CN118240075A - TSG101 single domain antibody and application thereof - Google Patents

Abstract

The invention discloses an anti-TSG101 single domain antibody, wherein a complementarity determining region CDR comprises a CDR1, a CDR2 and a CDR3, wherein the CDR1 sequence is shown as SEQ ID NO. 2, the CDR2 sequence is shown as SEQ ID NO.4, and the CDR3 sequence is shown as SEQ ID NO. 6; through immunization of alpaca, the anti-TSG101 nano antibody with high affinity is obtained by screening through phage surface display technology. The single domain antibody expression system obtained by the invention has flexible selection and low production cost. The multi-combination form of the antibody is easy to modify, has low immune heterogeneity, and can not generate stronger immune response under the condition of not being modified by humanization. The single domain antibody obtained by the invention has high affinity, can be used alone or used as a drug carrying system to carry related drugs, and has very broad prospect and great significance in the fields of exosome drug application, clinical diagnosis and the like.

Description

TSG101 single domain antibody and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an anti-TSG 101 single domain antibody and application thereof.

Background

Exosomes are extracellular vesicles with diameters of 30-150nm released outside cells after fusion of the multivesicular bodies with cell membranes, and naturally exist in body fluids, including blood, saliva, urine, breast milk and the like. The exosomes are discovered for 50 years, are reported to participate in the intercellular communication of various physiological and pathological functions, are one of hot spots in the current biomedical research, and are also 'potential new' of cell therapies and accurate medical treatment with great prospect in the future. Currently, exosomes prove to have good application prospects in diagnosis and treatment of various diseases, for example, micrornas carried by exosomes in blood circulation can be used for diagnosis, prognosis and even treatment of cardiovascular diseases; the central nervous system-derived exosomes are capable of crossing the blood brain barrier and carry biomolecules between cells, useful for early diagnosis of neurodegenerative diseases; in Human Immunodeficiency Virus (HIV), exosomes also act as carriers of potential biomarkers and show potential drug delivery pathways.

The identification of exosomes is an essential basis for their widespread use, ensuring the quality of exosomes is critical to fully exploiting their potential as disease biomarkers and therapeutics. According to the proposal of the international society of extracellular vesicles (International Society for ExtracellularVesicles, ISEV) in 2014 and the supplementary proposal in 2018, the isolated exosomes need to be identified from three layers of size, morphology and surface markers, wherein the identification of the surface markers needs to comprehensively judge the specificity of the extracted exosome protein by three exosome specific expression proteins and an exosome negative index. As MISEV2018 suggests, in exosome-specific expressed proteins, at least one transmembrane or glycosyl phosphatidylinositol anchor must be displayed in order to demonstrate the presence of a lipid bilayer in the assay material; to demonstrate that the material analyzed contains more than just open cell fragments, it is necessary to demonstrate at least one cytoplasmic/periplasmic protein with lipid or membrane protein binding capacity.

Tumor susceptibility gene 101 (Tumor susceptibility gene, TSG 101) is a classical exosome protein identification marker. Exosomes are transported by ceramide or transport endocytosis to the origin of the necessary sorting complex (endosomal sorting complex required for transport, esctr) and are secreted into various body fluids by exocytosis after multi-vesicle fusion. TSG101 protein, which is a core component of the ESCRT-I complex subunit, is closely related to membrane remodeling, and thus TSG101 is widely used as an exosome protein marker related to membrane binding. At the gene level, the coding sequences of mouse and human TSG101 mRNAs are 77% identical at the nucleotide level, the human TSG101 gene is located in the region p15.1-p15.2 of chromosome 11 short arm, and the encoded protein contains 390 amino acids and has a molecular weight of 42.84kDa and 85% similarity to the mouse. At the subcellular level, most of TSG101 is present in the cytoplasm. Functionally, TSG101 proteins promote a variety of molecular and biological processes, including regulation of transcription, cell proliferation and division, ubiquitination, and regulation of vesicle trafficking.

The most common methods for identifying current exosome markers include Westernblot, ELISA, which all use the principle of specific binding of antigen and antibody. In other words, the specificity and sensitivity of antibody recognition are key elements of the quality of identification of the exosome surface markers. However, most anti-TSG 101 antibodies developed on the market at present are murine/rabbit antibodies, which have poor antibody specificity and low sensitivity, and are prone to "false positives" and "false negatives" during use, which increase the difficulty in separation and identification of exosomes. In addition, antibodies and their functional domains play a key role in exosome research, diagnosis and therapy. The development of highly specific and sensitive antibodies is a popular and highly urgent need in the exosome industry.

Single-domain antibodies (sdabs), also known as nanobodies (Nb), are a naturally occurring class of antibodies that contain only heavy chains but lack light chains entirely, mainly from camels, such as dromedaries, alpaca, etc., in common alpaca blood antibodies in amounts up to 50% -80%. For a typical antibody, antigen binding is determined by the variable domains of its heavy (VH) and light (VL/VK) chains, whereas such an unconventional heavy chain antibody (HEAVY CHAIN anti-body, hcAb) binds antigen based solely on the variable domain VHH domain of its heavy chain. Compared with the traditional antibody, the single domain antibody has unique physicochemical properties. The specific expression is as follows: 1) The transportation capability is strong. Because of the absence of light chains, single domain antibodies are relatively small in molecular weight (about 15 kDa) and thus readily carry targeted drugs into cells and even across the blood brain barrier. 2) The solubility and stability are good. The framework 2 region of the conventional antibody has a plurality of conserved amino acids with hydrophobic effect, and the hydrophobic amino acids of the framework 2 region of the single domain antibody are replaced by hydrophilic amino acids after evolution, so that the single domain antibody has better reversible folding capability and protein degradation resistance. In addition, the denaturation temperature of the single-domain antibody can reach 60-80 ℃, and some single-domain antibodies have better tolerance to guanidine hydrochloride of 2.3-3.3 mol/L. Meanwhile, the additional disulfide bond in the single-domain antibody enhances the stability of the VHH conformational structure, so that the VHH conformational structure is not degraded by pepsin and chymotrypsin, and therefore, some single-domain antibodies can be used as oral medicines. 3) Antigen recognition is more sensitive. The long complementarity determining regions (Complementarity-DETERMINING REGION, CDRs) of the single domain antibody are longer, making the binding mode with the antigen more flexible, and the exposed convex paratope can enter into the surface cavity or slit of the antigen which is difficult to access by the traditional antibody, so that the antigen can be easily recognized by the long complementarity determining region CDR3 of Nb. 4) The immunogenicity is small. Traditional murine monoclonal antibodies are anti-human.

Disclosure of Invention

The invention aims to design a TSG101 single domain antibody and application thereof, and uses TSG101 protein to immunize alpaca cells, and anti-TSG101 antibody is obtained through phage surface display.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

An anti-TSG 101 single domain antibody, its complementarity determining region CDR includes CDR1, CDR2 and CDR3, wherein, the CDR1 sequence is shown in SEQ ID NO. 2, the CDR2 sequence is shown in SEQ ID NO. 4, the CDR3 sequence is shown in SEQ ID NO. 6.

Further, the CDRs are separated by four framework regions, FR1, FR2, FR3 and FR4, wherein FR1 shown in SEQ ID NO. 1, FR2 shown in SEQ ID NO. 3, FR3 shown in SEQ ID NO. 5 and FR4 shown in SEQ ID NO. 7.

Furthermore, the anti-TSG 101 single domain antibody is an antibody specifically binding to TSG101 and has an amino acid sequence shown in SEQ ID NO. 8.

Further, at least 80% of the amino acid sequence is identical or similar to the amino acid sequence shown in SEQ ID NO. 8.

The amino acid sequences described above are specifically shown below:

And (3) a sequence table:

The invention also provides a fusion protein of an anti-TSG 101 single domain antibody, which comprises the anti-TSG 101 single domain antibody of claim 1 and an immunoglobulin Fc region, which is a human immunoglobulin Fc region.

The invention also provides a nucleic acid encoding the anti-TSG 101 single domain antibody of claim 1.

The invention also proposes an expression vector comprising a nucleic acid according to claim 6.

The invention also proposes a host cell comprising an expression vector according to claim 7.

The invention also provides any one of the following applications of the single domain antibody and the biological material:

(1) Use in the preparation of a product for exosome identification, isolation or purification, or use in exosome identification, isolation or purification;

(2) Use in the manufacture of a product for diagnosing or treating a disease, or use in diagnosing or treating a disease;

(3) Use in detecting TSG101 protein or in preparing a product for detecting TSG101 protein;

(4) Use in the preparation of a product for binding to TSG101 protein;

(5) Use in mediating drug specific recognition of expressed TSG101 antigen or in the preparation of a product mediating drug specific recognition of expressed TSG101 antigen;

(6) Use in vivo imaging of TSG101 protein or in the preparation of a product for in vivo imaging of TSG101 protein.

Further, anti-TSG 101 single domain antibodies are antibodies that bind to an epitope of TSG101 and can block interactions with TSG 101.

The following beneficial effects can be obtained through the technical scheme:

According to the invention, firstly, a single-domain antibody gene is obtained by immunizing alpaca with TSG101 recombinant protein, a single-domain antibody expression library is constructed, and then, a high-affinity anti-TSG101 single-domain antibody is obtained by screening from the single-domain antibody expression library through a phage display screening technology. After sequencing to obtain antibody gene sequence, preparing anti-TSG101 single domain antibody by adopting a genetic engineering method, and further carrying out affinity performance experiment on the obtained antibody. Experimental results prove that the Half maximum effect concentration (Half-maximal effective concentration, EC 50) of the anti-TSG101 nano antibody obtained by immunization of alpaca and screening and the affinity of human TSG101-His protein is only 2.97nM, and the anti-TSG101 nano antibody has higher affinity with the TSG101 protein.

(1) The single domain antibody obtained by the invention has flexible expression system selection, can be expressed in a prokaryotic system or a eukaryotic system of yeast cells or mammal cells, has low expression cost in the prokaryotic expression system, and can reduce the post production cost.

(2) The single-domain antibody obtained by the invention has simple reconstruction of the multi-combination form of the antibody, can obtain multivalent and multi-specific antibodies through simple serial connection in a genetic engineering mode, has low immune heterogeneity and can not generate stronger immune response under the condition of not carrying out humanized reconstruction.

(3) The single domain antibody obtained by the invention has high affinity and can be used for different later uses.

(4) The single domain antibody obtained by the invention can be used alone or used as an exosome drug carrying system to carry related drugs, and has very broad prospect and great significance in the fields of exosome drug application, clinical diagnosis and the like.

Drawings

Fig. 1 is: immune library insertion rate plots.

Fig. 2 is: TSG101 antibody affinity profile.

Detailed Description

Terminology:

Unless otherwise indicated or defined, all terms used have the usual meaning in the art, which will be understood by those skilled in the art. Reference is made, for example, to the standard handbook, sambrook et al,

"Molecular cloning: A Laboratory Manual" (2 nd edition), volumes 1-3, cold Spring Harbor LaboratoryPress (1989); the general prior art cited herein; moreover, unless otherwise indicated, all methods, steps, techniques and operations not specifically detailed may be, and have been, performed in a manner known per se, which will be appreciated by those skilled in the art. Reference is also made to, for example, standard handbooks, the above-mentioned general prior art and other references cited therein.

The terms "antibody" or "immunoglobulin" are used interchangeably herein to refer to either heavy chain antibodies or conventional 4 chain antibodies, unless otherwise indicated, as general terms to include full length antibodies, individual chains thereof, and all portions, domains, or fragments thereof (including but not limited to antigen binding domains or fragments, e.g., VHH domains or VH/VL domains, respectively). Furthermore, the term "sequence" (e.g. in terms of "immunoglobulin sequence", "antibody sequence", "single variable domain sequence", "VHH sequence" or "protein sequence", etc.) as used herein is generally understood to include both the relevant amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding the sequence, unless the context requires a more defined interpretation.

The term "nanobody (VHH)" refers to an immunoglobulin domain comprising four "framework regions" referred to in the art and hereinafter as "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3", and "framework region 4" or "FR4", respectively, wherein the framework regions are separated by three "complementarity determining regions" or "CDRs" referred to in the art and hereinafter as "complementarity determining region 1" or "CDR1", "complementarity determining region 2" or "CDR2", and "complementarity determining region 3" or "CDR3", respectively. Thus, the general structure or sequence of a single domain antibody (VHH) can be expressed as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Single domain antibodies (VHH) confer specificity to an antigen by possessing an antigen binding site.

The terms "single domain antibody", "heavy chain single domain antibody", "VHH domain", "VHH", "nanobody" are used interchangeably.

For amino acid residues used in the VHH domain of the family Camelidae, numbering is according to the general numbering of the VH domain given by Kabat et al ("Sequence ofproteins ofimmunological interest", US PublicHealth Services, NIH Bethesda, MD, publication No. 91). According to this numbering process, the number of the cells is,

FR1 comprises amino acid residues at positions 1-30,

CDR1 comprising amino acid residues at positions 31-35,

FR2 comprises the amino acids at positions 36-49,

CDR2 comprises amino acid residues at positions 50-65,

FR3 comprises amino acid residues at positions 66-94,

CDR3 comprises amino acid residues at positions 95-102,

FR4 comprises amino acid residues at positions 103-113.

It should be noted, however, that the total number of amino acid residues in each CDR may be different and may not correspond to the total number of amino acid residues indicated by Kabat numbering (i.e., one or more positions according to Kabat numbering may not be occupied in the actual sequence or the actual sequence may contain more amino acid residues than the Kabat numbering allows), as is well known in the art for VH domains and VHH domains. This means that in general, numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence.

Alternative methods of numbering amino acid residues of VH domains are known in the art, which may also be similarly applied to VHH domains. However, unless otherwise indicated, in the present description, claims and figures, numbering according to Kabat and as appropriate for VHH domains as described above will be followed. The total number of amino acid residues in the VHH domain will typically range from 110 to 120, often between 112 and 115. It should be noted, however, that smaller and longer sequences may also be suitable for the purposes described herein. Other structural and functional properties of VHH domains and polypeptides containing them can be summarized as follows:

VHH domains (which have been naturally "designed" to functionally bind to an antigen in the absence of and without interaction with a light chain variable domain) can be used as single and relatively small functional antigen binding building blocks, domains, or polypeptides. This distinguishes VHH domains from the VH and VL domains of conventional 4-chain antibodies, which are not themselves generally suitable for practical use as a single antigen-binding protein or immunoglobulin single variable domain, but need to be combined in some form or another to provide a functional antigen-binding unit (e.g., in the form of a conventional antibody fragment such as a Fab fragment; or in the form of an scFv consisting of a VH domain covalently linked to a VL domain).

Constructs

The present invention provides a construct, the construction method of which should be known to those skilled in the art, for example, the construct may be constructed by in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombinant technology, etc., and more specifically, may be constructed by inserting the isolated polynucleotide into a multiple cloning site of an expression vector. Expression vectors in the present invention generally refer to various commercially available expression vectors and the like well known in the art, and may be, for example, bacterial plasmids, phages, phage plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors. The vector may also include one or more regulatory sequences operably linked to the polynucleotide sequence, which may include a suitable promoter sequence. The promoter sequence is typically operably linked to the coding sequence for the amino acid sequence to be expressed. The promoter may be any nucleotide sequence that exhibits transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell. Regulatory sequences may also include suitable transcription terminator sequences, sequences recognized by a host cell to terminate transcription. A terminator sequence is attached to the 3' terminus of the nucleotide sequence encoding the polypeptide and any terminator which is functional in the host cell of choice may be used in the present invention.

In general, a suitable vector may comprise an origin of replication functional in at least one organism, a promoter sequence, a convenient restriction enzyme site and one or more selectable markers. For example, these promoters may be lac or trp promoters including, but not limited to, E.coli; a lambda phage PL promoter; eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the methanol oxidase promoter of Pichia pastoris, and some other known promoters that control the expression of genes in prokaryotic or eukaryotic cells or viruses thereof. Marker genes can be used to provide phenotypic traits for selection of transformed host cells, and can be, for example, dihydrofolate reductase, neomycin resistance, and Green Fluorescent Protein (GFP), including but not limited to eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli, and the like. When the polynucleotide is expressed, an enhancer sequence may also be included in the expression vector, which, if inserted into the vector, will enhance transcription, the enhancer being a cis-acting element of DNA, typically about 10 to 300 base pairs, acting on the promoter to enhance transcription of the gene.

Expression system

The present invention provides an expression system for a single domain antibody comprising a construct provided in the fourth aspect of the invention or a polynucleotide provided in the third aspect of the invention integrated into the genome. Any cell suitable for expression of an expression vector may be used as a host cell, e.g., the host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as phage cells; or higher eukaryotic cells, such as mammalian cells, which may specifically include, but are not limited to, E.coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as phage, filamentous fungi, plant cells; insect cells of Drosophila S2 or Sf 9; CHO, COS, HEK293 cells, or animal cells of Bowes melanoma cells, and the like. Methods of constructing the expression system should be known to those of skill in the art and may be, for example, a combination of one or more of the methods including, but not limited to, microinjection, particle gun, electroporation, virus-mediated transformation, electron bombardment, calcium phosphate precipitation, and the like.

Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.

Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention.

Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed in the present invention employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques, and related arts. These techniques are well described in the prior art.

The invention is further described with reference to fig. 1-2:

EXAMPLE 1 mammalian cell preparation of TSG101-LaFc fusion proteins

The TSG101 gene (Met 1-Pro140, SEQ ID NO: 8) was ligated with the human alpaca LaFc sequence (SEQ ID NO: 9) to construct a cell expression plasmid fused with TSG101-LaFc and LaFc, the cell expression plasmid was extracted with a deiotoxin plasmid large extraction kit (Biomiga), the plasmid was mixed with a transfection reagent PEI (Polysciences, inc.) 1:3 uniformly and allowed to stand for 30min, and then added to HEK293F cells, which were cultured in a 5% CO2 shaker incubator at 37℃for 7 days, and the supernatant was centrifuged. The supernatant was adjusted to pH7.0 and applied to a ProteinA affinity column (Boglan Biotechnology Co., ltd.) and eluted with 100%0.1M Gly-HCl (pH 3.0); the eluate was pre-added with 10%1M Tris-HCl (pH 8.5).

EXAMPLE 2TSG101-LaFc recombinant protein immunization of alpaca

TSG101-LaFc recombinant protein immunized alpaca, every 21 days, immunization 1 time, total immunization 4 times, blood collection 50ml, using QIAGEN company provided RNA extraction kit to extract total RNA, using Super-SCRIPT III FIRST STRANDSUPERMIX kit according to the instruction to the extracted RNA reverse transcription into cDNA. PCR amplification of the VHH antibody encoding nucleic acid fragment (FIG. 1), recovery of the target heavy chain single domain antibody nucleic acid fragment, phage display plasmid ligation, electrotransformation into bacterial TG1 electrotransformation competence, construction of heavy chain single domain antibody phage display library against TSG 101. The reservoir size was determined to be 1×10 ¹⁰ by gradient dilution plating.

Example 3 phage display library screening and ELSIA identification

3.1 Phage library screening

Phage display library screening, according to PHAGE DISPLAY _ Methods andProtocols-Humana Press; editedby Michael screening is performed by the general method. Briefly, phage library bacteria were taken at 10-fold stock capacity, incubated at 37℃for about 2h, rescued by adding helper phage M13KO7 (NEB), incubated at 30℃overnight, and phagemids obtained using PEG8000 pellet for antibody screening. TSG101-His protein coated ELISA plate at 5ug/ml concentration, 3% BSA blocked at 37℃for 2h, 2X10 ^11/ well phagemid was added and allowed to act at 37℃for 1.5h. The phage were then washed 6 times with PBST (0.1% Tween 20 in PBS) to remove non-specifically bound phage. The washed ELISA wells were eluted by incubation with 100. Mu.L of 0.1M gly-HCl 1mg/ml BSA (pH 2.2) buffer for 10 min and neutralized with 10% by volume of 1 mM Tris-HCl, pH 8.0. Phage titers were determined to be 1X 10 ⁷ PFU/ml. The phage eluate was amplified and the titer was determined to be 2.3X10 ¹³ PFU/ml. TSG101-His protein was coated on an ELISA plate at a concentration of 2ug/ml, 3% Ovalbumin (OVA) was blocked, and the same screening procedure was followed by a second round of screening, and the phage titer determination for the second round of panning was 1.92X 10 ⁸ PFU/ml.

3.2 Enzyme-Linked immunosorbent assay (ELISA) screening

96 Individual clones were picked from the phage titer plate eluted from the second round of panning and cultured in 96 well plates, and infected with M13KO7 helper phage and packaged to obtain accumulation of recombinant phage in the supernatant. TSG101-His was blocked at 200 ng/Kong Baoban and with skimmed milk powder at 37℃for 1 hour, and the monoclonal recombinant phage supernatant was diluted twice with PBS and incubated at 100 ul/Kong Jiaru for 1 hour at 37 ℃. After 5 washes of PBST, 100. Mu.l of Anti-M13Antibody (HRP) (Yiqiao Shenzhou) diluted 1:10000 was added to each well and incubated for 1 hour at 37 ℃. After PBST is washed for 5 times, TMB chromogenic working solution is added, after incubation is carried out for 5 minutes at room temperature for color development, 1M sulfuric acid is added for stopping reaction, OD450nm reading is displayed, positive binding is displayed, and clone with the highest OD450 reading value is selected for sequencing analysis. The inventors finally obtained 1 strongly positive clone Nb19-17, SEQ ID NO:10.

Example 4anti-TSG101 nanobody expression and analysis

4.1Anti-TSG101 nanobody expression

The competition positive clone Nb19-17 nanobody gene of example 4 was ligated with human IgG1 Fc (SEQ ID NO: 11) and ligated into pcDNA3.1 vector, and transiently expressed using HEK293F cells. The plasmids were extracted with a endotoxin-free plasmid large-extraction kit (Biomiga), mixed with a transfection reagent PEI (Polysciences, inc.) 1:3, allowed to stand for 30min, then added to HEK293F cells, cultured in a shaking incubator at 37℃for 7 days with 5% CO ₂, and the supernatants were centrifuged. The supernatant was adjusted to pH7.0 and applied to a ProteinA affinity column (Boglan Biotechnology Co., ltd.) and eluted with 100%0.1M Gly-HCl (pH 3.0); the eluate was pre-added with 10%1M Tris-HCl (pH 8.5).

4.2Anti-TSG101 nanobody binding specificity

TSG101-His (Yiqiaoshenzhou) protein 0.1 μg/well overnight coated plate at 4 ℃. After that, TSG101 single domain antibodies Nb19-17 were added at different concentrations (100 nM, 20nM, 4nM, 0.8nM, 0.16nM, 0.032nM, 0.0064 nM) and reacted at 37℃for 1 hour. After washing, goat anti-human IgG-Fc horseradish peroxidase labeled antibody was added and reacted at 37℃for 45min. After washing, the absorbance was read at 450nM by adding a chromogenic solution to generate an affinity binding curve, see FIG. 2, with EC50 values of 2.97nM for Nb19-17 and TSG 101. The nano antibody obtained by screening has good binding specificity with human TSG101-His protein.

And (3) a sequence table:

The foregoing is a preferred embodiment of the present application, and modifications, obvious to those skilled in the art, of the various equivalent forms of the present application can be made without departing from the principles of the present application, are intended to be within the scope of the appended claims.

Claims (10)

1. An anti-TSG 101 single domain antibody, characterized in that: the CDRs of the complementarity determining regions comprise CDR1, CDR2 and CDR3, wherein the CDR1 sequence is shown as SEQ ID NO. 2, the CDR2 sequence is shown as SEQ ID NO. 4, and the CDR3 sequence is shown as SEQ ID NO. 6.

2. An anti-TSG 101 single domain antibody according to claim 1, wherein: the CDRs are separated by four framework regions FR1, FR2, FR3 and FR4, wherein FR1 shown in SEQ ID NO. 1, FR2 shown in SEQ ID NO. 3, FR3 shown in SEQ ID NO. 5 and FR4 shown in SEQ ID NO. 7.

3. A TSG101 single domain antibody according to claim 1 or 2, characterized in that: the anti-TSG 101 single domain antibody is an antibody specifically binding to TSG101 and has an amino acid sequence shown in SEQ ID NO. 8.

4. An anti-TSG 101 single domain antibody according to claim 1, wherein: at least 80% of the amino acid sequence is identical or similar to the amino acid sequence shown in SEQ ID NO. 8.

5. A fusion protein of an anti-TSG 101 single domain antibody, characterized in that: comprising the anti-TSG 101 single domain antibody of claim 1 and an immunoglobulin Fc region, said immunoglobulin Fc region being a human immunoglobulin Fc region.

6. A nucleic acid, characterized in that: a nucleic acid encoding the anti-TSG 101 single domain antibody of claim 1.

7. An expression vector, characterized in that: comprising the nucleic acid of claim 6.

8. A host cell, characterized in that: comprising the expression vector of claim 7.

9. Use of an anti-TSG 101 single domain antibody according to claim 1 or 2 or a biomaterial according to any one of claims 5 to 8: characterized in that the application has any one of the following:

(1) Use in the preparation of a product for exosome identification, isolation or purification, or use in exosome identification, isolation or purification;

(2) The application in preparing a product for diagnosing exosomes as detection targets or in preparing a medicament with exosomes as carriers;

(3) Use in detecting TSG101 protein or in preparing a product for detecting TSG101 protein;

(4) Use in the preparation of a product for binding to TSG101 protein;

(5) Use in mediating drug specific recognition of expressed TSG101 antigen or in the preparation of a product mediating drug specific recognition of expressed TSG101 antigen;

(6) Use in vivo imaging of TSG101 protein or in the preparation of a product for in vivo imaging of TSG101 protein.

10. The use according to claim 9, characterized in that: anti-TSG 101 single domain antibodies are antibodies that bind to an epitope of TSG101 and can block interactions with TSG 101.