CN112239505B

CN112239505B - Method for capturing recombinant anti-CD 171 octavalent antibody and nerve-derived exosome

Info

Publication number: CN112239505B
Application number: CN202011088282.7A
Authority: CN
Inventors: 孙玉龙; 郑昌欣; 杨亚云; 王弢
Original assignee: Shanghai Liangrun Biomedical Technology Co ltd
Current assignee: Shanghai Liangrun Biomedical Technology Co ltd
Priority date: 2020-10-13
Filing date: 2020-10-13
Publication date: 2022-05-20
Anticipated expiration: 2040-10-13
Also published as: WO2022078364A1; CN112239505A

Abstract

The invention relates to a method for capturing a recombinant anti-CD 171 octavalent antibody and a nerve-derived exosome, which comprises two same polypeptide chains, wherein the two polypeptide chains are combined through a covalent bond; the polypeptide chain comprises, in order from one end to the other, the variable regions of two first antibodies, a first linker peptide and the variable regions of two second antibodies; the heavy chain variable region sequence of the first antibody is shown as SEQ ID NO.1, and the light chain variable region sequence of the first antibody is shown as SEQ ID NO. 2; the heavy chain variable region sequence of the second antibody is shown as SEQ ID NO. 3, the light chain variable region sequence of the second antibody is shown as SEQ ID NO. 4, and the first linker peptide comprises a hinge region sequence and a flexible linker sequence connected to one end or both ends of the hinge region sequence. The antibody for capturing the exosome derived from the nerve has the advantages of high specificity, rapidness, stability and the like, can ensure high abundance of the captured exosome, and maintains the specificity of the nerve tissue.

Description

Method for capturing recombinant anti-CD 171 octavalent antibody and nerve-derived exosome

Technical Field

The invention relates to the technical field of biochemical molecules, in particular to a method for capturing a recombinant anti-CD 171 octreous antibody and a nerve-derived exosome.

Background

A nervous system disease represented by Alzheimer Disease (AD) is a neurodegenerative disease commonly seen in the elderly, and its main pathological changes are diffuse atrophy of cerebral cortex, neurofibrillary tangles, formation of a large number of senile plaques between nerve cells, and the like, and progressive cognitive impairment and memory hypofunction are its main clinical symptoms. Dementia is a human problem and is also a global problem. Early diagnosis and timely intervention are the only effective measures at present for delaying the progress of diseases. Early diagnosis of AD relies primarily on clinical history, imaging measures, scale assessment, and detection of CSF (cerebrospinal fluid) markers. The examination in the aspect of PET and other images is not only expensive, but also has high requirements for equipment, and cannot be performed on a large scale in general hospitals. Although the assessment of the mental scale is convenient to detect, the psychological scale is influenced by a plurality of factors, such as the cultural degree of a subject, the subjective judgment of an assessor or the environmental influence. The CSF markers Abeta, Tau and the like are verified to have extremely high specificity and sensitivity, but clinical application is limited because lumbar puncture is invasive and sample specimens are not easy to obtain. Plasma is more readily available than cerebrospinal fluid and is easier to be clinically developed, however, the results of studies on A beta 1-42 (human beta amyloid 1-42) in peripheral blood are different, and the reason for this may be related to the fact that A beta 1-42 in plasma is derived from more than central nervous system.

The detection of plasma nervous system-derived exosome markers facilitates non-invasive early diagnosis of AD. Exosomes are used as an intercellular communication mode, participate in forming a neuron-glial signal network in the central nervous system, brain glial cells penetrate through a blood brain barrier to enter blood in a mode of secreting exosomes, and meanwhile, the exosomes carry a plurality of marker proteins related to AD pathogenesis, such as Tau protein, Abeta and the like. However, the current methods for capturing exosomes still have poor effects and are difficult to perform efficient enrichment.

Disclosure of Invention

Based on this, there is a need for a recombinant anti-CD 171 multivalent antibody that can be used to capture neurosource exosomes with high efficiency.

A recombinant anti-CD 171 octavalent antibody comprising two identical polypeptide chains, wherein the two polypeptide chains are covalently bound to each other; the polypeptide chain comprises, in order from one end to the other, the variable regions of two first antibodies, a first linker peptide and the variable regions of two second antibodies; the variable region comprises a light chain variable region and a heavy chain variable region which are connected through a second linker peptide; the heavy chain variable region sequence of the first antibody is shown as SEQ ID NO.1, and the light chain variable region sequence of the first antibody is shown as SEQ ID NO. 2; the heavy chain variable region sequence of the second antibody is shown as SEQ ID NO. 3, the light chain variable region sequence of the second antibody is shown as SEQ ID NO. 4, and the first linker peptide comprises a hinge region sequence and a flexible linker sequence connected to one end or two ends of the hinge region sequence.

The recombinant anti-CD 171 octavalent antibody is an octavalent antibody which is formed by covalently binding two identical bispecific scFv polypeptide chains and can be combined with the 1 st domain of Ig-like C2 (Ig-like C2-type 1) on the CD171 protein and the 5 th domain of Fibronectin 3 (Fibronectin type-III 5) on the CD171 protein. The recombinant antibody for capturing the nerve-derived exosome has the advantages of high specificity, rapidness, stability and the like, can ensure the high abundance of the captured exosome, and maintains the superstrong nerve tissue specificity. The recombinant anti-CD 171 octavalent antibody provides more powerful support for development of clinical diagnosis of a nerve source marker of an exosome, and can be used for detecting various applications such as early screening, progress monitoring or treatment effect evaluation of nervous system diseases characterized by abnormal changes of biomarkers of the nerve source exosome.

In one embodiment, the hinge region sequence is shown in SEQ ID NO 5 and the flexible linker sequence is (GGGGS)_nAnd n is a natural number of 1 to 6.

In one embodiment, the amino acid sequence of the first linker peptide is shown in SEQ ID NO. 6 and the amino acid sequence of the second linker peptide is shown in SEQ ID NO. 7.

In one embodiment, the amino acid sequence of the polypeptide chain is set forth in SEQ ID NO 8.

The invention also provides application of the recombinant anti-CD 171 eight-valent antibody in preparation of a product for capturing the nerve-derived exosome.

The invention also provides a method for capturing the nerve-derived exosome for non-disease diagnosis and treatment purposes, and the nerve-derived exosome is enriched by utilizing the specific binding reaction of the recombinant anti-CD 171 octavalent antibody and the nerve-derived exosome.

In one embodiment, the method comprises the following steps: coupling the recombinant anti-CD 171 octavalent antibody with a solid phase carrier, mixing and incubating the coupled solid phase carrier with a sample containing the exosomes derived from nerves, allowing the recombinant anti-CD 171 octavalent antibody to be specifically bound with the exosomes derived from nerves, and then separating the solid phase carrier.

In one embodiment, the solid support is selected from the group consisting of magnetic beads and resins, and the sample containing exosomes of neural origin is selected from the group consisting of serum, plasma, blood, urine, saliva and cerebrospinal fluid.

The invention also provides an exosome-capturing kit, which comprises the recombinant anti-CD 171 bivalent antibody, a buffer solution and a solid phase carrier.

The invention also provides an expression vector comprising a gene sequence encoding said polypeptide chain.

In one embodiment, the gene sequence encoding the polypeptide chain is set forth in SEQ ID NO 9.

The invention also provides a host cell containing the expression vector.

In one embodiment, the host cell is a COS cell, a CHO cell, a NS0 cell, an sf9 cell, an sf21 cell, a DH5 a cell, or a BL21 cell.

Drawings

FIG. 1 is a schematic structural diagram of a recombinant anti-CD 171 multivalent antibody according to one embodiment of the present invention;

FIG. 2 is an SDS-PAGE pattern of the recombinant anti-CD 171 octavalent antibody of example 1, which has a molecular weight of about 200 KD;

FIG. 3 is a Westernblot chart of epitope validation of recombinant anti-CD 171 bivalent antibody in example 1, which reacts with both CD171 epitope 1(Ig-like C2-type 1 domain) and CD171 epitope 2 (fibrin type III5 domain) compared to CD171 antibody 1 and CD171 antibody 2, indicating that it has double epitopes;

FIG. 4 is an NTA assay of exosomes captured using recombinant anti-CD 171 octavalent antibody in example 3, showing particle diameters between 58nm and 182 nm;

figure 5 is the capture effect of different capture antibodies on exosomes at WB level in example 3, showing that more exosomes were captured using recombinant anti-CD 171 octavalent antibody compared to CD171 antibody (5G3) and CD63 antibody;

FIG. 6 is a graph correlating the measured concentration of A.beta.42 in paired cerebrospinal fluid and plasma CD171 octavalent antibody capture exosomes of example 4.

Detailed Description

In order that the invention may be more fully understood, a more particular description of the invention will now be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

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. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items. It will be understood that when an element is referred to as being "connected" to another element, it can be directly connected to the other element or intervening elements may also be present.

In order that the disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meaning given below, unless explicitly specified otherwise herein.

Interpretation of terms

An "antibody" is a class of immunoglobulins that specifically bind to an antigen. Antibodies exist as one or more wye-shaped monomers, each of which consists of 4 polypeptide chains, comprising two identical heavy chains and two identical light chains, the light and heavy chains being named according to their molecular weight size. The variable region is located at the top of the Y-shaped structure and is an antigen binding site. Each heavy chain has two regions, a constant region and a variable region, and all antibodies of the same type have the same constant region and differ from one type to another. Each light chain also has two domains, a constant region and a variable region, connected in tandem.

"vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma polyoma vacuolatum viruses (e.g., SV 40).

"host cell" means a cell which can be used for introducing a vector, and includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

"exosomes" refer to small membrane vesicles containing complex RNAs and proteins, which are derived primarily from multivesicular bodies formed by invagination of intracellular lysosomal microparticles, and are released into the extracellular matrix after fusion with the cell membrane via the multivesicular body outer membrane. The function of exosomes depends on the cell type from which they are derived, and they can participate in aspects such as immune response, antigen presentation, cell migration, cell differentiation, tumor invasion, etc.

As shown in fig. 1, the recombinant anti-CD 171 eight-valent antibody according to one embodiment of the present invention comprises two identical polypeptide chains, wherein the two polypeptide chains are bonded to each other through a covalent bond, and the polypeptide chains comprise, from one end to the other end, two variable regions of a first antibody, a first linker peptide, and two variable regions of a second antibody. The variable region comprises a light chain variable region and a heavy chain variable region which are connected by a second linker peptide. The heavy chain variable region (VH1) sequence of the first antibody is shown as SEQ ID NO.1, and the light chain variable region (VL1) sequence of the first antibody is shown as SEQ ID NO. 2; the heavy chain variable region (VH2) sequence of the second antibody is shown in SEQ ID NO:3, the light chain variable region (VL2) sequence of the second antibody is shown in SEQ ID NO:4, and the first linker peptide comprises a hinge region sequence and a flexible linker sequence attached to one or both ends of the hinge region sequence.

In a specific example, the two polypeptide chains of the recombinant anti-CD 171 octavalent antibody described above are bilaterally symmetric.

In a specific example, the variable region comprises a heavy chain variable region and a light chain variable region in sequence from N-terminus to C-terminus, i.e., the polypeptide chain comprises VH1, a second linker peptide, VL1, a second linker peptide, VH1, a second linker peptide, VL1, a first linker peptide, VL2, a second linker peptide, VH2, a second linker peptide, VL2, a second linker peptide, VH2 in sequence from N-terminus to C-terminus.

In one specific example, the hinge region sequence is shown in SEQ ID NO:5 and the flexible linker sequence is (GGGGS)_nAnd n is a natural number of 1 to 6. It is understood that the hinge region sequence and the flexible linker sequence are not limited thereto and may be adjusted as desired.

In one particular example, the first linker peptide may also include part or all of the sequence of the CH2 domain of an immunoglobulin, and the CH2 domain is linked C-terminal to the hinge region sequence.

In a specific example, the amino acid sequence of the first linker peptide is shown as SEQ ID NO. 6 and the amino acid sequence of the second linker peptide is shown as SEQ ID NO. 7. It is to be understood that the specific amino acid sequences of the first and second connecting peptides are not limited thereto, and others may be selected as needed.

In one specific example, the amino acid sequence of the polypeptide chain is shown in SEQ ID NO 8.

In the method for capturing a nerve-derived exosome according to an embodiment of the present invention, the nerve-derived exosome is enriched by using a specific binding reaction between the recombinant anti-CD 171 octavalent antibody and the nerve-derived exosome. It is understood that the method can only isolate the nerve-derived exosomes, does not include the step of detecting and analyzing the markers on the nerve-derived exosomes, and cannot determine whether a patient has a certain disease or health condition without detecting and analyzing the exosome markers, so the method is not a disease diagnosis or treatment method in the meaning of patent law. Furthermore, the captured exosomes may also be used for other purposes, such as drug carriers for the preparation of drugs, etc.

In a specific example, the method for capturing the neural-derived exosomes specifically comprises the following steps: coupling the recombinant anti-CD 171 octavalent antibody with a solid phase carrier, mixing and incubating the coupled recombinant anti-CD 171 octavalent antibody with a sample containing the nerve-derived exosomes to ensure that the recombinant anti-CD 171 octavalent antibody is specifically combined with the nerve-derived exosomes, and then separating the solid phase carrier. It is understood that other methods utilizing antigen-antibody specific binding reactions may be selected as desired, such as biotin-avidin labeling methods, and the like.

In one specific example, the solid support is selected from the group consisting of magnetic beads and resins, and the sample containing exosomes of neural origin is selected from the group consisting of serum, plasma, blood, urine, saliva, and cerebrospinal fluid, but is not limited thereto.

The exosome-capturing kit of an embodiment of the present invention includes the recombinant anti-CD 171 octahedral antibody, a buffer solution, and a solid-phase carrier. Optionally, the buffer comprises phosphate buffer and Gly solution as sample dilution buffer and elution buffer, respectively.

An expression vector according to an embodiment of the present invention contains a gene sequence encoding the polypeptide chain.

In one specific example, the gene sequence encoding the polypeptide chain is shown in SEQ ID NO 9. It will be appreciated that due to codon degeneracy, nucleic acid sequences capable of expressing the same polypeptide may take many forms, and that the above are codon optimized nucleic acid sequences, but are not limited thereto.

The host cell according to an embodiment of the present invention comprises the above expression vector, or has a genome into which a gene sequence encoding the polypeptide chain amino acid is integrated.

In a specific example, the host cell is a COS cell, a CHO cell, a NS0 cell, an sf9 cell, an sf21 cell, a DH5 α cell or a BL21 cell.

The following are specific examples.

Example 1 recombinant anti-CD 171 octreovalent antibody expression

The clone number of a mouse hybridoma cell strain secreting anti-CD 171 protein Ig-like C2-type 1 structural domain is 2C1, the mouse hybridoma cell strain is preserved in China general microbiological culture collection management center (Beijing) 11.15.2019, the address is No. 3 of Xilu No.1 of Beijing Kogyo-oriented region in Beijing, and the preservation number is CGMCC No. 18893. The heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence of the secretory antibody are respectively shown as SEQ ID NO. 3 and SEQ ID NO. 4. The clone number of a mouse hybridoma cell strain secreting the anti-CD 171 protein Fibronectin type-III5 structural domain is 9G12, the mouse hybridoma cell strain is preserved in China general microbiological culture collection management center (Beijing) 11.15 days in 2019, the address is No. 3 of the Xilu No.1 of Beijing Kogyo of the republic of Beijing, and the preservation number is CGMCC No. 18894. The heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence of the secretory antibody are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2. The heavy chain subtype of the above two domain epitope antibodies is IgG 1.

FIG. 1 shows a schematic structural diagram of a recombinant anti-CD 171 octavalent antibody, wherein one polypeptide chain is VH 1-second linker peptide-VL 1-second linker peptide-VH 1-second linker peptide-VL 1 from N-terminus to C-terminus-a first linker peptide-VL 2-a second linker peptide-VH 2-a second linker peptide-VL 2-a second linker peptide-VH 2. Wherein VH1 is the heavy chain variable region sequence of the epitope antibody of the original Fibronectin type-III5 domain, VH2 is the heavy chain variable region sequence of the epitope antibody of the original Ig-like C2-type 1 domain, VL1 is the light chain variable region sequence of the epitope antibody of the original Fibronectin type-III5 domain, and VL2 is the light chain variable region sequence of the epitope antibody of the original Ig-like C2-type 1 domain. The amino acid sequence of the first linker peptide is (GGGGS)₃-VPRDCGCKPCICT-(GGGGS)₃The amino acid sequence of the second linker peptide is (GGGGS)₃. Thus, the amino acid sequence of one polypeptide chain of the recombinant anti-CD 171 eight-valent antibody is shown in SEQ ID NO: 8.

Transiently transfecting an expression vector containing a nucleotide sequence of the recombinant anti-CD 171 bivalent antibody to CHO cells, culturing for several days, and then carrying out affinity purification on cell culture supernatant by using Protein L to obtain the recombinant anti-CD 171 bivalent antibody. The purity of the purified antibody is more than 90% as verified by SDS-PAGE, and the SDS-PAGE picture of the recombinant anti-CD 171 octavalent antibody is shown in FIG. 2. Meanwhile, the recognition capability of the recombinant antibody to the two epitopes is verified by WB through two fragment proteins of Ig-like C2-type 1 and Fibronectin type-III5, and WB (FIG. 3) shows that the recombinant anti-CD 171 octahedral antibody specifically reacts with both Ig-like C2-type 1 and Fibronectin type-III 5.

Example 2 verification of binding ability of recombinant anti-CD 171 octavalent antibody and CD171 protein

Binding of recombinant anti-CD 171 octavalent antibody to CD171 protein was detected by Biacore 3000. After coating the CD171 protein on a Biacore 3000 chip at various concentrations, its affinity was examined. The affinity of the recombinant anti-CD 171 octavalent antibody for CD171 protein was significantly higher than that of the parent antibody, see table 1 in particular.

TABLE 1 affinity of anti-CD 171 octreous antibodies for CD171

Antibody epitopes	Ka(1/Ms)	Kd(1/s)	KD(nM)
				Epitope antibody of original Ig-like C2-type 1 structural domain	3.5×10⁴	2.6×10^-4	7.4
Epitope antibody of original fibrinectin type-III5 structural domain	1.2×10⁴	3.8×10^-4	32
				Recombinant anti-CD 171 bivalent antibody	6.3×10⁵	1.6×10^-5	0.025

Example 3 isolation and characterization of exosomes of neural origin

Mu.g of recombinant anti-CD 171 octavalent antibody and commercially available anti-human CD171 monoclonal antibody (clone No. 5G3, Thermo, recognition epitope is Ig-like C2-type 1 domain), anti-human CD171 monoclonal antibody (clone No. UJ127, Thermo, Fibronectin type III5 domain), anti-human CD63 antibody, normal mouse IgG were coupled to M-270 magnetic beads, respectively. 0.5mL plasma samples were centrifuged at 2000g for 15min, then at 12000g for 30min, then diluted 4-fold with Phosphate Buffered Saline (PBS), spun incubated with a set of antibody-magnetic bead complexes for 12 hours, and then eluted with glycine solution at pH 2.8 to obtain exosomes.

Quantitative detection of exosomes by NS500 nanoparticle analyzer: mu.L of exosome solution was taken and added to 1mL of PBS solution at a ratio of 1:200, and the procedure was followed in the NS500 instrument. Particle size analysis of the nanoparticles was performed with NTA software, and a particle distribution curve was drawn, showing that the particle diameter was between 58nm and 182nm, as shown in FIG. 4.

WB detection of exosome surface marker proteins: and (3) carrying out SDS-PAGE electrophoresis on the exosome sample, then transferring the membrane, incubating the CD171 antibody and the CD81 antibody, and then incubating a secondary antibody to detect the protein marker. And simultaneously, the CD81 protein detection kit and the CD171 protein detection kit are used for respectively detecting the capture effect of different antibodies on exosomes on the ELISA level. Fig. 5 and table 2 show the capture effect of different capture antibodies on exosomes at WB level and ELISA level, respectively, and it can be seen that the recombinant anti-CD 171 octavalent antibody has more efficient capture effect on the neural-derived exosomes.

TABLE 2 exosome CD81 and CD171 protein content

Example 4 detection of the amount of Abeta in neuro-derived exosomes

60 matched samples of plasma and CSF from AD patients, 20 matched samples of plasma and CSF from normal persons, and plasma neuro-derived exosomes were captured using the recombinant anti-CD 171 octavalent antibody of example 1 according to the method of example 3. The marker protein content in the plasma exosome and CSF samples were measured separately using the Α β 42 assay kit. And (4) counting the content distribution of the exosome marker in each group of samples, and analyzing the consistency of the plasma exosome detection result and the CSF detection result. And (3) carrying out statistical analysis on the content distribution of the exosome marker in samples of normal persons and AD patients to obtain cuf-off values, and analyzing the AD detection sensitivity, specificity and overall detection coincidence rate. The results show that: the content of the plasma exosome sample and the content of the cerebrospinal fluid sample Abeta 42 have better correlation, R²Greater than 0.99, as shown in FIG. 6, plasma exosome samples had higher agreement with cerebrospinal fluid samples, AD patientsThe a β 42 content in plasma exosome samples was higher than normal. The AD detection sensitivity, specificity, and overall detection compliance analysis results are shown in table 3. The content of Abeta 42 in the nerve-derived exosome can reflect the brain injury state and can be used as a marker for diagnosing Alzheimer disease and monitoring the disease progression.

TABLE 3 statistical index for the detection of Abeta 42 content in neuro-derived exosomes

	Cutoff value	Sensitivity of the probe	Specificity of	Overall rate of agreement	AUC
						Normal person&AD patients	13.7U/mL	92.5％	91.8％	90.2％	0.907

The additional sequence information is as follows:

SEQ ID NO:1：

EFQLQQSGPELVKPGASVKISCKASGYQFTQYNMNWVKQSNGKSLEWIGVINPNYGTTSYNQKFKGKATLTVDQSSSTAYMQLNSLTSEDSAVYYCARYYAMDYWGQGTSVTVSS

SEQ ID NO:2：

DIVMTQTPASLAVSLGQRATISYRASKSQSTSGQSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWKNGLMLHQLY

SEQ ID NO:3：

EVQLEESGPGLVQPSQSLSITCTVSGQSLTQYGVHWVRQSPGEGLEWLGVIWSGGSTDYDAVFISRLSISKDNSKSQVFFRMNSLQPNDTAIYYCARNWGDGPMDYWGQGTSVTVSSAKTTPPSVY

SEQ ID NO:4：

DIVLTQSPASLAVSLGQRATISCRASQSVQTSSQSYMHWYQQKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLNIHPVEEVDTATYYCQHSWEIPWTFGGGTKLDIKRADAAPTVS

SEQ ID NO:5：

VPRDCGCKPCICT

SEQ ID NO:6：

GGGGSGGGGSGGGGSVPRDCGCKPCICTGGGGSGGGGSGGGGS

SEQ ID NO:7：

GGGGSGGGGSGGGGS

SEQ ID NO:8：

EFQLQQSGPELVKPGASVKISCKASGYQFTQYNMNWVKQSNGKSLEWIGVINPNYGTTSYNQKFKGKATLTVDQSSSTAYMQLNSLTSEDSAVYYCARYYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQTPASLAVSLGQRATISYRASKSQSTSGQSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWKNGLMLHQLYGGGGSGGGGSGGGGSEFQLQQSGPELVKPGASVKISCKASGYQFTQYNMNWVKQSNGKSLEWIGVINPNYGTTSYNQKFKGKATLTVDQSSSTAYMQLNSLTSEDSAVYYCARYYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQTPASLAVSLGQRATISYRASKSQSTSGQSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWKNGLMLHQLYGGGGSGGGGSGGGGSVPRDCGCKPCICTGGGGSGGGGSGGGGSDIVLTQSPASLAVSLGQRATISCRASQSVQTSSQSYMHWYQQKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLNIHPVEEVDTATYYCQHSWEIPWTFGGGTKLDIKRADAAPTVSGGGGSGGGGSGGGGSEVQLEESGPGLVQPSQSLSITCTVSGQSLTQYGVHWVRQSPGEGLEWLGVIWSGGSTDYDAVFISRLSISKDNSKSQVFFRMNSLQPNDTAIYYCARNWGDGPMDYWGQGTSVTVSSAKTTPPSVYGGGGSGGGGSGGGGSDIVLTQSPASLAVSLGQRATISCRASQSVQTSSQSYMHWYQQKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLNIHPVEEVDTATYYCQHSWEIPWTFGGGTKLDIKRADAAPTVSGGGGSGGGGSGGGGSEVQLEESGPGLVQPSQSLSITCTVSGQSLTQYGVHWVRQSPGEGLEWLGVIWSGGSTDYDAVFISRLSISKDNSKSQVFFRMNSLQPNDTAIYYCARNWGDGPMDYWGQGTSVTVSSAKTTPPSVY

SEQ ID NO:9：

GAATTTCAACTACAACAAAGTGGTCCAGAACTGGTCAAGCCCGGCGCCTCAGTGAAGATCTCCTGCAAGGCCTCCGGCTACCAGTTCACCCAGTACAACATGAACTGGGTTAAGCAGAGCAATGGCAAGTCTCTGGAGTGGATCGGCGTGATCAATCCCAACTACGGCACAACTTCCTACAACCAAAAGTTCAAGGGTAAAGCTACACTTACCGTGGACCAGTCCTCCTCCACCGCCTACATGCAGCTGAACTCTCTGACCTCCGAGGACTCCGCTGTGTACTATTGTGCTAGATACTACGCCATGGATTATTGGGGCCAAGGCACCTCCGTGACCGTGTCCAGCGGCGGCGGCGGCAGTGGCGGCGGCGGCTCCGGAGGCGGCGGCTCTGATATAGTGATGACCCAGACCCCTGCTTCTCTGGCTGTTTCCCTGGGTCAGCGGGCCACCATCTCGTACCGGGCCTCCAAGTCCCAGTCTACAAGCGGCCAGTCTTACATGCACTGGAACCAGCAGAAGCCTGGCCAGCCTCCCCGGCTGCTGATCTACCTGGTGTCTAACCTGGAGTCCGGCGTCCCGGCTCGCTTCTCTGGGAGCGGCTCTGGCACCGACTTCACACTGAACATCCACCCCGTGGAGGAAGAGGACGCTGCTACCTACTACTGCCAGCATATCCGTGAACTGACCCGGTCCGAGGGCGGACCTTCTTGGAAGAACGGCCTGATGCTGCACCAGCTGTACGGCGGCGGAGGTAGTGGGGGCGGAGGCAGTGGGGGCGGCGGCTCCGAGTTCCAGCTCCAGCAAAGCGGACCTGAGCTGGTGAAGCCAGGCGCTTCCGTGAAAATCTCCTGCAAGGCTTCTGGCTACCAGTTCACACAGTACAACATGAACTGGGTGAAGCAGTCTAACGGCAAGTCTCTGGAGTGGATCGGCGTGATCAACCCTAACTACGGCACCACCTCCTACAACCAGAAGTTCAAGGGCAAGGCTACCCTGACCGTGGACCAGTCTTCCTCTACCGCTTACATGCAGCTGAACTCTCTGACCTCTGAGGACTCTGCAGTGTACTACTGCGCCCGGTACTACGCCATGGACTACTGGGGCCAGGGCACATCCGTCACGGTGTCATCCGGCGGCGGTGGCTCTGGAGGCGGCGGCAGCGGCGGCGGCGGCTCCGATATCGTGATGACCCAGACCCCCGCTAGCCTGGCTGTGAGCCTGGGCCAGCGGGCTACCATCTCCTACAGAGCTAGCAAATCACAGAGCACCTCTGGCCAGAGCTACATGCACTGGAACCAGCAGAAACCTGGCCAACCACCTAGACTGCTGATCTACCTGGTGAGCAACCTGGAATCTGGCGTGCCTGCTAGATTCTCCGGCTCCGGGTCCGGCACCGATTTCACCCTGAATATCCATCCTGTGGAAGAGGAGGATGCCGCCACCTATTACTGTCAGCACATCAGAGAGCTCACCCGGTCCGAAGGCGGCCCTTCTTGGAAGAACGGCCTGATGCTGCACCAGCTGTACGGAGGAGGCGGCTCTGGCGGCGGCGGCTCTGGAGGCGGTGGCTCCGTGCCTAGAGACTGCGGCTGCAAGCCTTGCATCTGCACCGGCGGAGGCGGCTCCGGCGGCGGGGGATCCGGCGGCGGAGGCAGCGACATCGTGCTGACCCAGAGCCCAGCCTCCCTGGCCGTGTCCCTGGGCCAGAGAGCCACCATCTCCTGCCGGGCCAGCCAGTCCGTGCAAACATCCTCCCAGTCCTACATGCACTGGTACCAGCAGAAGCCAGGACAGCCTCCTAAGCTGCTGATCAAGTACGCCTCCAACCTGGAGTCGGGAGTGCCTGCCCGGTTTTCCGGCTCGGGATCTGGTACCGATTTCACCCTGAACATCCACCCCGTGGAAGAGGTGGACACCGCGACCTACTACTGTCAGCACTCCTGGGAAATCCCTTGGACCTTCGGCGGCGGAACCAAGCTGGACATCAAAAGAGCCGACGCCGCCCCAACCGTGTCTGGGGGCGGCGGAAGCGGCGGCGGTGGCTCCGGCGGCGGCGGGTCAGAGGTGCAGCTGGAGGAATCCGGACCTGGCCTGGTGCAGCCATCCCAGTCCCTGTCCATCACATGTACCGTGTCAGGCCAATCTCTTACTCAGTACGGCGTGCACTGGGTGCGGCAGTCTCCTGGCGAGGGACTGGAGTGGCTGGGCGTGATCTGGTCTGGAGGTTCCACTGACTACGACGCCGTCTTTATCTCCAGACTGTCTATTTCTAAGGACAACAGCAAGTCACAGGTGTTCTTTCGAATGAACTCCTTGCAACCTAACGACACCGCTATCTATTACTGCGCCCGGAACTGGGGCGACGGCCCCATGGACTACTGGGGGCAGGGCACCTCTGTGACCGTATCCTCTGCCAAGACCACACCTCCATCTGTGTACGGCGGAGGCGGCTCCGGGGGCGGCGGCTCCGGTGGCGGCGGATCCGACATCGTGCTGACACAATCCCCCGCTTCCTTGGCCGTGTCCCTGGGCCAGAGAGCTACAATCTCTTGCAGAGCCTCACAATCCGTGCAGACCTCTTCTCAGTCCTACATGCACTGGTATCAGCAAAAACCTGGCCAACCTCCTAAGCTCTTAATCAAGTACGCTTCTAATCTGGAATCTGGTGTGCCTGCCAGATTCTCCGGCTCCGGCTCAGGAACCGACTTCACCCTGAACATCCACCCTGTCGAGGAAGTGGATACCGCCACTTACTACTGCCAGCACAGCTGGGAGATCCCCTGGACCTTTGGAGGGGGCACCAAGCTGGACATCAAGAGAGCCGATGCCGCTCCTACCGTAAGCGGCGGCGGAGGCTCTGGAGGCGGCGGCAGCGGGGGCGGGGGCTCTGAGGTGCAGCTGGAGGAATCCGGCCCCGGCCTGGTGCAGCCTTCCCAGTCTCTGTCTATCACCTGCACCGTGTCTGGCCAGTCACTGACACAGTACGGCGTGCATTGGGTCAGACAGTCCCCTGGAGAAGGTTTAGAGTGGCTGGGCGTGATTTGGTCCGGCGGCAGCACCGACTACGACGCCGTGTTCATCTCCAGACTGAGCATCTCTAAGGACAATTCCAAGTCTCAAGTGTTCTTCCGGATGAACTCCCTGCAGCCTAACGACACCGCCATCTACTATTGTGCCAGAAACTGGGGCGATGGCCCTATGGATTACTGGGGTCAGGGAACATCCGTGACAGTTTCCTCTGCCAAAACCACCCCACCTAGCGTGTAC

the technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

<110> Shanghai Liangrun biomedical science and technology Limited

<120> capture method of recombinant anti-CD 171 octavalent antibody and nerve-derived exosome

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 115

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Glu Phe Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Gln Phe Thr Gln Tyr

20 25 30

Asn Met Asn Trp Val Lys Gln Ser Asn Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Val Ile Asn Pro Asn Tyr Gly Thr Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Gln Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr

100 105 110

Val Ser Ser

115

<210> 2

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Asp Ile Val Met Thr Gln Thr Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser Gln Ser Thr Ser

20 25 30

Gly Gln Ser Tyr Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg

85 90 95

Glu Leu Thr Arg Ser Glu Gly Gly Pro Ser Trp Lys Asn Gly Leu Met

100 105 110

Leu His Gln Leu Tyr

115

<210> 3

<211> 126

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Glu Val Gln Leu Glu Glu Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Gln Ser Leu Thr Gln Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ser Pro Gly Glu Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asp Ala Val Phe Ile

50 55 60

Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Phe

65 70 75 80

Arg Met Asn Ser Leu Gln Pro Asn Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Arg Asn Trp Gly Asp Gly Pro Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr

115 120 125

<210> 4

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Gln Thr Ser

20 25 30

Ser Gln Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Val Asp Thr Ala Thr Tyr Tyr Cys Gln His Ser Trp

85 90 95

Glu Ile Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Asp Ile Lys Arg

100 105 110

Ala Asp Ala Ala Pro Thr Val Ser

115 120

<210> 5

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr

1 5 10

<210> 6

<211> 43

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Val

1 5 10 15

Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Gly Gly Gly Gly

20 25 30

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

35 40

<210> 7

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 8

<211> 1089

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Glu Phe Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Gln Phe Thr Gln Tyr

20 25 30

Asn Met Asn Trp Val Lys Gln Ser Asn Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Val Ile Asn Pro Asn Tyr Gly Thr Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Gln Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr

100 105 110

Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Asp Ile Val Met Thr Gln Thr Pro Ala Ser Leu Ala Val Ser

130 135 140

Leu Gly Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser Gln Ser

145 150 155 160

Thr Ser Gly Gln Ser Tyr Met His Trp Asn Gln Gln Lys Pro Gly Gln

165 170 175

Pro Pro Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val

180 185 190

Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn

195 200 205

Ile His Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His

210 215 220

Ile Arg Glu Leu Thr Arg Ser Glu Gly Gly Pro Ser Trp Lys Asn Gly

225 230 235 240

Leu Met Leu His Gln Leu Tyr Gly Gly Gly Gly Ser Gly Gly Gly Gly

245 250 255

Ser Gly Gly Gly Gly Ser Glu Phe Gln Leu Gln Gln Ser Gly Pro Glu

260 265 270

Leu Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly

275 280 285

Tyr Gln Phe Thr Gln Tyr Asn Met Asn Trp Val Lys Gln Ser Asn Gly

290 295 300

Lys Ser Leu Glu Trp Ile Gly Val Ile Asn Pro Asn Tyr Gly Thr Thr

305 310 315 320

Ser Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Gln

325 330 335

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

340 345 350

Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Ala Met Asp Tyr Trp Gly

355 360 365

Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

370 375 380

Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Thr Pro

385 390 395 400

Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Tyr Arg

405 410 415

Ala Ser Lys Ser Gln Ser Thr Ser Gly Gln Ser Tyr Met His Trp Asn

420 425 430

Gln Gln Lys Pro Gly Gln Pro Pro Arg Leu Leu Ile Tyr Leu Val Ser

435 440 445

Asn Leu Glu Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly

450 455 460

Thr Asp Phe Thr Leu Asn Ile His Pro Val Glu Glu Glu Asp Ala Ala

465 470 475 480

Thr Tyr Tyr Cys Gln His Ile Arg Glu Leu Thr Arg Ser Glu Gly Gly

485 490 495

Pro Ser Trp Lys Asn Gly Leu Met Leu His Gln Leu Tyr Gly Gly Gly

500 505 510

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Val Pro Arg Asp

515 520 525

Cys Gly Cys Lys Pro Cys Ile Cys Thr Gly Gly Gly Gly Ser Gly Gly

530 535 540

Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Leu Thr Gln Ser Pro

545 550 555 560

Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg

565 570 575

Ala Ser Gln Ser Val Gln Thr Ser Ser Gln Ser Tyr Met His Trp Tyr

580 585 590

Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Lys Tyr Ala Ser

595 600 605

Asn Leu Glu Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly

610 615 620

Thr Asp Phe Thr Leu Asn Ile His Pro Val Glu Glu Val Asp Thr Ala

625 630 635 640

Thr Tyr Tyr Cys Gln His Ser Trp Glu Ile Pro Trp Thr Phe Gly Gly

645 650 655

Gly Thr Lys Leu Asp Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser

660 665 670

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

675 680 685

Val Gln Leu Glu Glu Ser Gly Pro Gly Leu Val Gln Pro Ser Gln Ser

690 695 700

Leu Ser Ile Thr Cys Thr Val Ser Gly Gln Ser Leu Thr Gln Tyr Gly

705 710 715 720

Val His Trp Val Arg Gln Ser Pro Gly Glu Gly Leu Glu Trp Leu Gly

725 730 735

Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asp Ala Val Phe Ile Ser

740 745 750

Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Phe Arg

755 760 765

Met Asn Ser Leu Gln Pro Asn Asp Thr Ala Ile Tyr Tyr Cys Ala Arg

770 775 780

Asn Trp Gly Asp Gly Pro Met Asp Tyr Trp Gly Gln Gly Thr Ser Val

785 790 795 800

Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Gly Gly Gly

805 810 815

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Leu

820 825 830

Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Thr

835 840 845

Ile Ser Cys Arg Ala Ser Gln Ser Val Gln Thr Ser Ser Gln Ser Tyr

850 855 860

Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile

865 870 875 880

Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala Arg Phe Ser Gly

885 890 895

Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His Pro Val Glu Glu

900 905 910

Val Asp Thr Ala Thr Tyr Tyr Cys Gln His Ser Trp Glu Ile Pro Trp

915 920 925

Thr Phe Gly Gly Gly Thr Lys Leu Asp Ile Lys Arg Ala Asp Ala Ala

930 935 940

Pro Thr Val Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

945 950 955 960

Gly Gly Ser Glu Val Gln Leu Glu Glu Ser Gly Pro Gly Leu Val Gln

965 970 975

Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Gln Ser Leu

980 985 990

Thr Gln Tyr Gly Val His Trp Val Arg Gln Ser Pro Gly Glu Gly Leu

995 1000 1005

Glu Trp Leu Gly Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asp Ala

1010 1015 1020

Val Phe Ile Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln

1025 1030 1035 1040

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

1045 1050 1055

Tyr Cys Ala Arg Asn Trp Gly Asp Gly Pro Met Asp Tyr Trp Gly Gln

1060 1065 1070

Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val

1075 1080 1085

Tyr

<210> 9

<211> 3267

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gaatttcaac tacaacaaag tggtccagaa ctggtcaagc ccggcgcctc agtgaagatc 60

tcctgcaagg cctccggcta ccagttcacc cagtacaaca tgaactgggt taagcagagc 120

aatggcaagt ctctggagtg gatcggcgtg atcaatccca actacggcac aacttcctac 180

aaccaaaagt tcaagggtaa agctacactt accgtggacc agtcctcctc caccgcctac 240

atgcagctga actctctgac ctccgaggac tccgctgtgt actattgtgc tagatactac 300

gccatggatt attggggcca aggcacctcc gtgaccgtgt ccagcggcgg cggcggcagt 360

ggcggcggcg gctccggagg cggcggctct gatatagtga tgacccagac ccctgcttct 420

ctggctgttt ccctgggtca gcgggccacc atctcgtacc gggcctccaa gtcccagtct 480

acaagcggcc agtcttacat gcactggaac cagcagaagc ctggccagcc tccccggctg 540

ctgatctacc tggtgtctaa cctggagtcc ggcgtcccgg ctcgcttctc tgggagcggc 600

tctggcaccg acttcacact gaacatccac cccgtggagg aagaggacgc tgctacctac 660

tactgccagc atatccgtga actgacccgg tccgagggcg gaccttcttg gaagaacggc 720

ctgatgctgc accagctgta cggcggcgga ggtagtgggg gcggaggcag tgggggcggc 780

ggctccgagt tccagctcca gcaaagcgga cctgagctgg tgaagccagg cgcttccgtg 840

aaaatctcct gcaaggcttc tggctaccag ttcacacagt acaacatgaa ctgggtgaag 900

cagtctaacg gcaagtctct ggagtggatc ggcgtgatca accctaacta cggcaccacc 960

tcctacaacc agaagttcaa gggcaaggct accctgaccg tggaccagtc ttcctctacc 1020

gcttacatgc agctgaactc tctgacctct gaggactctg cagtgtacta ctgcgcccgg 1080

tactacgcca tggactactg gggccagggc acatccgtca cggtgtcatc cggcggcggt 1140

ggctctggag gcggcggcag cggcggcggc ggctccgata tcgtgatgac ccagaccccc 1200

gctagcctgg ctgtgagcct gggccagcgg gctaccatct cctacagagc tagcaaatca 1260

cagagcacct ctggccagag ctacatgcac tggaaccagc agaaacctgg ccaaccacct 1320

agactgctga tctacctggt gagcaacctg gaatctggcg tgcctgctag attctccggc 1380

tccgggtccg gcaccgattt caccctgaat atccatcctg tggaagagga ggatgccgcc 1440

acctattact gtcagcacat cagagagctc acccggtccg aaggcggccc ttcttggaag 1500

aacggcctga tgctgcacca gctgtacgga ggaggcggct ctggcggcgg cggctctgga 1560

ggcggtggct ccgtgcctag agactgcggc tgcaagcctt gcatctgcac cggcggaggc 1620

ggctccggcg gcgggggatc cggcggcgga ggcagcgaca tcgtgctgac ccagagccca 1680

gcctccctgg ccgtgtccct gggccagaga gccaccatct cctgccgggc cagccagtcc 1740

gtgcaaacat cctcccagtc ctacatgcac tggtaccagc agaagccagg acagcctcct 1800

aagctgctga tcaagtacgc ctccaacctg gagtcgggag tgcctgcccg gttttccggc 1860

tcgggatctg gtaccgattt caccctgaac atccaccccg tggaagaggt ggacaccgcg 1920

acctactact gtcagcactc ctgggaaatc ccttggacct tcggcggcgg aaccaagctg 1980

gacatcaaaa gagccgacgc cgccccaacc gtgtctgggg gcggcggaag cggcggcggt 2040

ggctccggcg gcggcgggtc agaggtgcag ctggaggaat ccggacctgg cctggtgcag 2100

ccatcccagt ccctgtccat cacatgtacc gtgtcaggcc aatctcttac tcagtacggc 2160

gtgcactggg tgcggcagtc tcctggcgag ggactggagt ggctgggcgt gatctggtct 2220

ggaggttcca ctgactacga cgccgtcttt atctccagac tgtctatttc taaggacaac 2280

agcaagtcac aggtgttctt tcgaatgaac tccttgcaac ctaacgacac cgctatctat 2340

tactgcgccc ggaactgggg cgacggcccc atggactact gggggcaggg cacctctgtg 2400

accgtatcct ctgccaagac cacacctcca tctgtgtacg gcggaggcgg ctccgggggc 2460

ggcggctccg gtggcggcgg atccgacatc gtgctgacac aatcccccgc ttccttggcc 2520

gtgtccctgg gccagagagc tacaatctct tgcagagcct cacaatccgt gcagacctct 2580

tctcagtcct acatgcactg gtatcagcaa aaacctggcc aacctcctaa gctcttaatc 2640

aagtacgctt ctaatctgga atctggtgtg cctgccagat tctccggctc cggctcagga 2700

accgacttca ccctgaacat ccaccctgtc gaggaagtgg ataccgccac ttactactgc 2760

cagcacagct gggagatccc ctggaccttt ggagggggca ccaagctgga catcaagaga 2820

gccgatgccg ctcctaccgt aagcggcggc ggaggctctg gaggcggcgg cagcgggggc 2880

gggggctctg aggtgcagct ggaggaatcc ggccccggcc tggtgcagcc ttcccagtct 2940

ctgtctatca cctgcaccgt gtctggccag tcactgacac agtacggcgt gcattgggtc 3000

agacagtccc ctggagaagg tttagagtgg ctgggcgtga tttggtccgg cggcagcacc 3060

gactacgacg ccgtgttcat ctccagactg agcatctcta aggacaattc caagtctcaa 3120

gtgttcttcc ggatgaactc cctgcagcct aacgacaccg ccatctacta ttgtgccaga 3180

aactggggcg atggccctat ggattactgg ggtcagggaa catccgtgac agtttcctct 3240

gccaaaacca ccccacctag cgtgtac 3267

Claims

1. A recombinant anti-CD 171 octavalent antibody comprising two identical polypeptide chains; the polypeptide chain comprises two variable regions of a first antibody, a first linker peptide and two variable regions of a second antibody in sequence from N-terminus to C-terminus; the variable region comprises a light chain variable region and a heavy chain variable region which are connected through a second linker peptide; the heavy chain variable region sequence of the first antibody is shown as SEQ ID NO.1, and the light chain variable region sequence of the first antibody is shown as SEQ ID NO. 2; the heavy chain variable region sequence of the second antibody is shown as SEQ ID NO. 3, the light chain variable region sequence of the second antibody is shown as SEQ ID NO. 4, and the first linker peptide comprises a hinge region sequence and a flexible linker sequence connected to one end or two ends of the hinge region sequence; the two polypeptide chains are joined by a covalent bond at the hinge region; the amino acid sequence of the polypeptide chain is shown as SEQ ID NO. 8.

2. Use of the recombinant anti-CD 171 octavalent antibody of claim 1 in the manufacture of a product for capturing CD 171-expressing neurogenic exosomes.

3. A method for capturing neurogenic exosomes for non-disease diagnosis and treatment purposes, wherein the neurogenic exosomes are enriched by using a specific binding reaction of the recombinant anti-CD 171 octavalent antibody of claim 1 with the neurogenic exosomes.

4. The method of capturing of claim 3, comprising the steps of: the recombinant anti-CD 171 octavalent antibody of claim 1 coupled to a solid support, followed by incubation in admixture with a sample containing a neurogenic exosome to allow the recombinant anti-CD 171 octavalent antibody to specifically bind to the neurogenic exosome, followed by isolation of the solid support.

5. The capture method of claim 4, wherein the solid support is selected from magnetic beads or a resin, and the sample containing the exosomes of neural origin is selected from serum, plasma, blood, urine, saliva or cerebrospinal fluid.

6. An exosome-capture kit comprising the recombinant anti-CD 171 octavalent antibody of claim 1, a buffer, and a solid support.

7. An expression vector comprising a gene sequence encoding the polypeptide chain of claim 1.

8. The expression vector of claim 7, wherein the gene sequence encoding the polypeptide chain is set forth in SEQ ID NO 9.

9. A host cell comprising the expression vector of claim 7 or 8.

10. The host cell of claim 9, wherein the host cell is a COS cell, a CHO cell, a NS0 cell, an sf9 cell, an sf21 cell, a DH5 a cell, or a BL21 cell.