Specific antibody for Rab11 protein and preparation method and application thereof

Abstract

The present invention relates to an antigen binding protein, antibody or antibody active fragment obtained by immunizing camelids with Rab11 protein. The present invention relies on the immune system of camelids to screen, identify and prepare antibodies that specifically recognize and bind to Rab11 protein. The obtained antibodies have strong specificity and can be used for target antigen detection, with potential clinical diagnosis and treatment value; and the antibody provided by the present invention has a simple structure, is easy to be genetically engineered, is easy to be humanized, has high stability, and has low mass production cost, which is conducive to large-scale production.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a specific antibody of Rab11 protein and a preparation method and application thereof. Background Art

[0002] Antibodies are proteins that are mainly secreted by plasma cells and used by the immune system to identify and neutralize foreign substances such as bacteria and viruses, which are called antigens. The binding of antibodies and antigens relies entirely on non-covalent interactions. This specific binding mechanism allows antibodies to capture foreign microorganisms and infected cells, further inducing other immune mechanisms to attack them, or directly neutralize their targets. Antibodies and antibody-related products have been widely used in research fields such as life sciences and medicine. Many experimental techniques derived from antigen-antibody specific binding have laid an important foundation for scientific research and clinical treatment, such as immunodiagnosis, immunoblotting, enzyme-linked immunosorbent assay, flow cytometry, etc.

[0003] Rab11 (Ras-related protein 11) is a member of the Ras superfamily and a key regulator of intracellular membrane transport, involved in the formation of transport vesicles and their fusion with the membrane. Rab family protein members cycle between inactive GDP-bound and active GTP-bound forms, and are able to recruit different sets of downstream effectors to the membrane, directly responsible for the formation, movement, binding and fusion of vesicles. Rab11 is mainly involved in regulating the endocytic cycle, and is also a major regulator of membrane transport during cytokinesis. Rab11 also participates in epithelial cell polarization and promotes cell transcytosis together with various proteins such as Rab8, and is involved in the transport and sorting of various important cell membrane proteins from the Golgi apparatus to the cell membrane.

[0004] In the process of studying Rab11, antibodies are a very important research tool, which has great value and significance for patient diagnosis, virus analysis and research. However, traditional animals such as mice and rabbits can only recognize flat peptides on the surface of antigens. Therefore, it is necessary to develop a nano-antibody that can recognize the complex spatial structure of the antigen surface and produce highly specific and high-affinity nano-antibodies. Summary of the invention

[0005] In order to solve one of the above technical problems existing in the prior art, the present invention provides a specific antibody of Rab11 protein and a preparation method and application thereof.

[0006] In a first aspect, the present invention provides an antigen binding protein, an antibody or an antibody active fragment obtained by immunizing a camelid with a Rab11 protein.

[0007] In some embodiments, the camelid is selected from the group consisting of dromedary, Bactrian camel, llama, llama, alpaca and vicuña, preferably alpaca.

[0008] In some embodiments, the antibody is a nanobody and the antibody active fragment is a nanobody active fragment.

[0009] In some embodiments, the antibody is a monoclonal antibody or a polyclonal antibody.

[0010] In some embodiments, the kd value of the antigen binding protein, antibody or antibody active fragment binding to the Rab11 protein is below 500 nM, preferably below 300 nM, more preferably below 200 nM, more preferably below 100 nM, and more preferably below 50 nM.

[0011] In some embodiments, the Rab11 protein has an amino acid sequence as shown in SEQ ID NO: 1. Further preferably, the Rab11 protein is prepared by a method comprising the following steps: constructing a nucleotide sequence encoding the Rab11 protein into a vector plasmid; transfecting the vector plasmid into a eukaryotic cell line for expression, and purifying.

[0012] In some embodiments, the Rab11 protein can be obtained through commercial channels.

[0013] In a second aspect, the present invention provides a method for constructing an antibody library, the method comprising the following steps:

[0014] (1) Using Rab11 protein as an antigen to immunize camelid animals, collecting venous peripheral blood from the immunized animals, and separating lymphocytes;

[0015] (2) extracting total mRNA from the lymphocytes, reverse transcribing it into cDNA and amplifying it;

[0016] (3) Inserting the amplified DNA into a viral expression vector, transforming into bacteria, collecting colonies, and obtaining an antibody library.

[0017] In some embodiments, the camelid is selected from the group consisting of dromedary, Bactrian camel, llama, llama, alpaca and vicuña, preferably alpaca.

[0018] In some embodiments, the immunization in step (1) is performed by subcutaneous injection. The number of immunizations is preferably 3 to 5 times. The venous peripheral blood is preferably collected before and after the last immunization.

[0019] In some embodiments, the viral expression vector in step (3) is a phage expression vector.

[0020] In some embodiments, the bacteria in step (3) are TG1 competent bacteria.

[0021] In a third aspect, the present invention provides an antibody library obtained by the above method for constructing an antibody library, or a polyclonal antibody expressed by the antibody library.

[0022] In a fourth aspect, the present invention provides a method for constructing an antigen-specific antibody library, the method comprising the following steps: screening the antibody library described in the third aspect to obtain an antigen-specific antibody library.

[0023] In some embodiments, the method for constructing an antigen-specific antibody library comprises the following steps:

[0024] (i) culturing the antibody library to release viruses;

[0025] (ii) incubating the virus with the antigen to remove the virus that non-specifically binds to the antigen and retain the virus that specifically binds to the antigen;

[0026] (iii) infecting bacteria with the virus that specifically binds to the antigen, collecting colonies, and obtaining an antigen-specific antibody library.

[0027] In some embodiments, the bacteria in step (iii) is Escherichia coli.

[0028] In a fifth aspect, the present invention provides an antigen-specific antibody library obtained by the above method for constructing an antigen-specific antibody library, or a polyclonal antibody that specifically binds to an antigen and is expressed and produced by the antigen-specific antibody library.

[0029] In a sixth aspect, the present invention provides a method for preparing an antigen-binding protein, an antibody or an active fragment of an antibody, the method comprising the following steps: screening the antibody library described in the third aspect to obtain an antigen-binding protein, an antibody or an active fragment of an antibody that specifically binds to an antigen.

[0030] In some embodiments, the method for preparing an antigen binding protein, an antibody or an antibody active fragment comprises the following steps:

[0031] (a) culturing the antibody library to release viruses;

[0032] (b) incubating the virus with the antigen to remove the virus that non-specifically binds to the antigen and retain the virus that specifically binds to the antigen;

[0033] (c) Infecting bacteria with the virus that specifically binds to the antigen, smearing the infected bacteria on a plate culture medium for culture, and selecting a single colony.

[0034] In some embodiments, the bacteria in step (c) is Escherichia coli.

[0035] In some embodiments, the single colony can be expanded and then identified for antigen-specific binding.

[0036] In some embodiments, the single colony can be expanded and then subjected to step (d): DNA extraction, transformation into host cells and expression to obtain monoclonal antibodies.

[0037] In a seventh aspect, the present invention provides an antigen-binding protein, an antibody or an antibody active fragment obtained by the above-mentioned method for preparing an antigen-binding protein, an antibody or an antibody active fragment.

[0038] In an eighth aspect, the present invention provides an antigen-binding protein, an antibody or an antibody active fragment that specifically recognizes and / or binds to Rab11 protein; the antigen-binding protein, antibody or antibody active fragment comprises at least one heavy chain variable region; the heavy chain variable region has:

[0039] CDR1 as shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6;

[0040] The CDR2 shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11; and

[0041] A CDR3 as set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16;

[0042] In some embodiments, the heavy chain variable region has: a CDR1 as shown in SEQ ID NO:2, a CDR2 as shown in SEQ ID NO:7, and a CDR3 as shown in SEQ ID NO:12.

[0043] In some embodiments, the heavy chain variable region has: a CDR1 as shown in SEQ ID NO:3, a CDR2 as shown in SEQ ID NO:8, and a CDR3 as shown in SEQ ID NO:13.

[0044] In some embodiments, the heavy chain variable region has: a CDR1 as shown in SEQ ID NO:4, a CDR2 as shown in SEQ ID NO:9, and a CDR3 as shown in SEQ ID NO:14.

[0045] In some embodiments, the heavy chain variable region has: a CDR1 as shown in SEQ ID NO:5, a CDR2 as shown in SEQ ID NO:10, and a CDR3 as shown in SEQ ID NO:15.

[0046] In some embodiments, the heavy chain variable region has: a CDR1 as shown in SEQ ID NO:6, a CDR2 as shown in SEQ ID NO:11, and a CDR3 as shown in SEQ ID NO:16.

[0047] In some embodiments, the heavy chain variable region has: an amino acid sequence as shown in SEQ ID NO: 17, or a conservative variant of the amino acid sequence as shown in SEQ ID NO: 17 obtained by adding, deleting, replacing or modifying one or more amino acids.

[0048] In some embodiments, the heavy chain variable region has: an amino acid sequence as shown in SEQ ID NO: 18, or a conservative variant of the amino acid sequence as shown in SEQ ID NO: 18 obtained by adding, deleting, replacing or modifying one or more amino acids.

[0049] In some embodiments, the heavy chain variable region has: an amino acid sequence as shown in SEQ ID NO: 19, or a conservative variant of the amino acid sequence as shown in SEQ ID NO: 19 obtained by adding, deleting, replacing or modifying one or more amino acids.

[0050] In some embodiments, the heavy chain variable region has: an amino acid sequence as shown in SEQ ID NO: 20, or a conservative variant of the amino acid sequence as shown in SEQ ID NO: 20 obtained by adding, deleting, replacing or modifying one or more amino acids.

[0051] In some embodiments, the heavy chain variable region has: an amino acid sequence as shown in SEQ ID NO: 21, or a conservative variant of the amino acid sequence as shown in SEQ ID NO: 21 obtained by adding, deleting, replacing or modifying one or more amino acids.

[0052] In some embodiments, the antigen binding protein, antibody or antibody active fragment comprises one of the heavy chain variable regions and lacks a light chain.

[0053] In some embodiments, the antibody is a nanobody and the antibody active fragment is a nanobody active fragment.

[0054] In a ninth aspect, the present invention provides a nucleotide sequence encoding the amino acid sequence shown in any one of SEQ ID NO: 2 to SEQ ID NO: 21, or the antigen-binding protein, antibody or antibody active fragment described above.

[0055] In some embodiments, the nucleotide sequence encoding the antigen binding protein, antibody or antibody active fragment is shown in SEQ ID NO:22.

[0056] In some embodiments, the nucleotide sequence encoding the antigen binding protein, antibody or antibody active fragment is shown in SEQ ID NO:23.

[0057] In some embodiments, the nucleotide sequence encoding the antigen binding protein, antibody or antibody active fragment is shown in SEQ ID NO:24.

[0058] In some embodiments, the nucleotide sequence encoding the antigen binding protein, antibody or antibody active fragment is shown in SEQ ID NO:25.

[0059] In some embodiments, the nucleotide sequence encoding the antigen binding protein, antibody or antibody active fragment is shown as SEQ ID NO:26.

[0060] In a tenth aspect, the present invention provides an expression vector containing the nucleotide sequence described above.

[0061] In some embodiments, the expression vector is a phage expression vector, preferably a phage surface display screening vector.

[0062] In some embodiments, the expression vector further contains a nucleotide sequence encoding the bacteriophage envelope protein pill.

[0063] In an eleventh aspect, the present invention provides a virus into which the above-mentioned expression vector is exogenously introduced.

[0064] In some embodiments, the virus is a bacteriophage.

[0065] In a twelfth aspect, the present invention provides a host cell into which the above-mentioned expression vector is exogenously introduced, or a host cell infected by the above-mentioned virus.

[0066] In some embodiments, the host cell is Escherichia coli.

[0067] In a thirteenth aspect, the present invention provides a method for expressing an antigen-binding protein, an antibody or an active fragment of an antibody using the host cell described above.

[0068] In a fourteenth aspect, the present invention provides an antigen-binding protein, an antibody or an antibody active fragment obtained by expressing the host cell described above.

[0069] In a fifteenth aspect, the present invention provides a humanized antigen-binding protein, antibody or antibody active fragment obtained by humanizing the antigen-binding protein, antibody or antibody active fragment described above.

[0070] In a sixteenth aspect, the present invention provides a protein conjugate comprising the antigen-binding protein, antibody or antibody active fragment described above or the humanized antigen-binding protein, antibody or antibody active fragment described above and a ligand.

[0071] In some embodiments, the ligand is selected from the group consisting of a radioisotope, a fluorescent group, and a delivery vehicle.

[0072] In a seventeenth aspect, the present invention provides a kit for detecting the content of Rab11 protein in a sample, which comprises the antigen-binding protein, antibody or antibody active fragment described above or the humanized antigen-binding protein, antibody or antibody active fragment described above.

[0073] In some embodiments, the antigen binding protein, antibody or antibody active fragment is labeled with a marker. Preferably, the marker is selected from an enzyme, a chemiluminescent group and an isotope group.

[0074] In some embodiments, the sample is animal serum, preferably human serum.

[0075] In an eighteenth aspect, the present invention provides use of the above-mentioned antigen-binding protein, antibody or antibody active fragment, the above-mentioned humanized antigen-binding protein, antibody or antibody active fragment, the above-mentioned protein conjugate or the above-mentioned kit in detecting the content of Rab11 protein in a sample.

[0076] In some embodiments, the sample is animal serum, preferably human serum.

[0077] In a nineteenth aspect, the present invention provides a method for detecting the content of Rab11 protein in a sample using the antigen binding protein, antibody or antibody active fragment described above, the humanized antigen binding protein, antibody or antibody active fragment described above, the protein conjugate described above or the kit described above.

[0078] In some embodiments, the sample is animal serum, preferably human serum.

[0079] Compared with the prior art, the technical solution provided by the present invention has the following significant advantages: the antibodies provided by the present invention can be used to detect the content of target antigens in serum, and further used to detect proto-oncogenes, judge lesions, and have potential clinical diagnosis and treatment value; the antibodies provided by the present invention have a simple structure, are easy to be genetically engineered, and have mature optimization strategies for enhancing the affinity of nano antibodies, prolonging the half-life in vivo, and coupling with other molecules for drug development, such as connecting radioactive isotopes, coupling delivery drugs, CART and fluorescent labeling high-resolution imaging, etc.; the antibody sequence provided by the present invention has high homology with the VH region sequence of human IgG, and a few amino acid mutations can achieve the humanization of single-domain antibodies; and the antibodies provided by the present invention have high stability, which can avoid the requirements of conventional antibodies for low-temperature storage and transportation, and are conducive to large-scale popularization and application, with low mass production costs and easy large-scale recombinant preparation. The monoclonal nano antibodies designed by the present invention can be well recombinantly expressed in a low-cost Escherichia coli expression system, with low mass production costs and yields of up to tens of milligrams per liter of Escherichia coli. The Escherichia coli recombinant expression system technology is mature and the quality control is simple, which is conducive to reducing production costs and achieving large-scale production.

[0080] Unlike traditional technologies that rely on classic model animals such as mice, rabbits, monkeys, and sheep, the technical solution of the present invention relies on antibodies produced by the immune system of alpacas, which are called "nanoantibodies". Nanoantibodies are tiny antibody fragments separated from immunoglobulins in animals such as camels and sharks. They have the same antigen binding ability and structural stability as complete antibodies. They are the smallest units that can bind to target antigens, with a relative molecular mass of only 15kD. Compared with traditional animals such as mice and rabbits that can only recognize flat polypeptides on the surface of antigens, the immune systems in animals such as alpacas can recognize the complex spatial structure of the antigen surface and can produce highly specific and high-affinity nanoantibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 Schematic diagram of the affinity test results between monoclonal antibody Rab11_5C8 and antigen.

[0082] Figure 2 Schematic diagram of the affinity test results between monoclonal antibody Rab11_5A10 and antigen.

[0083] Figure 3 This is a schematic diagram of the affinity test results between monoclonal antibody Rab11_5H11 and antigen.

[0084] Figure 4 Schematic diagram of the affinity test results between monoclonal antibody Rab11_6H3 and antigen.

[0085] Figure 5 Schematic diagram of the affinity test results between monoclonal antibody Rab11_6G1 and antigen. DETAILED DESCRIPTION

[0086] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0087] definition

[0088] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0089] Unless the context clearly dictates otherwise, the expressions "a", "an" and "an" as used herein include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0090] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0091] Rab11 : Rab11 (Ras-related protein 11) is a member of the Ras superfamily and plays a role in protein transport and membrane reorganization. Rab family proteins cycle between inactive GDP-bound and active GTP-bound forms, can recruit different groups of downstream effectors to the membrane, and are directly responsible for the formation, movement, binding and fusion of vesicles. The cellular physiological functions of Rab11 include participating in polarized vesicle transport and neurotransmitter release, promoting transcytosis with a variety of important proteins, regulating the compact morphology of the Golgi apparatus, participating in epithelial cell polarization, participating in membrane transport to cilia and ciliogenesis, and regulating adherens junction components.

[0092] kd value :The dissociation constant (kd) is a specific type of equilibrium constant used to measure the tendency of a larger object to separate (dissociate) from another smaller component. It is the reciprocal of the association constant and is expressed in mol / L (M) or nmol / L (nM). The smaller the kd value, the stronger the binding ability of the two substances.

[0093] Nanobodies : An antibody that naturally lacks light chains and exists in the peripheral blood of camelids. This antibody contains only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, but it is not as easy to stick to each other or even aggregate into clumps as artificially modified single-chain antibody fragments; the VHH structure cloned and expressed separately has the same structural stability and antigen binding activity as the original heavy chain antibody, and is the smallest unit known to bind to the target antigen; the VHH crystal is 2.5nm, 4nm long, and has a molecular weight of only 15KDa, so it is also called a nanobody (Nanobody, Nb). Compared with traditional animals such as mice and rabbits that can only recognize flat polypeptides on the surface of antigens, the immune system in camelids can recognize the complex spatial structure of the antigen surface and can produce highly specific and high-affinity nanobodies.

[0094] According to the technical solution of the present invention, certain amino acids in the amino acid sequence can be conservatively substituted without changing the activity or function of the protein, as shown in Table 1 below:

[0095] Table 1

[0096] Residue Conservative substitution Residue Conservative substitution Ala Ser Leu Ile; Val Arg Lys Lys Arg; Gln Asn Gln; His Met Leu; Ile Asp Glu Phe Met; Leu; Tyr Gln Asn Ser Thr; Gly Cys Ser Thr Ser; Val Glu Asp Trp Tyr Gly Pro Tyr Trp; Phe His Asn; Gln Val Ile; Leu Ile Leu; Val

[0097] In addition, due to the degeneracy of the bases, the bases of the polynucleotide sequence can be substituted without changing the activity or function of the polynucleotide sequence, as shown in Table 2 below:

[0098] Table 2

[0099]

[0100]

[0101] Examples and drawings are provided below to help understand the present invention. However, it should be understood that these examples and drawings are only used to illustrate the present invention, but do not constitute any limitation. The actual protection scope of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0102] Example 1. Preparation of antigens

[0103] Recombinant human Rab11 protein was purchased from PROSPEC (Cat. No. PRO-826).

[0104] The recombinant human Rab11 protein antigen has an amino acid sequence as shown in SEQ ID NO: 1, specifically:

[0105] MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDRWGSMGTRDDEYDYLFKVVLIGDSGVGKSNLLSRFTRNEFNLESKSTIGVEFATRSIQVDGKTIKAQIWDTAGQERYRAITSAYYRGAVGALLV YDIAKHLTYENVERWLKELRDHADSNIVIMLVGNKSDLRHLRAVPTDEARAFAEKNGLSFIETSALDSTNVEAAFQTILTEIYRIVSQKQMSDRRENDMSPSNNVVPIHVPPTTENKPKVQCC(SEQ ID NO:1).

[0106] Example 2. Alpaca Immunization

[0107] In this example, the antigen of Example 1 was used to immunize alpacas. The specific steps are as follows:

[0108] (1) The antigen in Example 1 was evenly divided into 4 portions, each portion was about 0.25 mg; the alpacas were immunized 4 times in total, and the antigen was injected subcutaneously into the animal body, with the first immunization being recorded as the first day, and the subsequent immunizations were on the 10th day, the 19th day, and the 28th day respectively;

[0109] (2) On day 28, before the fourth immunization, approximately 200 mL of peripheral venous blood was collected from the alpaca;

[0110] (3) On day 42, i.e., 14 days after the fourth immunization, approximately 200 mL of peripheral venous blood was collected from the alpaca.

[0111] Compared with traditional immunization technology solutions for animal antibodies such as mice and rabbits, the advantage of this technology is that it collects a large amount of alpaca venous peripheral blood, which is conducive to subsequent screening to obtain highly diverse nano-antibodies.

[0112] Example 3. Construction of Alpaca Nanobody Library

[0113] The two batches of alpaca venous peripheral blood collected in Example 2 were used as raw materials to construct a highly diverse nanoantibody library. The two batches of alpaca venous peripheral blood were processed in the same way, and the specific steps were as follows:

[0114] (1) Lymphocytes were isolated from alpaca venous peripheral blood using density gradient centrifugation and other methods;

[0115] (2) extracting total mRNA from lymphocytes and reverse transcribed into cDNA;

[0116] (3) using appropriate DNA primers and the above cDNA as a template, amplifying the VHH fragments of alpaca immunoglobulins IgG2 and IgG3, i.e., the DNA fragments of the nanobody, by polymerase chain reaction (PCR);

[0117] (4) connecting the VHH DNA to a phage surface display screening vector to form a VHH-pIII fusion protein expression vector plasmid library; wherein pIII is a protein present on the flagella on the surface of the phage;

[0118] (5) The DNA ligation product is transformed into TG1 competent bacteria by electroporation, and all colonies are collected after appropriate cultivation, which is the alpaca nanoantibody library.

[0119] Compared with the traditional method of isolating antibodies from the serum or lymphocytes of animals such as mice and rabbits, this technical solution can preserve all the nano-antibody fragments (i.e., the library) of alpacas for a long time, and can continuously support the subsequent screening and development of nano-antibodies.

[0120] Example 4. Screening of specific nanobodies by phage surface display

[0121] This example uses the nanobody library obtained in Example 3 as a source, and obtains antigen-specific nanobodies through phage surface display screening. The specific steps are as follows:

[0122] (1) taking an appropriate amount of frozen nanobody library, inoculating it into bacterial culture medium, adding an appropriate amount of helper phage after appropriate cultivation, and continuing to cultivate under appropriate conditions;

[0123] (2) extracting the amplified phages from the bacterial culture supernatant using the PEG-NaC method;

[0124] (3) phages were appropriately incubated with antigens, and Rab11 antigen was pre-immobilized in an immunotube (Maxisorp immunotube, ThermoFisher Scientific);

[0125] (4) Panning: discard the phages, rinse the antigen with an appropriate buffer (such as PBS) for an appropriate number of times, remove the phages that non-specifically bind to the antigen, and retain the phages that specifically bind to the antigen;

[0126] (5) Elution: Treat the phages that specifically bind to the antigen with an appropriate method (such as acidic glycine solution, etc.) to dissociate the phages from the antigen and retain them.

[0127] At this point, phages expressing specific nanobodies have been obtained, and these phages can be used for the following operations:

[0128] (6) Transformation into a specific nanoantibody library: The phage is again infected with the E. coli cultured to a suitable state, but no helper phage is added. After the phage infection is complete, the specific nanoantibody exists in the E. coli in the form of a DNA plasmid. Collect all these E. coli to form an antigen-specific nanoantibody library. This library can be used as a raw material and returned to step (1) for the next round of phage surface display screening;

[0129] (7) Transformation into monoclonal nanoantibody colonies: Take a small amount of the phage obtained in step (5) (e.g., 0.5%), dilute it, and infect the E. coli cultured to a suitable state again, but no helper phage is added. After the phage infection is complete, the E. coli are evenly spread on a bacterial culture dish, and monoclonal colonies containing nanoantibody DNA plasmids can be obtained by culturing under appropriate conditions. These monoclonal colonies can be used as raw materials for the identification of positive monoclonal nanoantibodies.

[0130] Example 5. Identification of positive monoclonal nanobodies

[0131] This example uses step (7) of Example 4 to obtain a bacterial culture dish with monoclonal colonies, and conducts identification of positive monoclonal nanoantibodies. The specific steps are as follows:

[0132] (1) Pick a single clone and culture it in a microplate;

[0133] (2) adding IPTG to induce the expression of VHH-pIII (i.e., the fusion protein containing the nanobody);

[0134] (3) The bacterial culture supernatant containing nanobodies was collected and incubated with the antigen Rab11, which was pre-immobilized in a 96-well microplate (Maxisorp transparent microplate, ThermoFisher Scientific);

[0135] (4) using enzyme-linked immunosorbent assay (ELISA) to detect whether the monoclonal nanoantibody binds to the Rab11 antigen;

[0136] (5) For the monoclonal nanoantibody microbial colonies that can bind to the antigen, after appropriate culturing again, the DNA plasmid is extracted and DNA sequencing is performed to obtain the nanoantibody nucleic acid sequence. After translation, the complete amino acid sequence of the nanoantibody can be obtained, as shown in Table 3-7.

[0137] Table 3. Amino acid sequence and nucleotide sequence of monoclonal antibody Rab11_5C8

[0138]

[0139] Table 4. Amino acid sequence and nucleotide sequence of monoclonal antibody Rab11_5A10

[0140]

[0141] Table 5. Amino acid sequence and nucleotide sequence of monoclonal antibody Rab11_5H11

[0142]

[0143] Table 6. Amino acid sequence and nucleotide sequence of monoclonal antibody Rab11_6H3

[0144]

[0145]

[0146] Table 7. Amino acid sequence and nucleotide sequence of monoclonal antibody Rab11_6G1

[0147]

[0148] Example 6. Small batch monoclonal nanobody recombinant expression and purification

[0149] (1) A monoclonal nanobody capable of specifically recognizing and binding to an antigen was obtained in Example 5. The DNA plasmid of the nanobody was transformed into BL21 (DE3) competent cells. The monoclonal nanobody can be expressed and purified in small batches using the E. coli expression system, with a batch production capacity of about several milligrams.

[0150] (2) Using the ELISA method, different concentrations of nanoantibodies were incubated, and the affinity of the nanoantibody to the antigen was measured based on the binding ability of the nanoantibody to Rab11.

[0151] The results of the affinity test of the antibodies corresponding to the two groups of monoclonal colonies and the Rab11 protein antigen are as follows: Figures 1 to 5 The results of affinity values ​​kd are shown in Table 8 below.

[0152] Table 8. Affinity test results

[0153] kd(nM) Rab11_5C8 99.28 Rab11_5A10 145.8 Rab11_5H11 248.4 Rab11_6H3 37.54 Rab11_6G1 36.06

[0154] From the above results, it can be seen that the kd value of the antibody obtained by selecting the monoclonal colony in this example for binding to the Rab11 protein is below 500 nM, preferably below 300 nM, more preferably below 200 nM, more preferably below 100 nM, and more preferably below 50 nM.

[0155] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A nanobody or an active fragment thereof that specifically recognizes and / or binds to Rab11 protein, characterized in that: The Nanobody or its active fragment comprises a heavy chain variable region, and the heavy chain variable region has: CDR1 as shown in SEQ ID NO:6, CDR2 as shown in SEQ ID NO:11 and CDR3 as shown in SEQ ID NO:

16.

2. The nanobody or active fragment thereof according to claim 1, characterized in that The heavy chain variable region has the amino acid sequence shown in SEQ ID NO:

21.

3. A nucleic acid molecule encoding the Nanobody or active fragment thereof according to claim 1 or 2. An expression vector comprising the nucleic acid molecule according to claim 3.

5. The expression vector according to claim 4, characterized in that The expression vector is a phage expression vector.

6. The expression vector according to claim 4, characterized in that The expression vector is a phage surface display screening vector.

7. The expression vector according to claim 4, characterized in that The expression vector also contains a nucleotide sequence encoding phage envelope protein pill.

8. A host cell into which the expression vector according to any one of claims 4 to 7 is exogenously introduced.

9. The host cell according to claim 8, characterized in that The host cell is Escherichia coli.

10. A method for expressing a nanobody or an active fragment thereof using the host cell according to claim 8 or 9.

11. A humanized Nanobody or an active fragment thereof obtained by humanizing the Nanobody or an active fragment thereof according to claim 1 or 2.

12. A protein conjugate, characterized in that The protein conjugate comprises the Nanobody or its active fragment according to claim 1 or 2 or the humanized Nanobody or its active fragment according to claim 11, and the protein conjugate further comprises a ligand, wherein the ligand is selected from a radioactive isotope and a fluorescent group.

13. A kit for detecting the content of Rab11 protein in a sample, characterized in that: Comprising the Nanobody or its active fragment according to claim 1 or 2 or the humanized Nanobody or its active fragment according to claim 11.

14. The kit according to claim 13, characterized in that The nanobody or its active fragment is labeled with a marker.

15. The kit according to claim 14, characterized in that The label is an enzyme, a chemiluminescent group or an isotope group.

16. The kit according to claim 13, characterized in that The sample is animal serum.

17. The kit according to claim 16, characterized in that The sample was human serum.

18. Use of the nanobody or active fragment thereof according to claim 1 or 2, the humanized nanobody or active fragment thereof according to claim 11, or the protein conjugate according to claim 12 in the preparation of a kit for detecting the content of Rab11 protein in a sample.

19. The use according to claim 18, characterized in that The sample is animal serum.

20. The use according to claim 19, characterized in that The sample was human serum.

Images