阅读说明：本技术 特异性针对MMP9蛋白ZnMc结构域的单域抗体及产品与应用 (Single-domain antibody specifically aiming at ZnMc structural domain of MMP9 protein, product and application ) 是由 苏志鹏 孟巾果 赵泽英 于 2019-12-20 设计创作，主要内容包括：本发明涉及生物医学技术领域,具体而言,提供了一种特异性针对MMP9蛋白ZnMc结构域的单域抗体及产品与应用。本发明提供的单域抗体包括互补决定区CDR包括CDR1、CDR2和CDR3,CDR1的氨基酸序列如SEQ ID NO.29-SEQ ID NO.34中的任一种；CDR2的氨基酸序列如SEQ ID NO.35-SEQ ID NO.41中的任一种；CDR3的氨基酸序列如SEQ ID NO.42-SEQ ID NO.50中的任一种。该单域抗体的亲和力明显,可替代传统单克隆抗体快速表达用于检测MMP-9蛋白或改变其活性。(The invention relates to the technical field of biomedicine, and particularly provides a single domain antibody specifically aiming at a ZnMc structural domain of MMP9 protein, a product and application thereof. The single domain antibody provided by the invention comprises Complementarity Determining Regions (CDRs) including CDR1, CDR2 and CDR3, wherein the amino acid sequence of the CDR1 is any one of SEQ ID NO.29-SEQ ID NO. 34; the amino acid sequence of the CDR2 is any one of SEQ ID NO.35-SEQ ID NO. 41; the amino acid sequence of the CDR3 is any one of SEQ ID NO.42-SEQ ID NO. 50. The single-domain antibody has obvious affinity, can replace the traditional monoclonal antibody to quickly express and be used for detecting MMP-9 protein or changing the activity of the MMP-9 protein.)

1. A single domain antibody specific for the ZnMc domain of the MMP9 protein, characterized in that it comprises complementarity determining region CDRs comprising CDR1, CDR2 and CDR 3;

the amino acid sequence of the CDR1 is any one of SEQ ID NO.29-SEQ ID NO. 34;

the amino acid sequence of the CDR2 is any one of SEQ ID NO.35-SEQ ID NO. 41;

the amino acid sequence of the CDR3 is any one of SEQ ID NO.42-SEQ ID NO. 50.

2. The single domain antibody of claim 1, wherein the CDR of the single domain antibody is as follows:

CDR1 shown in SEQ ID NO.29, CDR2 shown in SEQ ID NO.35 and CDR3 shown in SEQ ID NO. 42;

or, the CDR1 shown in SEQ ID NO.29, the CDR2 shown in SEQ ID NO.35 and the CDR3 shown in SEQ ID NO. 43;

or, CDR1 shown in SEQ ID NO.30, CDR2 shown in SEQ ID NO.36 and CDR3 shown in SEQ ID NO. 44;

or, the CDR1 shown in SEQ ID NO.31, the CDR2 shown in SEQ ID NO.37 and the CDR3 shown in SEQ ID NO. 45;

or, the CDR1 shown in SEQ ID NO.32, the CDR2 shown in SEQ ID NO.38 and the CDR3 shown in SEQ ID NO. 46;

or, the CDR1 shown in SEQ ID NO.33, the CDR2 shown in SEQ ID NO.39 and the CDR3 shown in SEQ ID NO. 47;

or, the CDR1 shown in SEQ ID NO.34, the CDR2 shown in SEQ ID NO.40 and the CDR3 shown in SEQ ID NO. 48;

or, CDR1 shown in SEQ ID NO.34, CDR2 shown in SEQ ID NO.41 and CDR3 shown in SEQ ID NO. 49;

or, the CDR1 shown in SEQ ID NO.34, the CDR2 shown in SEQ ID NO.41 and the CDR3 shown in SEQ ID NO. 50.

3. The single domain antibody of claim 2, further comprising a framework region FR;

preferably, the framework regions FR include FR1, FR2, FR3 and FR 4;

the amino acid sequence of FR1 is any one of SEQ ID NO.51-SEQ ID NO. 59;

the amino acid sequence of FR2 is any one of SEQ ID NO.60-SEQ ID NO. 65;

the amino acid sequence of FR3 is any one of SEQ ID NO.66-SEQ ID NO. 72;

the amino acid sequence of FR4 is SEQ ID NO. 73;

preferably, the framework regions FR are as follows:

FR1 shown in SEQ ID NO.51, FR2 shown in SEQ ID NO.60, FR3 shown in SEQ ID NO.66 and FR4 shown in SEQ ID NO. 73;

or FR1 shown in SEQ ID NO.52, FR2 shown in SEQ ID NO.60, FR3 shown in SEQ ID NO.66 and FR4 shown in SEQ ID NO. 73;

or FR1 shown in SEQ ID NO.53, FR2 shown in SEQ ID NO.61, FR3 shown in SEQ ID NO.67 and FR4 shown in SEQ ID NO. 73;

or FR1 shown in SEQ ID NO.54, FR2 shown in SEQ ID NO.62, FR3 shown in SEQ ID NO.68 and FR4 shown in SEQ ID NO. 73;

or FR1 shown in SEQ ID NO.55, FR2 shown in SEQ ID NO.62, FR3 shown in SEQ ID NO.68 and FR4 shown in SEQ ID NO. 73;

or FR1 shown in SEQ ID NO.56, FR2 shown in SEQ ID NO.63, FR3 shown in SEQ ID NO.69 and FR4 shown in SEQ ID NO. 73;

or FR1 shown in SEQ ID NO.57, FR2 shown in SEQ ID NO.64, FR3 shown in SEQ ID NO.70 and FR4 shown in SEQ ID NO. 73;

or FR1 shown in SEQ ID NO.56, FR2 shown in SEQ ID NO.64, FR3 shown in SEQ ID NO.71 and FR4 shown in SEQ ID NO. 73;

or FR1 shown in SEQ ID NO.57, FR2 shown in SEQ ID NO.65, FR3 shown in SEQ ID NO.72 and FR4 shown in SEQ ID NO. 73;

or FR1 shown in SEQ ID NO.58, FR2 shown in SEQ ID NO.65, FR3 shown in SEQ ID NO.72 and FR4 shown in SEQ ID NO. 73;

or FR1 shown in SEQ ID NO.59, FR2 shown in SEQ ID NO.65, FR3 shown in SEQ ID NO.72 and FR4 shown in SEQ ID NO. 73.

4. The single domain antibody of any one of claims 1 to 3, characterized in that the amino acid sequence of said single domain antibody is any one of SEQ ID No.1 to SEQ ID No. 14.

5. A biological material associated with the single domain antibody of any one of claims 1 to 4, wherein the biological material is any one of:

(a) a nucleic acid molecule encoding the single domain antibody of any one of claims 1-4;

(b) an expression cassette comprising the nucleic acid molecule of (a);

(c) a recombinant vector comprising the nucleic acid molecule of (a) or the expression cassette of (b);

(d) a recombinant eukaryotic cell comprising the nucleic acid molecule of (a), the expression cassette of (b), or the recombinant vector of (c);

(e) a recombinant prokaryotic cell comprising the nucleic acid molecule of (a), the expression cassette of (b), or the recombinant vector of (c).

6. A biomaterial according to claim 5, wherein the nucleic acid molecule is as defined in (a) or (b):

(a) the nucleotide sequence is any one of SEQ ID NO.15-SEQ ID NO. 28;

(b) DNA having 75% or more identity to the nucleotide sequence defined in (a) and encoding the single domain antibody of any one of claims 1 to 4.

7. The derivative antibody of the single domain antibody of any one of claims 1 to 4, wherein said derivative antibody is any one of (a) to (e) below:

(a) a single chain antibody comprising the single domain antibody of any one of claims 1-4;

(b) a fusion antibody comprising a single chain antibody of (a) or a single domain antibody of any one of claims 1-4;

(c) a Fab comprising the single domain antibody of any one of claims 1-4;

(d) a heavy chain antibody comprising a single domain antibody of any one of claims 1-4;

(e) a whole antibody comprising a single domain antibody according to any one of claims 1 to 4.

8. The method for producing a single domain antibody according to any one of claims 1 to 4, wherein a nucleic acid molecule encoding the single domain antibody according to any one of claims 1 to 4 is introduced into a recipient cell to obtain a transgenic cell, and the transgenic cell is cultured to obtain the single domain antibody.

9. The method according to claim 8, wherein the recipient cell is a microbial cell or a mammalian cell.

10. The use of any one of the following (a) to (h):

(a) use of a single domain antibody according to any one of claims 1 to 4 in the manufacture of a tumor suppressor, a tumor cell suppressor, an agent for inflammatory diseases or an agent for autoimmune diseases;

(b) use of the biomaterial of claim 5 or 6 in the preparation of a tumor suppressor, a tumor cell suppressor, a medicament for inflammatory diseases or a medicament for autoimmune diseases;

(c) use of the derivatized antibody of claim 7 for the preparation of a tumor suppressor, a tumor cell suppressor, a drug for inflammatory diseases, or a drug for autoimmune diseases;

(d) the use of the process according to claim 8 or 9 for the preparation of a tumor suppressor, a tumor cell suppressor, a medicament for inflammatory diseases or a medicament for autoimmune diseases;

(e) use of a single domain antibody according to any one of claims 1 to 4 in the manufacture of a product for inhibiting MMP-9 activity or binding to MMP-9;

(f) use of a biomaterial according to claim 5 or 6 in the manufacture of a product for inhibiting MMP-9 activity or binding to MMP-9;

(g) use of a derivatized antibody according to claim 7 for the preparation of a product inhibiting MMP-9 activity or binding to MMP-9;

(h) use of the process of claim 8 or 9 for the preparation of a product for inhibiting MMP-9 activity or binding to MMP-9.

Technical Field

The invention relates to the technical field of biomedicine, in particular to a single domain antibody specifically aiming at a ZnMc structural domain of MMP9 protein, a product and application thereof.

Background

Matrix Metalloproteinases (MMPs) belong to a family of extracellular enzymes involved in extracellular matrix formation and remodeling. These enzymes contain a conserved catalytic domain in which the zinc atom is coordinated by three histidine residues. Members of this family are organized into groups including collagenases, gelatinases, stromelysins, amelysins and membrane MMPs.

MMP-9 (matrix metalloproteinase-9) belongs to the gelatinase group of matrix metalloproteinases. The MMP-9 gene is located on chromosome 20q 11.1-13.1, 26-27 kbp, and has 13 exons and 9 introns. MMP-9 has a major function in the dynamic equilibrium of degradation and remodelling of the extracellular matrix (extracellular matrix), and the matrix metalloproteinase family comprises a plurality of mechanistic metalloproteinases, respectively responsible for the hydrolysis and equilibrium of different substrates.

MMP-9 is a zinc-dependent endopeptidase, and hydrolysis of matrix proteins is an important role in leukocyte migration. MMP-9 also plays a role in the bone resorption process, hydrolyzing type I and V gelatin, as well as type IV and V collagen. The cofactor for this enzyme is generally Zn²⁺Or Ca²⁺One MMP-9 can bind 2 Zn²⁺Or 3 Ca²⁺MMP-9 can break down structural complexes in the respiratory tract and lung such as ECM and basement membrane, and thus can participate in the remodeling of the respiratory tract and lung, and can also regulate the activity of other proteases and cytokines, degrading α antitrypsin, protecting neutrophil elastase activity, and at the same time, can enhance the collagenolytic activity of collagen cells and MMP-13 in collagen colloid, and MMP-9 can also break down a 62 amino acid peptide from interleukin 8(CXCL8/CL8), increasing its chemotactic activity towards neutrophils 10-fold, but it also inhibits chemokines of other neutrophils. in addition, MMP-9 binds to TGF- β stored in CD44, and in addition, MMP-9 can participate in angiogenesis by releasing Vascular Endothelial Growth Factor (VEGF).

MMP-9 has relevance to many diseases, such as vascular diseases (heart and brain) and respiratory system diseases, and researches show that MMP-9 is abnormally highly expressed in various malignant tumors such as lung cancer and is closely related to invasion and metastasis of cancer cells.

The prior MMP-9 antibodies are all traditional monoclonal antibodies, the traditional monoclonal antibody technology generally refers to a monoclonal antibody which is prepared based on mouse hybridoma cells and aims at target proteins, the technology is mature, the phage display technology is used for respectively displaying heavy chains and light chains of the antibodies or simultaneously displaying Fab fragments of the antibodies to replace the original hybridoma technology, but the finally obtained antibody structure is the same as or similar to the structure of the traditional monoclonal antibody. The specificity and the efficacy of the traditional monoclonal antibody are not completely satisfactory, and in addition, the immune heterogeneity is higher and the modification space is small.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The first object of the present invention is to provide a single domain antibody specific for the ZnMc domain of the MMP9 protein.

The second purpose of the invention is to provide a biological material related to the single domain antibody provided by the invention.

The third objective of the invention is to provide a derivative antibody of the single domain antibody of the invention.

The fourth object of the present invention is to provide a method for producing a single domain antibody.

The fifth purpose of the invention is the application of the product provided by the invention.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

a single domain antibody specific for the ZnMc domain of the MMP9 protein, comprising complementarity determining regions CDRs comprising CDR1, CDR2 and CDR 3;

the amino acid sequence of the CDR1 is any one of SEQ ID NO.29-SEQ ID NO. 34;

the amino acid sequence of the CDR2 is any one of SEQ ID NO.35-SEQ ID NO. 41;

the amino acid sequence of CDR3 is any one of SEQ ID NO.42-SEQ ID NO. 50.

In a preferred embodiment, the complementarity determining regions CDRs of the single domain antibody are as follows:

CDR1 shown in SEQ ID NO.29, CDR2 shown in SEQ ID NO.35 and CDR3 shown in SEQ ID NO. 42;

or, the CDR1 shown in SEQ ID NO.29, the CDR2 shown in SEQ ID NO.35 and the CDR3 shown in SEQ ID NO. 43;

or, the CDR1 shown in SEQ ID NO.30, the CDR2 shown in SEQ ID NO.36 and the CDR3 shown in SEQ ID NO. 44;

or, the CDR1 shown in SEQ ID NO.31, the CDR2 shown in SEQ ID NO.37 and the CDR3 shown in SEQ ID NO. 45;

or, the CDR1 shown in SEQ ID NO.32, the CDR2 shown in SEQ ID NO.38 and the CDR3 shown in SEQ ID NO. 46;

or, the CDR1 shown in SEQ ID NO.33, the CDR2 shown in SEQ ID NO.39 and the CDR3 shown in SEQ ID NO. 47;

or, the CDR1 shown in SEQ ID NO.34, the CDR2 shown in SEQ ID NO.40 and the CDR3 shown in SEQ ID NO. 48;

or, CDR1 shown in SEQ ID NO.34, CDR2 shown in SEQ ID NO.41 and CDR3 shown in SEQ ID NO. 49;

or, the CDR1 shown in SEQ ID NO.34, the CDR2 shown in SEQ ID NO.41 and the CDR3 shown in SEQ ID NO. 50.

In the present invention, the single domain antibody is composed of the complementarity determining region CDR and the framework region FR.

In a preferred embodiment, the framework regions FR comprise FR1, FR2, FR3 and FR4, wherein the amino acid sequence of FR1 is any one of SEQ ID No.51 to SEQ ID No. 59; the amino acid sequence of FR2 is any one of SEQ ID NO.60-SEQ ID NO. 65; the amino acid sequence of FR3 is any one of SEQ ID NO.66-SEQ ID NO. 72; the amino acid sequence of FR4 is SEQ ID NO. 73.

In a preferred embodiment, the framework regions FR are as follows:

FR1 shown in SEQ ID NO.51, FR2 shown in SEQ ID NO.60, FR3 shown in SEQ ID NO.66 and FR4 shown in SEQ ID NO. 73;

or, FR1 shown in SEQ ID NO.52, FR2 shown in SEQ ID NO.60, FR3 shown in SEQ ID NO.66 and FR4 shown in SEQ ID NO. 73;

or, FR1 shown by SEQ ID NO.53, FR2 shown by SEQ ID NO.61, FR3 shown by SEQ ID NO.67 and FR4 shown by SEQ ID NO. 73;

or, FR1 shown in SEQ ID NO.54, FR2 shown in SEQ ID NO.62, FR3 shown in SEQ ID NO.68 and FR4 shown in SEQ ID NO. 73;

or, FR1 shown by SEQ ID NO.55, FR2 shown by SEQ ID NO.62, FR3 shown by SEQ ID NO.68 and FR4 shown by SEQ ID NO. 73;

or, FR1 shown in SEQ ID NO.56, FR2 shown in SEQ ID NO.63, FR3 shown in SEQ ID NO.69 and FR4 shown in SEQ ID NO. 73;

or, FR1 shown by SEQ ID NO.57, FR2 shown by SEQ ID NO.64, FR3 shown by SEQ ID NO.70 and FR4 shown by SEQ ID NO. 73;

or, FR1 shown in SEQ ID NO.56, FR2 shown in SEQ ID NO.64, FR3 shown in SEQ ID NO.71 and FR4 shown in SEQ ID NO. 73;

or FR1 shown in SEQ ID NO.57, FR2 shown in SEQ ID NO.65, FR3 shown in SEQ ID NO.72 and FR4 shown in SEQ ID NO. 73;

or FR1 shown in SEQ ID NO.58, FR2 shown in SEQ ID NO.65, FR3 shown in SEQ ID NO.72 and FR4 shown in SEQ ID NO. 73;

or FR1 shown by SEQ ID NO.59, FR2 shown by SEQ ID NO.65, FR3 shown by SEQ ID NO.72 and FR4 shown by SEQ ID NO. 73.

The amino acid sequence of the single domain antibody provided by the invention is any one of SEQ ID NO.1-SEQ ID NO. 14.

The single domain antibody of the invention can be artificially synthesized, or can be obtained by synthesizing the coding gene and then performing biological expression. For example, the single domain antibody is obtained by performing biological expression using a biological material related to the single domain antibody, and preferably, the biological material is any one of the following:

(a) a nucleic acid molecule encoding a single domain antibody provided by the invention;

(b) an expression cassette comprising the nucleic acid molecule of (a);

(c) a recombinant vector comprising the nucleic acid molecule of (a) or the expression cassette of (b);

(d) a recombinant eukaryotic cell comprising the nucleic acid molecule of (a), the expression cassette of (b), or the recombinant vector of (c);

(e) a recombinant prokaryotic cell comprising the nucleic acid molecule of (a), the expression cassette of (b), or the recombinant vector of (c).

A "nucleotide molecule" may be DNA, such as cDNA or recombinant DNA, or RNA, such as mRNA or hnRNA, etc.

An "expression cassette" comprises a polynucleotide sequence encoding the polypeptide to be expressed (single domain antibody) and sequences controlling its expression such as a promoter and optionally enhancer sequences, including any combination of cis-acting transcriptional control units. Sequences that control the expression of a gene (i.e., its transcription and translation of the transcription product) are often referred to as regulatory units. Most of the regulatory units are located upstream of and operably linked to the coding sequence of the gene. The expression cassette may also contain a downstream 3' untranslated region comprising a polyadenylation site.

The vector of the "recombinant vector" may be a plasmid, a phage or a virus.

The host cell of the "recombinant eukaryotic cell" may be a yeast or mammalian cell, etc.

The host cell of the "recombinant prokaryotic cell" may be a bacterium, an alga or the like.

In preferred embodiments, the nucleotide sequence of the nucleic acid molecule is any one of SEQ ID No.15 to SEQ ID No. 28; alternatively, the nucleotide sequence obtained by deleting one or several amino acid residues from the above-mentioned DNA sequence and/or by carrying out missense mutation of one or several base pairs is a nucleotide sequence derived from and identical to the nucleotide sequence of the present invention as long as the nucleotide sequence encodes and has the activity of the single domain antibody provided by the present invention. Preferably, the nucleic acid molecule is a DNA having more than 75% identity with any one of SEQ ID No.15-SEQ ID No.28 and encoding a single domain antibody provided by the present invention. As used herein, "identity" refers to sequence similarity to a native nucleic acid sequence, and specifically refers to a nucleotide sequence that has 75% or greater, or 85% or greater, or 90% or greater, or 95% or greater identity to a nucleotide sequence of any one of SEQ ID No.15 to SEQ ID No. 28.

The derivative antibody of the single-domain antibody provided by the invention is any one of the following (a) to (e):

(a) single-chain antibodies, comprising the single-domain antibodies provided by the invention;

(b) a fusion antibody comprising a single chain antibody of (a) or a single domain antibody provided by the invention;

(c) fab, containing the single domain antibody provided by the invention;

(d) heavy chain antibodies comprising a single domain antibody provided herein;

(e) an intact antibody comprising a single domain antibody provided by the invention.

The preparation method of the single domain antibody provided by the invention is characterized in that a nucleic acid molecule for encoding the single domain antibody provided by the invention is introduced into a receptor cell to obtain a transgenic cell, and the transgenic cell is cultured to obtain the single domain antibody.

In a preferred embodiment, the recipient cell is a microbial cell or a mammalian cell. Such as E.coli, phage or CHO cells, etc.

The invention also protects the application of any one of the following (a) to (h):

(a) the single domain antibody provided by the invention is applied to the preparation of tumor inhibitors, tumor cell inhibitors, inflammatory disease drugs or autoimmune drugs;

(b) the biological material provided by the invention is applied to the preparation of tumor inhibitors, tumor cell inhibitors, inflammatory disease drugs or autoimmune drugs;

(c) the derivative antibody provided by the invention is applied to the preparation of tumor inhibitors, tumor cell inhibitors, inflammatory disease drugs or autoimmune drugs;

(d) the preparation method provided by the invention is applied to preparing tumor inhibitors, tumor cell inhibitors, inflammatory disease drugs or autoimmune drugs;

(e) the invention provides the application of the single domain antibody in preparing products for inhibiting MMP-9 activity or combining with MMP-9;

(f) the application of the biological material provided by the invention in preparing products for inhibiting the activity of MMP-9 or combining with MMP-9;

(g) the derivative antibody provided by the invention is applied to the preparation of products for inhibiting the activity of MMP-9 or combining with MMP-9;

(h) the preparation method provided by the invention is applied to preparing products for inhibiting the activity of MMP-9 or combining with MMP-9.

The above product can be medicine, etc.; tumors such as primary rectal cancer, gastric cancer, colon cancer, hepatocellular carcinoma and related adenocarcinoma; inflammatory and autoimmune diseases, such as rheumatoid arthritis, crohn's disease and other diseases associated with inflammation.

The primer pair for amplifying the nucleic acid molecule encoding the single-domain antibody provided by the invention also belongs to the protection scope of the invention.

Compared with the prior art, the invention has the beneficial effects that:

the invention uses biological genetic engineering technology to immunize inner Mongolia Alalashan bactrian camel, obtains a single domain antibody with specificity aiming at MMP-9 protein ZnMc structural domain by screening, limits the CDR sequence of the specific complementarity determining region of the single domain antibody, and simultaneously provides a gene sequence for coding the single domain antibody and a host cell capable of expressing the single domain antibody. The single-domain antibody has obvious affinity, has good binding activity through prokaryotic expression, and can replace the traditional monoclonal antibody to quickly express and be used for detecting related proteins.

The method has the following advantages:

(1) the expression system suitable for the single domain antibody is flexible to select, can be expressed in a prokaryotic system and also can be expressed in a eukaryotic system of a yeast cell or a mammalian cell, and the expression cost of the single domain antibody in the prokaryotic expression system is low, so that the later-stage production cost can be reduced.

(2) The single domain antibody is a single domain antibody, so that the multi-combination form of the antibody is simpler to modify, a multivalent and multi-specific antibody can be obtained by simply connecting the single domain antibody in series in a genetic engineering mode, the immune heterogeneity is very low, and stronger immune response can not be generated under the condition of not carrying out humanized modification.

(3) The single domain antibody has a wider affinity range, and before affinity maturation is carried out, the affinity range of the single domain antibody can be from a nM level to a pM level, so that multiple choices are provided for later antibodies with different purposes.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is an SDS-PAGE analysis picture of purified MMP9 protein and ZnMc protein expressed by the same company by itself in example 1 of the present invention;

FIG. 2 is a graph showing the results of fragment insertion rate analysis of the constructed nanobody library in example 1 of the present invention;

FIG. 3 is the data of target-specific panning of quality control-compliant libraries in example 2 of the present invention;

FIG. 4 is a SDS-PAGE analysis graph of the purification after expression of nanobody specific to MMP9 protein in example 4 of the present invention;

FIG. 5 shows the result of preliminary antigen affinity identification of the nanobody after obtaining the purified nanobody in example 9 of the present invention;

FIG. 6 is a diagram showing the multiple cloning sites of the fusion antibody expression vector in example 10 of the present invention.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.

Unless otherwise defined, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods or materials similar or equivalent to those described herein can also be used in the present invention.

The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.