, optionally linked by three hinge sequences. Multivalent polypeptides can bind to the same antigen epitope or to different antigen binding epitopes, the latter also being referred to as multispecific polypeptides.

[0062] About containing one or more V HH For multivalent and multispecific polypeptides of the structural domain and their preparation, reference may be made to the description in EP0822985.

[0063] The hinges used for multivalent and multispecific polypeptides should be well known to those skilled in the art, for example, including Gly-Ser, such as (Gly4Ser)3 or (Gly3Ser2)3 described in WO 99 / 42077; or naturally occurring heavy chain antibody hinge regions or partial regions thereof. For other suitable hinges, reference can also be made to the comprehensive background technology cited above.

[0064] Furthermore, in addition to the one or more Nanobodies, the polypeptides of the invention may also comprise functional groups, parts or residues, such as therapeutically active substances, and / or labels, such as fluorescent labels, isotope labels, biotin labels and enzyme catalytic labels, and the like.

[0065] In addition, the dissociation equilibrium constant (K) of the Nanobodies or polypeptides of the invention binding to VIM D ) is 10 -11 Mole / liter (M). The dissociation equilibrium constant of the present invention is measured using the plasma resonance technique.

[0066] The specific binding between the above antigen and the antigen binding molecule can be determined by any known appropriate method, including Sercatchard Analysis and / or competitive binding assays such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), as well as other new methods known in the art, such as plasma resonance technology (SPR) and / or biomembrane interferometry (BLI) technology, etc. It should be known to those skilled in the art that the affinity parameters measured by different methods vary greatly, and may even differ by 2 to 3 orders of magnitude.

[0067] The nanobodies, polypeptides, and nucleic acids encoding the same of the present invention can be prepared in a known manner, which will be clear to those skilled in the art from the further description herein. A particularly useful method for preparing the nanobodies, polypeptides, and nucleic acids generally comprises the following steps:

[0068] (1) expressing a nucleic acid encoding a Nanobody or polypeptide of the invention in a suitable host cell or host organism or in another suitable expression system, optionally followed by;

[0069] (2) Isolating and / or purifying the Nanobodies or polypeptides of the invention thus obtained.

[0070] Alternatively, other methods may include the following steps:

[0071] (3) culturing and / or maintaining the host of the invention under conditions such that the host of the invention expresses and / or produces a Nanobody and / or polypeptide of the invention; optionally followed by;

[0072] (4) Isolating and / or purifying the Nanobody or polypeptide of the invention thus obtained.

[0073] Nucleic acid of the present invention can be the form of single-stranded or double-stranded DNA or RNA, and preferably double-stranded DNA form.For example, nucleotide sequence of the present invention can be genomic DNA, cDNA or synthetic DNA (as DNA with codon usage that is particularly suitable for expressing in the host cell or host organism to be used, that is, codon optimized).

[0074] The nucleic acids of the invention can be prepared or obtained in a manner known per se, based on the information given herein on the amino acid sequences of the Nanobodies or polypeptides of the invention, and / or can be isolated from suitable natural sources. HH The nucleic acid sequence of the structural domain is subjected to site-directed mutagenesis to provide the nucleic acid of the present invention encoding the analog.

[0075] Nucleic acid of the present invention can also be in such a form, is present in and / or is the part of a genetic construct, which is well known to those skilled in the art. Such a genetic construct generally comprises at least one nucleic acid of the present invention, can be in the form of a vector, such as a plasmid, YAC, viral vector or transposon. Especially, the vector can be an expression vector, that is, a vector (such as in a suitable host cell, host organism and / or expression system) for external and in vivo expression can be provided.

[0076] The nucleic acids of the invention and / or the genetic constructs of the invention can be used to transform host cells or host organisms, i.e. for expressing and / or producing the Nanobodies or polypeptides of the invention. Suitable hosts or host cells are well known to those skilled in the art and may for example be any suitable fungal, prokaryotic or eukaryotic cell or organelle or organism, as well as all other hosts or host cells known per se for the expression and production of antibodies and antibody fragments (including but not limited to single domain antibodies and ScFv fragments), which are well known to those skilled in the art.

[0077] For production, the Nanobodies and polypeptides of the invention can be produced in the milk of transgenic mammals, for example in the milk of rabbits, cows, goats or sheep, but also in plants or parts of plants, including but not limited to their leaves, flowers, fruits, roots or seeds.

[0078] As mentioned above, one advantage of using nanobodies is that polypeptides based thereon can be expressed and prepared in prokaryotic systems, and suitable prokaryotic expression systems, vectors, host cells, etc. are well known to those skilled in the art, such as the references cited above. However, it should be noted that the present invention in its broadest sense is not limited to expression in bacterial systems.

[0079] Preferably, in the present invention, the Nanobodies or polypeptides are produced in bacterial cells, in particular in bacterial cells suitable for large-scale drug production, as described above.

[0080] When the Nanobodies or polypeptides of the invention are expressed for production in cells, the Nanobodies or polypeptides of the invention may be produced intracellularly (e.g. in the cytoplasm or periplasmic space), then isolated from the host cell, and optionally further purified; or may be produced extracellularly (i.e. secretory expression), then isolated from the culture medium, and optionally further purified.

[0081] Some preferred but non-limiting vectors for use with these host cells include vectors for expression in mammalian cells - pMANneo (Clonetech), pUCTtag (ATCC37460) and pMClneo (Stratagene); vectors for expression in bacterial cells - pET vectors (Novagen) and pQE vectors (Qiagen); vectors for expression in yeast or other fungal cells - pYES2 (Invitrogen) and Picha expression vector (Picha expression vector) (Invitrogen); vectors for expression in insect cells - pBlueBacⅡ (Invitrogen) and other baculovirus vectors; and the like.

[0082] The corresponding techniques for transforming the host or host cell of the present invention are well known to those skilled in the art.

[0083] After transformation, those hosts that can detect and select to have transformed nucleotide sequence / genetic construct of the present invention successfully.Converted host cell (it can be in the form of stable cell line) or host organism (it can be in the form of stable mutant line or strain) form another aspect of the present invention.

[0084] The amino acid sequence of the invention can then be isolated from the host cells / host organisms and / or from the culture medium in which the host cells or host organisms are cultivated, by means of protein separation and / or purification techniques known per se, such as (preparative) chromatography and / or electrophoresis techniques, differential precipitation techniques, affinity techniques (e.g. using specific / cleavable amino acid sequences fused to the amino acid sequence of the invention) and / or preparative immunological techniques (i.e. using antibodies directed against the amino acid sequence to be isolated).

[0085] The adsorbent of the present invention can be used to specifically identify VIM.

[0086] The nanobodies, polypeptides or adsorbents of the present invention can be used to purify and detect VIM, and can also be used to capture and detect VIM-positive cells.

[0087] The purpose of the present invention is to solve the above problems and provide a nanobody with an amino acid sequence of a specific structure, a polypeptide comprising the nanobody and its application, so as to solve the problems of complex antibody preparation process, high cost, poor antibody affinity and stability faced by the existing VIM enrichment, purification and detection.

[0088] In order to achieve the above technical objectives, the technical solutions adopted in this application are as follows:

[0089] In the first aspect, the present invention provides a nanobody that binds to VIM, wherein the variable region in the amino acid sequence of the nanobody includes a complementarity determining region CDR and a framework region FR, wherein the complementarity determining region CDR includes a complementarity determining region CDR1, a complementarity determining region CDR2 and a complementarity determining region CDR3, wherein CDR1 is a sequence of SEQ ID No.27 to SEQ ID No.32, CDR2 is a sequence of SEQ ID No.33 to SEQ ID No.38, and CDR3 is a sequence of SEQ ID No.39, and sequences with a homology of more than 75% thereto.

[0090] Preferably, the amino acid sequence of the Nanobody includes: SEQ ID No: 1 to SEQ ID No: 9.

[0091] Preferably, the nanobody is a humanized nanobody, preferably, the humanized nanobody comprises: SEQ ID No: 10 to SEQ ID No: 26.

[0092] In a second aspect, the present invention provides a polypeptide obtained by modifying the N-terminus and / or C-terminus of the above-mentioned Nanobody.

[0093] Preferably, the method of modifying the amino acids at the N-terminus and / or C-terminus of the Nanobody comprises:

[0094] Method 1: adding a tag to the N-terminal and / or C-terminal amino acid of the Nanobody;

[0095] Mode 2, after the N-terminal and / or C-terminal amino acids of the Nanobody are tagged, the tags are further connected to protect the amino acids via hinges;

[0096] Preferably, the tag includes at least one of His-tag, GST-tag, Myc-tag, SUMO-tag, Strep-tag, and Flag-tag, the hinge includes at least one of GS hinge, IgG hinge, IgA hinge, and PEG, and the protected amino acid includes Ala, Gln, Glu, Met or a combination of any two or more of the foregoing amino acids.

[0097] In a third aspect, the present invention provides a polypeptide obtained by multivalent synthesis of the above-mentioned nanoantibody.

[0098] In a fourth aspect, the present invention provides a nucleic acid encoding the above-mentioned Nanobody or the above-mentioned polypeptide.

[0099] In a fifth aspect, the present invention provides an expression vector comprising an expression cassette of the nucleic acid described above in claim 1.

[0100] In a sixth aspect, the present invention provides a host cell comprising the expression vector as claimed in claim 1.

[0101] In a seventh aspect, the present invention provides the use of the nanobody and / or the polypeptide in immunodetection, enrichment and / or purification.

[0102] Preferably, the nanobody and / or the polypeptide is used in the preparation of a VIM adsorbent, a VIM purification kit, or a VIM detection kit.

[0103] Preferably, the Nanobody and / or the polypeptide are used for capturing and detecting VIM-positive cells.

[0104] Example

[0105] The following examples are given to illustrate the specific implementation of the present invention, but the implementation of the present invention is not limited to the following examples, and any selection and change can be made within the scope that does not affect the technical effect to be achieved by the present invention.

[0106] Example 1

[0107] Construction of anti-VIM nanobody library.

[0108] The phage display library used in the present invention is an immune library with T7 phage as a carrier, and the establishment steps are as follows:

[0109] (1) Alpacas (numbered 2209-1 and 2209-2) were immunized with human VIM. Jugular vein blood was collected from two alpacas after four immunizations. Peripheral blood lymphocytes were isolated and total RNA was extracted (PuerLink TM RNA Mini Kit, Life Technologies: 12183018A);

[0110] (3) Total RNA was reverse transcribed into cDNA and amplified using two rounds of nested PCR. HH Gene;

[0111] In the first round of PCR, cDNA was used as a template, UP primer1 and DOWN primer1 were used as upstream and downstream primers, respectively. After amplification, a band of 650-750 bp was recovered, which was used as a template for the second round of PCR. The upstream and downstream primers were UP primer2 and DOWN primer2, respectively. A PCR product of 450-500 bp was recovered.

[0112] UP primer1:CTTGGTGGTCCTGGCTGCTCT,

[0113] DOWN primer1:GGTACGTGCTGTTGAACTGTTCC,

[0114] UP primer2:TATCTAGTC GAATTC CGCCCAGGTGCAGCTC,

[0115] DOWN primer2: AGCGACTAAGCTTTGAGGAGACGGTGAC;

[0116] (3) The PCR product was double-digested with EcoR Ι and Hind Ⅲ and subjected to agarose electrophoresis to recover the 350-500 bp gene band, which was V HH Gene fragments;

[0117] (4) Use T4 ligase to connect the T7 vector ( 10-3 Cloning Kit,MeterckMetillipore :70550-3) and V HH Gene fragments;

[0118] (5) mixing the ligation product with the packaging protein to form a complete T7 phage, and amplifying the mixture to obtain a phage original library;

[0119] (6) After testing, the titer of the original library was 5.32×10 9pfu / mL, with a diversity of 7.5×10 6 .

[0120] Example 2

[0121] Screening of nanobodies.

[0122] First, dilute the antigen VIM to 10 μg / mL with TBS, take 100 μL and add it to a 96-well plate, and incubate at 4°C for 12 hours. Aspirate the antigen dilution in the well, wash the plate 3 times with TBS, pat dry, add 1% protein-free blocking solution (purchased from Sangon Biotechnology Co., Ltd.), 300 μL / well, and incubate at room temperature for 2 hours (1% protein-free blocking solution and 1% BSA are used alternately during screening). Aspirate the blocking agent in the well, wash the plate 6 times with TBST, pat dry, add the amplified phage, 100 μL / well, and incubate at room temperature for 30 minutes. Wash the plate 10 times with TBST, add T7 elution buffer (1% SDS) to elute the phage, incubate at room temperature for 30 minutes, and amplify the eluate for the next round of screening.

[0123] Example 3

[0124] Construction of genetically engineered bacteria

[0125] (1) After four rounds of screening, the screening eluate was subjected to solid amplification, plaques were picked, and PCR amplification was performed using the plaque amplification solution as a template and UP primer 3 and DOWN primer 3 as upstream and downstream primers;

[0126] UP primer3: TTCCTTAA CATATG GCCCAGGTGCAGCTCGT,

[0127] DOWN primer3: TTAAGGAA CTCGAG CACGGTGACCAGGGTC;

[0128] (2) A portion of the PCR products were outsourced for sequencing to obtain the sequence information of the nanobody. Based on the length and homology of the CDR region, 9 monoclonal sequences belonging to the same family were selected. The naming and sequence number information of the nanobody are shown in Table 1.

[0129] (3) Another part of the PCR product was double-digested with NdeI and XhoI, and the digestion product was recovered. At the same time, the digestion was performed in the same way and the vector was recovered. The digestion product and the vector were ligated with T4 ligase, and the ligation product was transferred into Escherichia coli to obtain a genetically engineered bacterium expressing VIM-specific nanobodies.

[0130] Table 1 Comparison table of nanobody sequence numbers and CDR sequence information

[0131] <h2 style=";text-align:left;direction:ltr"> Antibody Name Antibody Serial Number CDR1 Sequence SEQ ID CDR2 Sequence SEQ ID CDR3 Sequence SEQ ID V1 SEQ ID No.1 GNAFSDNA No.27 SKNSNT No.33 AIGPRYG No.39 V2 SEQ ID No.2 GNAGSDNA No.28 SKNSNT No.33 AIGPRYG No.39 V3 SEQ ID No.3 GSTSSENA No.29 SRNTNT No.34 AIGPRYG No.39 V4 SEQ ID No.4 GSTSSDNA No.30 SWDSNT No.35 AIGPRYG No.39 V5 SEQ ID No.5 GSTSSDNA No.30 SWDSNT No.35 AIGPRYG No.39 V6 SEQ ID No.6 GFAFSVNA No.31 SNGGNT No.36 AIGPRYG No.39 V7 SEQ ID No.7 GFAFSVNA No.31 SNGGNT No.36 AIGPRYG No.39 V8 SEQ ID No.8 GFPSSVNA No.32 SWDENT No.37 AIGPRYG No.39 V9 SEQ ID No.9 GFPSSVNA No.32 SWDGNT No.38 AIGPRYG No.39 <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0132] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.1:QLQESGGGLVQPGGSLRLSCVTS-GNAFSDNA-MGWYRQAPGKERELVAHI-SKNSNT-NYPDS VKGRFIISRNNAQNTVYLQMNSLNPEDTAVYYCT-AIGPRYG-WGQGTQVTVSS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0133] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.2:QLQESGGGLVQPGGSLRLSCVAS-GNAGSDNA-MGWYRQAPGKERELVAHI-SKNSNT-NYPDS VKGRFIISRNNAQNTVYLQMNSLNPEDTAVYYCT-AIGPRYG-WGQGTQVTVSS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0134] <h2 style=";text-align:left;direction:ltr"> SEQ ID No. 3: QLQESGGGLVQPGGSLRLSCVAS-GSTSSENA-MGWYRQAPGKERELVATI-SRNTNT-NYPDSV KGRFIISRNNAQNTVYLQMNSLNPEDTAVYYCT-AIGPRYG-WGQGTQVTVSS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0135] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.4:QLQESGGGLVQPGGSLRLSCVAS-GSTSSDNA-MGWYRQAPGKEREFVATI-SWDSNT-NYPDSV KGRFIISRDNAQNTVYLQMNSLKPEDTAVYYCT-AIGPRYG-WGQGTQVTVSS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0136] <h2 style=";text-align:left;direction:ltr"> SEQ ID No. 5: QLQESGGGLAQPGGSLRLSCAAS-GSTSSDNA-MGWYRQAPGEQREFVATI-SWDSNT-NYPDSV KGRFIISRDNAQNTVYLQMNSLKPEDTAVYYCT-AIGPRYG-WGQGTQVTVSS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0137] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.6:QLQESGGGLVQAGGSLRLSCATS-GFAFSVNA-MGWYRQAPGKQRELVASI-SNGGNT-NYKDS VKGRFIISRDNAKNTVYLQMNSLKPEDTAVYYCT-AIGPRYG-WGQGTQVTVSS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0138] SEQ ID No.7:QLQESGGGLVQAGGSLRLSCATS-GFAFSVNA-MGWYRQAPGKQRELVASI-SNGGNT-NYKDSVKGRFTISRDNAQNTVYLQMNSLKPEDTAVYYCT-AIGPRYG-WGQGTQVTVSS

[0139] SEQ ID No.8:QLQESGGGLVQPGGSLRLSCVVS-GFPSSVNA-MGWYRRAPGKERELVAVI-SWDENT-YYKDLAKGRFIISRDNAQNTVFLQMNSLTPEDTAVYYCT-AIGPRYG-WGQGTQVTVSS

[0140] SEQ ID No.9:QLQESGGGLVQPGGSLRLSCVVS-GFPSSVNA-MGWYRQGPGKERELVAVI-SWDGNT-YYKDLAKGRFIISRDNAQNTVFLQMNSLTPEDTAVYYCT-AIGPRYG-WGQGTQVTVSS

[0141] Example 4

[0142] Preparation of VIM nanobodies

[0143] (1) The basic culture medium of the nanobody is TB medium, which is inoculated at a 5% inoculum and cultured at 37°C for 3-5 h, and the inducer IPTG (final concentration 0.25 mM, the same below) is added for overnight induction;

[0144] (2) After induction, the mixture was centrifuged at 4000 rpm for 20 min to obtain wet bacteria containing nanobodies.

[0145] (3) Add lysis buffer (10 mM imidazole, 500 mM NaCl, pH 7.40.02M PB) to the obtained wet bacteria at a ratio of 1:10, and use a 700 bar high-pressure homogenizer to disrupt the cells;

[0146] (4) Centrifuge at 4°C and 10,000 rpm for 20 min and collect the supernatant;

[0147] (5) The supernatant was filtered through a 0.45 μm filter and then passed through an affinity chromatography column (GE Healthcare, US) to separate and purify the VIM nanobody, wherein the filler of the affinity chromatography column was Ni Sepharose High Performance;

[0148] (6) The nanoantibodies purified by affinity chromatography were subjected to SDS-PAGE electrophoresis to determine the purity, and the protein solution with higher purity was selected to determine the protein concentration using the BCA method.

[0149] Example 5 Affinity Analysis

[0150] The SPR technique was used to analyze the binding ability of nanobodies to human VIM. VIM was amino-coupled to a CM5 sensor chip at a density of 500-800 RU, and nanobodies were injected at 7 different concentrations ranging from 1 to 100 nM, with a flow rate of 45 μL / min in all experiments. The chip regeneration condition was glycine-HCl pH 1.5. The binding curves obtained at different nanobody concentrations were used to calculate the kinetic parameter K a , K d and K D . Figure 1 The nine response curves of nanobodies V1 to V9 are shown in Figure 2. The response curves are from top to bottom the response curves of nanobodies at seven concentrations, namely, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, and 1.5625 nM. The kinetic parameters shown in Table 2 were calculated by equation fitting. Nanobodies have a high affinity for VIM, with K D The range is 10 -11 (M).

[0151] Table 2 Affinity of nanobodies for VIM

[0152] Nanobodies ka(1 / Ms) kd(1 / s) KD(M) V1 2.07E+04 4.05E-07 1.96E-11 V2 2.31E+04 3.05E-07 1.32E-11 V3 3.07E+04 4.88E-07 1.59E-11 V4 2.49E+04 3.08E-07 1.24E-11 V5 3.08E+04 4.27E-07 1.39E-11 V6 6.67E+03 5.78E-07 8.67E-11 V7 8.50E+03 5.27E-07 6.20E-11 V8 9.90E+03 3.22E-07 3.25E-11 V9 1.06E+04 6.14E-07 5.79E-11

[0153] Example 6: Nanobody humanization.

[0154] Humanization method 1

[0155] The protein sequences of nanobodies V3 to V9 were compared with those of human germlines, and the differential amino acids were marked in the framework region. Then, the differential amino acids of the two were replaced with human amino acids by site-directed mutagenesis to generate partially humanized nanobodies V11 to V18, with corresponding sequence numbers of SEQ ID No. 10 to SEQ ID No. 17. The humanized nanobodies were then expressed and purified.

[0156] Humanization method 2

[0157] CDR region transplantation based on commonly used humanized or highly stable nanobody frameworks: The complementary determining region sequence of the nanobody is transplanted to the humanized framework reported in the literature, and humanized nanobody sequences V1-ah, V2-ah, V3-com, V4-com, V5-FGLA, V6-FGLA, V7-hs, V8-hs and V9-hs are generated by gene synthesis, and the corresponding sequence numbers are SEQ ID No. 18 to SEQ ID No. 26. The humanized nanobody is then expressed and purified.

[0158] The four nano antibody backbones selected by the present invention are:

[0159] The ah skeleton is a universal fully humanized skeleton taken from the literature (Chi, XJ et al. Humanized single domain antibodies neutralize SARS-CoV-2 by targeting the spike receptor binding domain. Nature Communications 11, doi: 10.1038 / s41467-020-18387-8 (2020).).

[0160] FGLA is a universal fully humanized scaffold taken from the literature (Vincke, C. et al. General Strategy to Humanize a Camelid Single-domain Antibody and Identification of a Universal Humanized Nanobody Scaffold. J. Biol. Chem. 284, 3273-3284, doi: 10.1074 / jbc.M806889200 (2009).).

[0161] hs is a highly stable, highly expressed, partially humanized skeleton from the literature (Moutel, S. et al. NaLi-H1: Auniversal synthetic library of humanized nanobodies providing highlyfunctional antibodies and intrabodies. Elife 5, doi: 10.7554 / eLife.16228 (2016).).

[0162] com is a universal skeleton with high stability and high expression, which comes from the literature (Ferrari, D., Garrapa, V., Locatelli, M. & Bolchi, A. A Novel Nanobody Scaffold Optimized for Bacterial Expression and Suitable for the Construction of Ribosome Display Libraries. Molecular Biotechnology 62, 43-55, doi: 10.1007 / s12033-019-00224-z (2020).).

[0163] The expression and purification process is briefly described as follows:

[0164] (1) The basic culture medium of humanized nanobodies is TB medium, which is inoculated at 5% inoculation volume, cultured at 37° C. for 3 to 5 hours, and the inducer IPTG is added for overnight induction;

[0165] (2) After induction, centrifuge at 4000 rpm for 20 min to obtain wet bacteria containing humanized nanobodies;

[0166] (3) Add lysis buffer (10 mM imidazole, 500 mM NaCl, pH 7.40.02M PB) to the obtained wet bacteria at a ratio of 1:10, and use a 700 bar high-pressure homogenizer to disrupt the cells;

[0167] (4) Centrifuge at 4°C and 10,000 rpm for 20 min and collect the supernatant;

[0168] (5) The supernatant was filtered through a 0.45 μm filter and then passed through an affinity chromatography column (GE Healthcare, US) to separate and purify the humanized nanoantibodies, wherein the filler of the affinity chromatography column was Ni Sepharose High Performance;

[0169] (6) The nanoantibodies purified by affinity chromatography were subjected to SDS-PAGE electrophoresis to determine the purity, and the protein solution with higher purity was selected to determine the protein concentration using the BCA method.

[0170] The SPR technique was used to analyze the binding ability of humanized nanobodies to VIM. The analysis process was as described in Example 5. The affinity results are shown in Table 3.

[0171] Table 3 Affinity of humanized antibodies for VIM

[0172] Serial Number Nanobodies ka(1 / Ms) kd(1 / s) KD(M) SEQ ID No.10 V11 1.84E+04 6.69E-07 3.64E-11 SEQ ID No.11 V12 3.36E+04 5.59E-07 1.66E-11 SEQ ID No.12 V13 1.07E+04 8.18E-07 7.64E-11 SEQ ID No.13 V14 1.30E+04 4.28E-07 3.31E-11 SEQ ID No.14 V15 2.32E+04 5.77E-07 2.49E-11 SEQ ID No.15 V16 5.62E+04 7.97E-07 1.42E-11 SEQ ID No.16 V17 1.67E+04 6.86E-07 4.11E-11 SEQ ID No.17 V18 2.11E+04 7.86E-07 3.72E-11 SEQ ID No.18 V1-ah 8.73E+03 4.16E-07 4.76E-11 SEQ ID No.19 V2-ah 3.31E+04 5.05E-07 1.53E-11 SEQ ID No.20 V3-com 1.26E+04 1.17E-06 9.28E-11 SEQ ID No.21 V4-com 4.28E+04 7.72E-07 1.80E-11 SEQ ID No.22 V5-FGLA 3.81E+04 2.24E-06 5.87E-11 SEQ ID No.23 V6-FGLA 7.75E+03 5.55E-07 7.16E-11 SEQ ID No.24 V7-hs 4.49E+04 1.97E-06 4.39E-11 SEQ ID No.25 V8-hs 8.70E+03 4.58E-07 5.26E-11 SEQ ID No.26 V9-hs 2.17E+04 8.88E-07 4.09E-11

[0173] SEQ ID No.10:QLQESGGGLVQPGGSLRLSCAAS-GSTSSENA-MGWYRQAPGKERELVATI-SRNTNT-NYKDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCT-AIGPRYG-WGQGTQVTVSS

[0174] SEQ ID No.11:QLQESGGGLAQPGGSLRLSCAAS-GSTSSDNA-MGWYRQAPGEQREFVATI-SWDSNT-NYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCT-AIGPRYG-WGQGTQVTVSS

[0175] SEQ ID No.12:QLQESGGGLVQAGGSLRLSCATS-GFAFSVNA-MGWFRQAPGQGLEAVAAI-SNGGNT-NYKDSVKGRFTISRDNAQNTVYLQMNSLKPEDTAVYYCT-AIGPRYG-WGQGTQVTVSS

[0176] SEQ ID No.13:QLQESGGGLVQPGGSLRLSCAAS-GFPSSVNA-MGWYRQGPGKERELVAVI-SWDGNT-YYKDLAKGRFIISRDNAQNTVFLQMNSLTPEDTAVYYCT-AIGPRYG-WGQGTQVTVSS

[0177] SEQ ID No.14:QLQESGGGLVQPGGSLRLSCVAS-GSTSSENA-MGWYRQAPGKERELVATI-SRNTNT-NYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCT-AIGPRYG-WGQGTQVTVSS

[0178] SEQ ID No.15:QLQESGGGLAQPGGSLRLSCAAS-GSTSSDNA-MGWFRQAPGKEREFVAAI-SWDSNT-NYPDSVKGRFIISRDNAQNTVYLQMNSLKPEDTAVYYCT-AIGPRYG-WGQGTQVTVSS

[0179] SEQ ID No.16:QLQESGGGLVQAGGSLRLSCAAS-GFAFSVNA-MGWFRQAPGKEREFVSAI-SNGGNT-NYKDSVKGRFTISRDNAQNTVYLQMNSLKPEDTAVYYCT-AIGPRYG-WGQGTQVTVSS

[0180] SEQ ID No.17:QLQESGGGFVQPGGSLRLSCAAS-GFPSSVNA-MGWYRQGPGKERELVAVI-SWDGNT-YYKDLAKGRFIISRDNAQNTVFLQMNSLTPEDTAVYYCT-AIGPRYG-WGQGTQVTVSS

[0181] SEQ ID No.18:QLVESGGGLVQPGGSLRLSCAAS-GNAFSDNA-MGWFRQAPGKGLEAVAAI-SKNSNT-YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA-AIGPRYG-WGQGTLVTVSS

[0182] SEQ ID No.19:QLVESGGGLVQPGGSLRLSCAAS-GNAGSDNA-MGWFRQAPGKGLEAVAAI-SKNSNT-YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA-AIGPRYG-WGQGTLVTVSS

[0183] SEQ ID No.20:QLQESGGGLVQAGGSLRLSCAAS-GSTSSENA-MGWFRQAPGKEREFVAAI-SRNTNT-YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCA-AIGPRYG-WGQGTQVTVSS

[0184] SEQ ID No.21:QLQESGGGLVQAGGSLRLSCAAS-GSTSSDNA-MGWFRQAPGKEREFVAAI-SWDSNT-YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCA-AIGPRYG-WGQGTQVTVSS

[0185] SEQ ID No.22:QLVESGGGLVQPGGSLRLSCAAS-GSTSSDNA-MGWFRQAPGQGLEAVAAI-SWDSNT-YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA-AIGPRYG-WGQGTLVTVSS

[0186] SEQ ID No.23:QLVESGGGLVQPGGSLRLSCAAS-GFAFSVNA-MGWFRQAPGQGLEAVAAI-SNGGNT-YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA-AIGPRYG-WGQGTLVTVSS

[0187] SEQ ID No.24:QLQASGGGFVQPGGSLRLSCAAS-GFAFSVNA-MGWFRQAPGKEREFVSAI-SNGGNT-YYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCA-AIGPRYG-WGQGTQVTVSS

[0188] SEQ ID No.25:QLQASGGGFVQPGGSLRLSCAAS-GFPSSVNA-MGWFRQAPGKEREFVSAI-SWDENT-YYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCA-AIGPRYG-WGQGTQVTVSS

[0189] SEQ ID No.26:QLQASGGGFVQPGGSLRLSCAAS-GFPSSVNA-MGWFRQAPGKEREFVSAI-SWDGNT-YYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCA-AIGPRYG-WGQGTQVTVSS

[0190] Note: 1. The “-” between FR and CDR represents a connector, which is used to distinguish FR from CDR. There is no vacant site.

[0191] Compared with the original sequence, the affinity of the humanized nanobody to the antigen VIM did not decrease significantly, and the affinity to the relevant antigen remained at the same order of magnitude. The affinity level of the above humanized antibody was still 10 -11 M. This indicates that the humanized nanobody of the present invention has been successfully transformed.

[0192] Example 7: Antibody labeling

[0193] Antibody labeling techniques well known to those skilled in the art include enzyme labeling, biotin labeling, fluorescein labeling, colloidal gold labeling, and radioisotope labeling. Each labeling technique has its specific application scenarios and advantages. Choosing an appropriate labeling technique can improve the sensitivity and specificity of the experiment.

[0194] The present invention adopts two commonly used antibody labeling methods, as follows:

[0195] a) Fluorescence labeling: 1 mg / mL of V1 was dissolved in PBS and labeled with Abcam's FITC fluorescence coupling kit (Fast) -Lightning- (ab188285), and the nanobody was labeled with FITC according to the instructions. It is denoted as V1-FITC.

[0196] b) HRP labeling: Nanobody V1 was replaced with PBS (pH 7.4) to a concentration of about 10 mg / mL and conjugated using an HRP conjugation kit (abcam, Lightning- ab102890) and dialyzed thoroughly for replacement of the solution. It is denoted as V1-HRP.

[0197] c) Biotinylation: 1 mg / mL V1 was dissolved in PBS, 10 times the molar amount of NHS-biotin was added, mixed and incubated at 4°C in the dark overnight, and the solution was replaced by ultrafiltration. This was recorded as V1-biotin.

[0198] Example 8 VIM-positive cell capture

[0199] The streptavidin-coated magnetic beads were mixed with V1-biotin (0.1 mg / mL) and incubated for 30 minutes, and then washed thoroughly with PBST buffer for later use; MCF-7 cells (VIM-positive cells) were used as model cells, the experimental group was 5 mL of fresh healthy human whole blood with 100 MCF-7 cells added, and the control group was 5 mL of fresh healthy human whole blood without MCF-7 cells added.

[0200] 20 μL of the above magnetic beads were added to the experimental group and the control group, respectively, and mixed and incubated at 37°C for 30 min. The magnetic beads were fully washed with PBS buffer, and then DAPI was labeled by conventional experimental methods and observed under a fluorescence microscope. Figure 2 As shown, Figure 2 (a). Images taken in bright field and fluorescence channels, showing MCF-7 cells captured by magnetic beads; Figure 2 (b) Magnetic beads photographed under bright field; Figure 2 (c) is a cell photographed under the fluorescence channel, which proves that the nanobody of the present invention can be used for VIM-positive cell capture.

[0201] Example 9: Fluorescence immunoassay

[0202] Application (1): Verification of antibody cross-reactivity

[0203] The cross-species reactivity of antibodies refers to the property that the same antibody can react with the same or similar antigens from different species. This property is very important in research and clinical applications, especially when using animal models for disease research and drug development. For example, during drug development, researchers often need to evaluate the activity of antibodies in humans and animal models, and cross-reactivity determines the applicability of antibodies in these models.

[0204] The present invention uses V1-FITC to treat human MCF-7 cells ( Figure 3 (A)), African green monkey kidney cells Vero E6 ( Figure 3 (B)), mouse cell l929( Figure 3 (C) After culturing, fluorescent labeling was performed and confocal images were taken using a fluorescence microscope. The results are shown in Figure 3 As shown, the green fluorescence shows the cytoskeleton labeled by V1-FITC, and the blue shows the cell nucleus stained by DAPI. This shows that the nanobody of the present invention can effectively label the vimentin of the cytoskeleton of humans and commonly used experimental animals (monkeys, mice), has cross-reactivity, and is highly applicable.

[0205] Application (2): Cell sorting using flow cytometry

[0206] Take 2 mL of human peripheral whole blood, add 6 mL of red blood cell lysis buffer to remove red blood cells, and then centrifuge at 450 × g for 10 min to obtain white blood cells. Add ≈1×10 6 A431 cells.

[0207] Immunofluorescence labeling was performed directly on the cells. The amount of antibody (V1-FITC) added was 200 μL / 2 mL cell suspension. The working concentration of the antibody was 4 μg / mL. The cells were incubated at 4 °C for 30-60 min. After centrifugation and washing with cold PBS, 500 μL of cold PBS was added to the cells. The cells were mixed by pipetting and placed in a flow tube. At this time, the concentration of the test sample cells was 1×10 6 Pieces / mL.

[0208] The results are as follows Figure 4 As shown in the figure, the horizontal axis of the scatter plot represents FITC fluorescence, and the vertical axis represents side scatter (SSC), which is usually related to the size and internal complexity of the cell. The horizontal axis of the histogram represents FITC fluorescence, and the vertical axis represents the number of cells detected at this fluorescence intensity. Figure 4As can be seen in the figure, most cells (blue dots) have low VIM expression, while cells in the red area show high VIM expression. This indicates that there is a specific cell population whose VIM expression is significantly higher than other cells, and this cell population is A431.

[0209] This indicates that the nanoantibody of the present invention can effectively mark cells with high expression of VIM, supporting the application of flow cytometry technology for cell sorting.

[0210] Example 10: Immunohistochemical analysis

[0211] MCF-7 cells (VIM positive cells) were inoculated into the tail vein of mice and fed normally until tumor tissue could be observed. The tumor tissue was cut and paraffin sections were prepared. The paraffin sections were labeled with V1-HRP and developed using conventional experimental methods. Figure 5 As shown, it can be seen that in the tumor tissue sections, the staining intensity of VIM protein is strongly positive, and the positively stained cells are diffusely distributed in the tumor tissue, covering most of the tumor cells. It is proved that the nanobody of the present invention can be used for the labeling and immunohistochemical analysis of VIM positive cells.

[0212] Industrial Applicability

[0213] The nanoantibody of the present invention is an anti-VIM nanoantibody with a new amino acid sequence discovered by screening a phage library. The nanoantibody and its polypeptide have high affinity and activity, and can specifically recognize and bind to VIM. The nanoantibody prepared by the present invention can be used for the capture and detection of VIM-positive cells, and can be applied to immunofluorescence analysis or immunohistochemistry analysis, etc. through appropriate antibody labeling technology.

[0214] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. An anti-VIM nanobody, characterized in that The complementary determining region CDR of the Nanobody includes CDR1, CDR2 and CDR3 sequences: (I): (1) the amino acid sequence of the CDR1 is shown in SEQ ID NO.27, (2) the amino acid sequence of the CDR2 is shown in SEQ ID NO.33; and (3) the amino acid sequence of the CDR3 is shown in SEQ ID NO.39; or (II): An amino acid sequence obtained by modifying, replacing, deleting or adding one or more amino acids to the amino acid sequence described in (1), (2) or (3) of (I), and having the same function as the amino acid sequence described in (I).

2. The Nanobody according to claim 1, characterized in that in: (II) The amino acid sequence obtained by replacing one or more amino acids in the amino acid sequences of (1), (2) and (3) of (I), comprising (II-1): The amino acid sequence of CDR1 is shown in SEQ ID NO.28, the amino acid sequence of CDR2 is shown in SEQ ID NO.33, and the amino acid sequence of CDR3 is shown in SEQ ID NO.39; or (II-2): The amino acid sequence of CDR1 is shown in SEQ ID NO.29, the amino acid sequence of CDR2 is shown in SEQ ID NO.34, and the amino acid sequence of CDR3 is shown in SEQ ID NO.39; or (II-3): The amino acid sequence of CDR1 is shown in SEQ ID NO.30, the amino acid sequence of CDR2 is shown in SEQ ID NO.35, and the amino acid sequence of CDR3 is shown in SEQ ID NO.39; or (II-4): The amino acid sequence of CDR1 is shown in SEQ ID NO.31, the amino acid sequence of CDR2 is shown in SEQ ID NO.36, and the amino acid sequence of CDR3 is shown in SEQ ID NO.39; or (II-5): The amino acid sequence of CDR1 is shown in SEQ ID NO.32, the amino acid sequence of CDR2 is shown in SEQ ID NO.37, and the amino acid sequence of CDR3 is shown in SEQ ID NO.39; or (II-6): The amino acid sequence of CDR1 is shown in SEQ ID NO.32, the amino acid sequence of CDR2 is shown in SEQ ID NO.38, and the amino acid sequence of CDR3 is shown in SEQ ID NO.

39.

3. The Nanobody according to claim 1, characterized in that in: (III) the framework region FR of the Nanobody comprises FR1, FR2, FR3 and FR4 sequences, wherein: (1) the amino acid sequence of the FR1 is as shown in SEQ ID NO.49, (2) the amino acid sequence of the FR2 is as shown in SEQ ID NO.57, (3) the amino acid sequence of the FR3 is as shown in SEQ ID NO.65, (4) the amino acid sequence of the FR4 is as shown in SEQ ID NO.72; or (IV) An amino acid sequence having 50% or more homology with the amino acid sequence described in (1), (2), (3) or (4) of (III).

4. The Nanobody according to claim 3, characterized in that (IV) an amino acid sequence having 50% or more homology with the amino acid sequences of (1), (2), (3) and (4) of (III), including (IV-1): the amino acid sequence of FR1 is shown in SEQ ID NO.40, the amino acid sequence of FR2 is shown in SEQ ID NO.57, the amino acid sequence of FR3 is shown in SEQ ID NO.65, and the amino acid sequence of FR4 is shown in SEQ ID NO.72; or (IV-2): the amino acid sequence of FR1 is shown in SEQ ID NO.40, the amino acid sequence of FR2 is shown in SEQ ID NO.53, the amino acid sequence of FR3 is shown in SEQ ID NO.65, and the amino acid sequence of FR4 is shown in SEQ ID NO.72; or (IV-3): the amino acid sequence of FR1 is shown in SEQ ID NO.40, the amino acid sequence of FR2 is shown in SEQ ID NO.59, the amino acid sequence of FR3 is shown in SEQ ID NO.64, and the amino acid sequence of FR4 is shown in SEQ ID NO.72; or (IV-4): the amino acid sequence of FR1 is shown in SEQ ID NO.43, the amino acid sequence of FR2 is shown in SEQ ID NO.56, the amino acid sequence of FR3 is shown in SEQ ID NO.64, and the amino acid sequence of FR4 is shown in SEQ ID NO.72; or (IV-5): the amino acid sequence of FR1 is shown in SEQ ID NO.45, the amino acid sequence of FR2 is shown in SEQ ID NO.58, the amino acid sequence of FR3 is shown in SEQ ID NO.68, and the amino acid sequence of FR4 is shown in SEQ ID NO.72; or (IV-6): the amino acid sequence of FR1 is shown in SEQ ID NO.45, the amino acid sequence of FR2 is shown in SEQ ID NO.58, the amino acid sequence of FR3 is shown in SEQ ID NO.63, and the amino acid sequence of FR4 is shown in SEQ ID NO.72; or (IV-7): the amino acid sequence of FR1 is shown in SEQ ID NO.47, the amino acid sequence of FR2 is shown in SEQ ID NO.60, the amino acid sequence of FR3 is shown in SEQ ID NO.62, and the amino acid sequence of FR4 is shown in SEQ ID NO.72; or (IV-8): the amino acid sequence of FR1 is shown in SEQ ID NO.47, the amino acid sequence of FR2 is shown in SEQ ID NO.54, the amino acid sequence of FR3 is shown in SEQ ID NO.62, and the amino acid sequence of FR4 is shown in SEQ ID NO.72; or (IV-9): the amino acid sequence of FR1 is shown in SEQ ID NO.46, the amino acid sequence of FR2 is shown in SEQ ID NO.53, the amino acid sequence of FR3 is shown in SEQ ID NO.69, and the amino acid sequence of FR4 is shown in SEQ ID NO.72; or (IV-10): the amino acid sequence of FR1 is shown in SEQ ID NO.43, the amino acid sequence of FR2 is shown in SEQ ID NO.56, the amino acid sequence of FR3 is shown in SEQ ID NO.71, and the amino acid sequence of FR4 is shown in SEQ ID NO.72; or (IV-11): the amino acid sequence of FR1 is shown in SEQ ID NO.45, the amino acid sequence of FR2 is shown in SEQ ID NO.52, the amino acid sequence of FR3 is shown in SEQ ID NO.63, and the amino acid sequence of FR4 is shown in SEQ ID NO.72; or (IV-12): the amino acid sequence of FR1 is shown in SEQ ID NO.46, the amino acid sequence of FR2 is shown in SEQ ID NO.54, the amino acid sequence of FR3 is shown in SEQ ID NO.62, and the amino acid sequence of FR4 is shown in SEQ ID NO.72; or (IV-13): the amino acid sequence of FR1 is shown in SEQ ID NO.40, the amino acid sequence of FR2 is shown in SEQ ID NO.53, the amino acid sequence of FR3 is shown in SEQ ID NO.70, and the amino acid sequence of FR4 is shown in SEQ ID NO.72; or (IV-14): the amino acid sequence of FR1 is shown in SEQ ID NO.43, the amino acid sequence of FR2 is shown in SEQ ID NO.51, the amino acid sequence of FR3 is shown in SEQ ID NO.64, and the amino acid sequence of FR4 is shown in SEQ ID NO.72; or (IV-15): the amino acid sequence of FR1 is shown in SEQ ID NO.44, the amino acid sequence of FR2 is shown in SEQ ID NO.50, the amino acid sequence of FR3 is shown in SEQ ID NO.63, and the amino acid sequence of FR4 is shown in SEQ ID NO.72; or (IV-16): the amino acid sequence of FR1 is shown in SEQ ID NO.48, the amino acid sequence of FR2 is shown in SEQ ID NO.54, the amino acid sequence of FR3 is shown in SEQ ID NO.62, and the amino acid sequence of FR4 is shown in SEQ ID NO.72; or (IV-17): the amino acid sequence of FR1 is shown in SEQ ID NO.41, the amino acid sequence of FR2 is shown in SEQ ID NO.55, the amino acid sequence of FR3 is shown in SEQ ID NO.61, and the amino acid sequence of FR4 is shown in SEQ ID NO.73; or (IV-18): the amino acid sequence of FR1 is shown in SEQ ID NO.44, the amino acid sequence of FR2 is shown in SEQ ID NO.51, the amino acid sequence of FR3 is shown in SEQ ID NO.67, and the amino acid sequence of FR4 is shown in SEQ ID NO.72; or (IV-19): the amino acid sequence of FR1 is shown in SEQ ID NO.41, the amino acid sequence of FR2 is shown in SEQ ID NO.52, the amino acid sequence of FR3 is shown in SEQ ID NO.61, and the amino acid sequence of FR4 is shown in SEQ ID NO.73; or (IV-20): The amino acid sequence of FR1 is shown in SEQ ID NO.42, the amino acid sequence of FR2 is shown in SEQ ID NO.50, the amino acid sequence of FR3 is shown in SEQ ID NO.66, and the amino acid sequence of FR4 is shown in SEQ ID NO.

72.

5. The Nanobody according to any one of claims 1 to 4, characterized in that The Nanobody has (V) an amino acid sequence as shown in SEQ ID NO.1; or (VI) An amino acid sequence obtained by modifying, replacing, deleting or adding one or more amino acids to the amino acid sequence described in (V), and having the same function as the amino acid sequence described in (I).

6. The Nanobody according to claim 5, characterized in that in, The amino acid sequence of the Nanobody of (VI) is shown in any one of SEQ ID NO.2 to SEQ ID NO.

26.

7. A polypeptide, characterized in that Comprising the Nanobody according to any one of claims 1 to 6.

8. A nucleic acid molecule encoding the Nanobody according to any one of claims 1 to 6.

9. An expression vector, characterized in that Comprising the nucleic acid molecule of claim 8.

10. A host cell transformed or transfected with the expression vector according to claim 9.

11. A conjugate or a coupling substance, characterized in that: The Nanobody according to any one of claims 1 to 6 is comprised of a chemically labeled or biologically labeled Nanobody.

12. An adsorbent, characterized in that: A nanobody comprising any one of claims 1 to 6; or a polypeptide according to claim 8; or the nucleic acid molecule according to claim 9; or the expression vector according to claim 10; or the host cell according to claim 11; Or the conjugate as claimed in claim 12; or the conjugate as claimed in claim 12, and a carrier.

13. A kit, characterized in that: A nanobody comprising any one of claims 1 to 6; or a polypeptide according to claim 7; or the nucleic acid molecule described in claim 8; or the expression vector described in claim 9; or the host cell described in claim 10; or the conjugate as claimed in claim 11; or the conjugate as claimed in claim 11; Or the adsorbent according to claim 12, and an auxiliary agent acceptable in the detection.

14. A device, characterized in that For capturing, adsorbing and / or detecting VIM, comprising the Nanobody according to any one of claims 1 to 6; or the polypeptide according to claim 7; or the nucleic acid molecule described in claim 8; or the expression vector described in claim 9; or the host cell described in claim 10; or the conjugate as claimed in claim 11; or the conjugate as claimed in claim 11; Or the adsorbent according to claim 12, or the kit according to claim 13.

15. The Nanobody according to any one of claims 1 to 6; or the polypeptide according to claim 7; or the nucleic acid molecule described in claim 8; or the expression vector described in claim 9; or the host cell described in claim 10; or the conjugate as claimed in claim 11; or the conjugate as claimed in claim 11; or the adsorbent according to claim 12, or the kit according to claim 13; Application in the preparation of specific capture, adsorption and / or detection of VIM preparations; or Application in the preparation of tumor detection preparations for specific capture, adsorption and / or detection of VIM; or Application in the preparation of enriched and / or purified cell preparations for specific capture, adsorption and / or detection of VIM; or Application of the invention in preparing immunofluorescence analysis or immunohistochemistry analysis reagents for heterogeneously capturing, adsorbing and / or detecting VIM.

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