SV2 receptor extracellular domain nano antibody and application thereof

Abstract

Neuroendocrine tumors (NETs) are highly heterogeneous tumors derived from neuroendocrine cells. The synaptic vesicle 2 (SV2) protein is a novel marker of NETs and has three subtypes of SV2A, SV2B and SV2C, the SV2C structure is more stable than the other two subtypes, and the maximum cavity ring L4 is the most important binding region with toxins. The nano antibody of the extracellular soluble part L4 of the SV2 is obtained by screening through a phage display technology, the obtained nano antibody is labeled by fluorescein, chemical toxin small molecules or nuclide to prepare a probe molecule, and the result can specifically bind to neuroendocrine tumor cells. The invention provides a nano antibody of an extracellular soluble part L4 of SV2. The nano antibody can be applied to preparation of a preparation for diagnosing and treating neuroendocrine tumors.

Description

Technical Field

[0001] The invention belongs to the field of medical technology and provides a nano antibody of the extracellular soluble part of SV2, which is used in the preparation of a neuroendocrine tumor nuclear imaging probe molecule and a radioactive or chemical toxin small molecule conjugate therapeutic drug targeting neuroendocrine tumors. Background Art

[0002] Radiopharmaceuticals are an important part of nuclear medicine. They are widely used in the diagnosis and treatment of cancer, myocardial imaging and diagnosis of heart diseases, as well as status monitoring of neurodegenerative diseases. Radiopharmaceuticals can be divided into diagnostic radiopharmaceuticals and therapeutic radiopharmaceuticals according to their uses. Diagnostic radiopharmaceuticals can be further divided into single-photon radiopharmaceuticals and positron radiopharmaceuticals. They can be combined with single-photon tomography (SPECT) or positron tomography (PET) to study the function and metabolic process of drugs in vivo at the molecular level, and achieve rapid, non-destructive and real-time imaging of physiological and pathological processes, providing new methods and means for truly early diagnosis and timely treatment, and providing possible ways to achieve preventive medicine, translational medicine and personalized medicine. [1] .

[0003] Neuroendocrine tumors (NETs) consist of a range of malignant tumors that may originate from neuroendocrine cells throughout the body. These tumors are characterized by their ability to produce peptides that lead to characteristic hormonal syndromes. The disease is mainly manifested by excessive secretion of neurotransmitters and hormones, causing related clinical symptoms such as skin flushing, rash, diarrhea, and heart failure. Neuroendocrine tumors occur in many parts of the human body. Neuroendocrine cells can form neuroendocrine tumors in many organs. Among them, female patients are more likely to develop primary tumors in the lungs, stomach, appendix or cecum, while male patients are more likely to develop primary tumors in the thymus, duodenum, pancreas, jejunum / ileum or rectum. The patient's prognosis and mortality rate depend on the degree of differentiation of NETs. The median life expectancy of patients with poorly differentiated malignant tumors is less than 1 year. [2] .

[0004] Neuroendocrine tumors can be divided into two categories based on whether the substances secreted by the tumor can cause typical clinical symptoms: functional and non-functional. Functional neuroendocrine tumors often show corresponding symptoms caused by excessive secretion of tumor-related substances, while non-functional neuroendocrine tumors do not cause corresponding clinical manifestations even if there is an increase in hormone or polypeptide concentrations. Due to the lack of typical clinical manifestations, it is often difficult to detect in the early stages. Patients often come to the hospital for treatment because of the large size of the tumor that can cause a mass in the abdomen or the corresponding complications caused by the tumor. When they come to the hospital for treatment, metastasis has often occurred. Therefore, somatostatin receptor imaging (SRI) is often used clinically to clearly locate the tumor. However, studies have shown that some patients have negative somatostatin receptor imaging, which makes somatostatin receptor imaging have a certain degree of defects. Therefore, more effective NETs nuclear imaging molecular probes are urgently needed in clinical practice to more accurately judge the size of NETs and their metastatic lesions. [3] . Synaptic vesicle 2 (SV2), synaptophysin, and chromogranin A have been shown to be useful clinically as diagnostic markers for NETs. In pituitary adenomas, SV2 immunoreactive cells were more numerous than the other two antibodies compared with the other two markers. In the gastric antrum and pancreas, SV2 and synaptophysin were superior to chromogranin A as NETs markers. Except for the gastric antrum and pancreas, SV2 immunoreactive cells were more numerous than synaptophysin immunoreactive cells in other gastrointestinal and other endocrine organs. In the adrenal medulla, SV2 immunoreactive cells were more numerous than chromogranin A antibody immunoreactive cells. In hindgut carcinoids, SV2 immunoreactive results were superior to synaptophysin, while chromogranin A was negative. [4] Therefore, SV2 is considered a more general marker for NETs.

[0005] SV2 protein is abundantly expressed in NETs tissues and serves as a Ca 2+ Triggered external osmoregulator, with three different isoforms (SV2A / B / C) [5-7] Since exocytosis plays an important role in regulating external messenger interactions and internal signaling, in cancerous tumors, SV2 may promote cellular machinery for antigen processing and presentation to evade the immune system. In cancer, SV2 may also be involved in regulating membrane markers, transdifferentiation and tumor angiogenesis, and inhibiting apoptosis. [8] However, currently only SV2 traditional antibodies are used clinically, which have poor penetration and are only used for in vitro tumor pathology detection and identification, and are not suitable for in vivo imaging molecules.

[0006] Nanobody (Nb) is a special antibody lacking light chain structure. It was first discovered in camelids by Belgian scientist C Hamers-Casterman in 1993, and was later found in some deep-sea fish such as nurse sharks and chimaeras. [9] The nanobody structure contains only one heavy chain variable region and two heavy chain constant regions, which has the advantages of not being easy to stick to each other, stable structure, and high hydrophilicity.

[10] The homology between nanobodies and the heavy chain variable region (VH) of human antibodies can reach 80%.

[11] , low immunogenicity, and a long complementarity determining region (CDR), of which the CDR3 region helps to recognize some antigenic epitopes that conventional antibodies cannot recognize.

[12] More importantly, nanobodies have a small molecular weight and strong penetrating power. Based on these special properties and advantages, nanobodies have been widely studied and applied in the fields of disease diagnosis and treatment, new drug development, etc.

[0007] Phage display technology was first created by Nobel Prize winner George P. Smith in 1985. In this technology, the obtained DNA fragment is connected to the gene encoding the coat protein of the phage by molecular cloning, so that the foreign DNA is expressed on the surface of the phage along with the proliferation of the phage, thereby achieving the effect of obtaining the coding gene through phenotypic screening.

[13] The antibody library constructed using phage display technology has a greatly increased library capacity. At the same time, since phage display technology can be completed during bacterial proliferation, the required cycle is short, which is conducive to the production of antibodies.

[14] Based on the above advantages and characteristics, the purpose of screening the corresponding antibodies of the target target can be achieved by constructing a phage display library, further analyzing the sequence information of the nano-antibodies obtained by the screening, constructing an expression vector and expressing the nano-antibodies.

[0008] The present invention expresses and purifies the extracellular soluble part of the SV2 receptor, uses it as an antigen to immunize alpacas, collects blood, separates lymphocytes to extract total RNA, reversely transcribes it into cDNA, amplifies the nanobody gene by nested PCR, inserts it into phagemid, constructs a nanobody library, and screens nanoantibodies targeting SV2-L4 based on the ELISA method, and finally provides a highly efficient nanoantibody against SV2 as a ligand targeting the SV2 target of neuroendocrine malignancies.

[15] , conventional methods can be used to use radionuclides 68 Ga, 99 Tc, 131 I, 89 Zr,111 In, 90 Y, 18 F etc. to make it a nuclear imaging probe molecule or a nuclear medicine molecule with therapeutic effect; it can also be coupled with chemical toxin small molecules such as auristatin, maytansine and its analogs, calicheamicin, duocarmycins, anthracyclines, pyrrolobenzodiazepine dimers, amatoxin and quinoline alkaloid (SN-38) to make it a nano-antibody chemical toxin molecule conjugate; it can also be labeled with fluorescein to make it a fluorescent imaging probe molecule as a research tool for studying neuroendocrine tumors.

[0009] References

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[0011] [2] Massironi S, Rossi RE, Casazza G, Conte D, Ciafardini C, Galeazzi M, et al. Chromogranin A in diagnosing and monitoring patients with gastroenteropancreatic neuroendocrine neoplasms: a large series from a singleinstitution. Neuroendocrinology. 2014; 100:240-249.

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[0019]

[10] Muyldermans S, Baral TN, Retamozzo VC, et al. Camelidimmunoglobulinsand nanobody technology. Vet Immunol Immunopathol, 2009; 128(1 / 3):178-183.

[0020]

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[0021]

[12] Wu Yue, Hao Xiujing, Li Min. Application of nanobodies in cancer therapy. Chinese Journal of Biotechnology, 2017; 33(7): 1085-1090.

[0022]

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[0023]

[14] Besnerais M, Veyradier A, Benhamou Y, et al. Caplacizumab: a change in the paradigm of thrombotic thrombocytopenic purpura treatment. Expert OpinBiol Ther, 2019; 19(11): 1127-1134.

[0024]

[15] Pardon Els et al. A general protocol for the generation of Nanobodies for structural biology. Nature protocols, 2014; 9(3): 674-693. Summary of the invention

[0025] The purpose of the present invention is to provide a nano antibody of the extracellular soluble part of SV2, which is used in the preparation of a neuroendocrine tumor nuclear imaging probe molecule and a radioactive or chemical toxin small molecule conjugate therapeutic drug targeting neuroendocrine tumors.

[0026] The present invention constructs the pGEX-4T-1-SV2C-L4 recombinant plasmid, wherein the SV2C-L4 gene sequence fragment is synthesized by outsourcing to Genewise Company and inserted into pET-28 a+ The SV2C-L4 gene sequence was Seq ID No: 1 and the protein sequence was Seq ID No: 14. First, the SV2C-L4 gene sequence was isolated from pET-28 by PCR. a+ -SV2C-L4 was amplified, and inserted into the pGEX-4T-1 vector after double enzyme digestion. Further, the recombinant pGEX-4T-1-SV2C-L4 was transformed into E. coli BL21 competent cells according to the heat shock method, coated with LB plates containing 100μg / mL ampicillin, cultured at 37°C overnight, and a single clone was picked in 20mL LB liquid culture medium containing 100μg / mL ampicillin, 37°C, 200r / min, and shaken overnight as a strain. The strain was inoculated into LB medium containing 100μg / mL ampicillin at 1%, cultured at 37°C until the OD600 value was about 0.6, IPTG with a final concentration of 0.4mmol / L was added, and induced at 16°C for 20h. The cells were harvested by centrifugation at 8000r / min for 5min, and 30 times the weight of the cells were added to the Binding Buffer, wherein the formula of the Binding Buffer was: 20mMPB, pH 7.4, 150mM NaCl, and the cells were broken by a homogenizer at 800bar until the bacterial solution was clear, 10% Triton X-100 was added, and the mixture was allowed to stand at 4°C for 30min, and centrifuged at 10000rpm for 10min. The supernatant was purified by GSH affinity chromatography, and the chromatography filler was balanced with 20 column volumes of Binding Buffer. After loading, the sample was washed with Binding Buffer until the UV detector read the baseline, and then eluted in stages with Elution Buffer, and the formula of the elution buffer was: Elution Buffer 1: 50mM Tris-HCl, pH 8.0, 5mM glutathione; Elution Buffer 2: 50mM Tris-HCl, pH 8.0, 10mM glutathione. The target protein is present in the elution component Elution 1. The elution sample 1 is collected and desalted by Sephadex G25 desalting column. The desalting buffer formula is: 50mM NaCl, 20mM PB, pH 7.4. After desalting, 5% glycerol is added to the collected protein, which is quickly frozen with liquid nitrogen and stored in a -80°C refrigerator.

[0027] The purified GST-SV2C-L4 protein was used as an antigen and placed in dry ice and sent to Shanxi Nami Biotechnology Development Co., Ltd. for outsourced alpaca immunization. After four immunizations and the alpaca antiserum quality test was qualified, alpaca blood was collected to separate lymphocytes, and total RNA was extracted from the returned alpaca lymphocytes. After RNA reverse transcription, cDNA amplification and PCR, pMES4-VHH plasmid was constructed, and after electroporation, GST-SV2C-L4 nanobody phage library was established. Then the nanobody phage library was screened by ELISA using the phage display method, and the coated target protein was SV2C-L4. Finally, after nano-antibody binding ability screening, nano-antibodies with strong binding ability were selected and outsourced to GeneWeichi for sequencing, and 12 nano-antibody gene sequences with different sequences were obtained, numbered: 1-A4, 1-A5, 1-A10, 1-C6, 1-F8, 1-H9, 2-A2, 2-A5, 2-C4, 2-F1, 2-G11, and 2-H4.

[0028] The SV2C-L4 nanoantibody strain obtained by screening was inoculated into LB medium at 1%, cultured at 37°C until the OD600 value was about 0.6, IPTG was added to a final concentration of 0.5mmol / L, induced at 20°C for 16h, and the bacteria were collected. Binding Buffer 20 times its weight was added to the bacteria, the bacteria were broken by a homogenizer, and centrifuged at 10000rpm for 10min. The supernatant was purified by Ni column (20 times column volume Binding Buffer balance, Binding Buffer: 20mM PB, pH 7.4, 20mM NaCl, 100mM imidazole; Elution Buffer: 20mM PB, pH7.4, 100mM NaCl, 500mM imidazole). The target protein is present in the Elution sample.

[0029] The purified protein was dialyzed, and the buffer was replaced with 0.1M NaHCO3 solution. According to the mass ratio of 10-200μg Super Flour SE per mg IgG antibody, SuperFlour 680SE dye solution was slowly added to 1mL protein solution for labeling, and slowly stirred at room temperature in the dark for 1 hour. The labeled protein solution was purified by Sephadex G25 column, eluted with PBS solution, and the eluted labeled protein solution was collected according to the reading of the UV detector.

[0030] The labeled nanoantibody solution was co-incubated with Neuro-2a cells to verify its cell targeting. Most of the labeled proteins bound to the receptors on the Neuro-2a cell membrane, so red fluorescence could be observed on the cell membrane. However, there were no receptors bound by nanoantibodies on the negative control Hela cells, so no fluorescence was observed.

[0031] The SV2 extracellular soluble part nano antibody provided by the present invention is an antibody naturally lacking light chains existing in the peripheral blood of alpacas, and has the advantages of small molecular weight, being able to penetrate the blood-brain barrier, being able to be highly expressed by using Escherichia coli, having strong specificity and high affinity, and having weak immunogenicity to humans.

[0032] In addition, different nuclides can be used to label the extracellular soluble part of SV2 nanobodies. The nuclide-labeled protein can become a nuclear imaging probe molecule or a nuclear medicine molecule with therapeutic effects, which can be understood by technicians in the same field. Generally, 89 Zr, 68 Ga, 99 Tc, 111 In, 90 Nuclides such as Y are used to label the extracellular soluble part of SV2 with nanobodies through bifunctional chelators such as DOPA, DTPA, and HYNIC; 18 F by first synthesizing 18 F precursor molecules are used to label the extracellular soluble part of SV2 with nanobodies; 131 I The SV2 extracellular soluble fraction was labeled with nanobodies using the chloramine T method.

[0033] The toxin molecules are coupled to the SV2 extracellular soluble part nanobody by conventional chemical methods. The SV2 extracellular soluble part nanobody coupled to the toxin small molecule can become a nuclear medicine molecule with therapeutic effect, which can be understood by technicians in the same field. Such toxin small molecules include: microtubule generation inhibitors such as auristatin, maytansine and its analogs; DNA damaging factors such as calicheamicin, duocarmycins, anthracyclines, pyrrolobenzodiazepine dimers; DNA transcription inhibitors such as amatoxin and quinoline alkaloid (SN-38). BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 .pGEX-4T-1-SV2C-L4 protein induction results, M in the figure is Protein Ladder, Lane 1 is the uninduced sample, and Lane 2 is the induced sample;

[0035] Figure 2.The results of pGEX-4T-1-SV2C-L4 protein purification. In the figure, M is Protein Ladder. Lane 1 is the precipitate sample after centrifugation of the lysis solution. Lane 2 is the pre-column sample of the supernatant of the lysis solution. Lane 3 is the chromatography breakthrough sample. Lane 4 is the Wash sample. Lane 5 is the Elution 1 sample. Lane 6 is the Elution 2 sample.

[0036] Figure 3 .ELISA affinity test results of the screened nanobodies;

[0037] Figure 4 .Comparison results of the selected nanobody protein sequences;

[0038] Figure 5 The purification results of the nanobodies obtained by screening, lanes 1-13 are nanobodies bands numbered 1-A4, 1-A5, 1-A10, 2-A5, 1-H9, 2-F1, 1-C6, 1-F8, 2-A2, 2-C4, 2-H4, 2-G11, and 2-A5, respectively;

[0039] Figure 6 .SF-680SE-labeled nanoantibodies bind to Neura-2a cells. Specific embodiments

[0040] Example 1 Expression and purification of pGEX-4T-1-SV2C-L4 protein

[0041] 1.1 Materials

[0042] Gold PCR Master Mix high-fidelity enzyme premix (Shanghai Yisheng Biotechnology Co., Ltd., catalog number 10149ES03), agarose (Shanghai Yisheng Biotechnology Co., Ltd., catalog number 10208ES60), low melting point agarose (Shanghai Yisheng Biotechnology Co., Ltd., catalog number 10214ES60), nucleic acid dye (Shanghai Yisheng Biotechnology Co., Ltd., catalog number 10202ES76), 1kb DNA ladder (Shanghai Yisheng Biotechnology Co., Ltd., catalog number 10510-A), 6×DNA loading buffer (Shanghai Yisheng Biotechnology Co., Ltd., catalog number 10213ES08), SpeedyCut BamHI fast-cutting enzyme, EcoRI endonuclease, and NdeⅠ endonuclease were purchased from Shanghai Bioengineering Co., Ltd. SanPrep column DNA gel recovery kit (Sangon Bioengineering Co., Ltd., catalog number B518131), SanPrep column plasmid DNA small-scale extraction kit (Sangon Bioengineering Co., Ltd., catalog number B518191), ampicillin sodium (Sangon Bioengineering Co., Ltd., catalog number A610028-0025), kanamycin sulfate (Sangon Bioengineering Co., Ltd., catalog number A506636-0025), IPTG (Sangon Bioengineering Co., Ltd., catalog number A600168-0025), imidazole (Sangon Bioengineering Co., Ltd., catalog number A600277-0025), and 1,2-dihydropyrrolidone (1,6-dihydropyrrolidone) ... 0100), SDS (Sangon Biotechnology Co., Ltd., Catalog No. A600485-0100), Glycine (Sangon Biotechnology Co., Ltd., Catalog No. A610235-0005), Tris (Sangon Biotechnology Co., Ltd., Catalog No. A610195-0005), Coomassie Brilliant Blue R-250 (Sangon Biotechnology Co., Ltd., Catalog No. A610037), Sephadex G-25 Resin chromatography medium (Sangon Biotechnology Co., Ltd., Catalog No. C510060-0026), BL21 (DE3) Escherichia coli competent cells (Sangon Biotechnology Co., Ltd., Catalog No. B528414), GST-Sefinose (TM) Resin (Settled Resin (Sangon Biotechnology Co., Ltd., Catalog No. C600031-0005), T4 DNA Ligase (Beijing Bio-Technology Co., Ltd., Catalog No. 2011A), Triton X-100, agar powder (Beijing Solebold Technology Co., Ltd., Catalog No. A8190), TEMED (Beijing Solebold Technology Co., Ltd., Catalog No. T8090), bromophenol blue (Beijing Solebold Technology Co., Ltd., Catalog No. B8120), sodium chloride, sodium hydroxide, sodium dihydrogen phosphate, sodium hydrogen phosphate, methanol, glacial acetic acid, anhydrous ethanol were purchased from Tianjin Best Chemical Co., Ltd., tryptone (Thermo Fisher Scientific,No. LP0042B), yeast extract (Thermo Fisher Scientific, Catalog No. LP0021), ammonium persulfate (Beijing Jinming Biotechnology Co., Ltd., Catalog No. 7727-54-0), acrylamide (Beijing Jinming Biotechnology Co., Ltd., Catalog No. 79-06-1), N,N-methylenebisacrylamide (Beijing Jinming Biotechnology Co., Ltd., Catalog No. 110-26-9), M5 HiClear Prestained Plus Protein Ladder (10-250kDa) (Beijing Juhemei Biotechnology Co., Ltd., Catalog No. MF028-plus-01), Ni Sepharose, TM 6FastFlow (GE Healthcare, cat. no. 17-5318-01).

[0043] 1.2 Implementation steps

[0044] 1.2.1 Construction of pGEX-4T-1-SV2C-L4 plasmid

[0045] The SV2C-L4 gene sequence fragment was synthesized by GeneWeiZ and inserted into pET-28 a+ The plasmid was sequenced and checked for quality. The SV2C-L4 gene sequence was Seq ID No: 1 and the protein sequence was Seq ID No: 14. Upstream and downstream PCR primers were designed, SV2C-F: CGCGGATCCTTTCCGGATGTGATTAAACCGC; SV2C-R:

[0046] CCGGAATTCTTACGCGCTATAATCGTCATCAAAGG, using these two primers from pET-28 a+ -SV2C-L4 plasmid to amplify SV2C-L4 sequence. PCR system was added according to Table 1, and PCR program was according to Table 2.

[0047] Table 1 SV2C-L4 sequence PCR system

[0048]

[0049] Table 2 SV2C-L4 sequence PCR program

[0050]

[0051] According to the instructions of the supplier, the PCR product was double-digested with EcoR I and BamH I, and the double-digested product fragment was obtained by gel recovery and purification. Then, the double-digested PCR product was ligated with the pGEX-4T-1 vector digested with the same two enzymes at a molar ratio of vector: fragment = 1:10 at 16°C overnight to obtain the recombinant plasmid pGEX-4T-1-SV2C-L4.

[0052] The ligation product was transformed into competent E. coli BL21 by conventional heat shock transformation method, spread on LB plate containing 100 μg / mL ampicillin, cultured at 37°C overnight, picked the positive clone grown on the plate, extracted the plasmid using a small plasmid extraction kit according to the kit instructions, sent for testing, selected the clone whose target gene sequence was consistent with the theoretical sequence, picked the single clone and inoculated it into 20 mL LB liquid culture medium containing 100 μg / mL ampicillin, cultured at 37°C overnight, and preserved the strain in 20% glycerol in a -80°C ultra-low temperature refrigerator.

[0053] 1.2.2 GST-SV2C-L4 protein expression

[0054] Take out the strain from the -80℃ ultra-low temperature freezer, inoculate 5μL into 20mL LB liquid culture medium containing 100μg / mL ampicillin, and culture at 37℃ overnight as the strain. Inoculate 1% of the BL21 strain carrying the above-mentioned recombinant plasmid pGEX-4T-1-SV2C-L4 into LB culture medium containing 100μg / mL ampicillin, and culture at 37℃ until the OD600 value is about 0.6. Take the uninduced sample, add IPTG to a final concentration of 0.4mmol / L, induce at 16℃ for 20h, and take the induced sample. After centrifugation, prepare the sample for SDS-PAGE electrophoresis to detect the expression of GST-SV2C-L4 protein. Results are shown in Figure 1 The induced sample had an obvious induced band at about 42 kDa, which was consistent with the theoretical size of GST-SV2C-L4.

[0055] 1.2.3 GST-SV2C-L4 protein purification

[0056] Add 30 times the weight of Binding Buffer to the bacteria, use a homogenizer at 800 bar to break the bacteria until the bacterial solution is clear, add 10% Triton X-100, let stand at 4°C for 30 minutes, and centrifuge at 10000rpm for 10 minutes. Take the supernatant and purify it through a GSH affinity chromatography column, equilibrate with 20 column volumes of Binding Buffer, Binding Buffer: 20mM PB, pH 7.4, 150mMNaCl; elution buffer gradient elution, Elution Buffer 1: 50mMTris-HCl, pH 8.0, 5mM glutathione; Elution Buffer 2: 50mM Tris-HCl, pH 8.0, 10mM glutathione. The target protein is mainly present in Elution 1, and the results of SDS-PAGE electrophoresis are shown in Figure 2 The collected samples were desalted by desalting column (desalting buffer: 50 mM NaCl; 20 mM PB, pH 7.4). After desalting, 5% glycerol was added to the collected target protein, which was quickly frozen with liquid nitrogen and stored in a -80°C refrigerator.

[0057] Example 2 Establishment of SV2C-L4 Nanobody Phage Library

[0058] 2.1 Materials

[0059] Stripwell TM Microplate (COSTAR, Catalog No. 42592), Tween 20 purchased from Tianjin Bailunsi Biotechnology Co., Ltd., Total RNA Extraction Reagent (Shanghai Yisheng Biotechnology Co., Ltd., Catalog No. 10606ES60), AEBSF (Beijing Solaibao Technology Co., Ltd., Catalog No. IA0110), (NEB, Product No. R3140V), (NEB Company, Catalog No. R3162V), pMES4 phagemid vector (Fenghui Biotechnology Co., Ltd., Catalog No. ZT642), TG1 Escherichia coli cell line (Rui Chu Biotechnology Co., Ltd., Catalog No. R09003), SanPrep column PCR product purification kit (Sangon Biotechnology Co., Ltd., Catalog No. B518141-0100).

[0060] 2.2 Implementation steps

[0061] 2.2.1 Extraction of total RNA from alpaca immune lymphocytes

[0062] The purified GST-SV2C-L4 protein was used as an antigen and placed in dry ice and sent to Shanxi Nami Biotechnology Development Co., Ltd. After the antigen was emulsified with adjuvant, the alpaca was immunized. After 4 immunizations, the alpaca antiserum quality test was qualified and the titer reached 10 5 At 10 pm, alpaca blood was collected and Ficoll was used to separate lymphocytes. Alpaca immune lymphocytes were collected and packaged, and each tube contained about 5×10 7 Extract total RNA as follows:

[0063] 1. Every 5×10 6 -1×10 7 Add 1 mL of Total RNA Extraction Reagent to each cell and pipette repeatedly.

[0064] 2. Shake the homogenate sample vigorously and place it at room temperature for 5 minutes to allow complete dissociation of ribosomes.

[0065] 3. Add 1 / 5 volume of chloroform to the above lysate (e.g., add 0.2 mL of chloroform to every 1 mL of Total RNA Extraction Reagent). Cover the centrifuge tube tightly, shake vigorously for 15 seconds, and let stand at room temperature for 2-3 minutes.

[0066] Centrifuge at 4.4°C, 12,000 g for 10-15 min.

[0067] After centrifugation, the mixture can be divided into three layers: the upper colorless water layer, the middle layer, and the lower red organic phenol chloroform layer. RNA exists in the water layer, and the upper layer volume is about 50-60% of the total amount of Total RNA Extraction Reagent added.

[0068] 5. Carefully pipette the upper aqueous phase into a new centrifuge tube, add 1 / 2 volume of isopropanol, invert to mix, and place at room temperature for 10 minutes.

[0069] Centrifuge at 6.4°C, 12000g for 10-15 min.

[0070] 7. Carefully discard the supernatant and add an equal volume of 75% ethanol (prepared with DEPC water, i.e., add 1 mL of 75% ethanol to every 1 mL of Total RNA Extraction Reagent). Vortex to wash thoroughly and flick the bottom of the tube to suspend the precipitate.

[0071] Centrifuge at 8.4°C, 7500g for 5 min, discard the supernatant, and carefully aspirate dry to prevent loss of RNA precipitate.

[0072] 9. Repeat steps 7 and 8 twice, i.e. wash the precipitate twice.

[0073] 10. Leave to air dry at room temperature for 10 minutes. Add 20 μL of RNase-free water to dissolve the RNA. After it is completely dissolved, take a small amount for testing and store the rest of the solution at -70°C.

[0074] 11. RNA integrity test: Take 2μL RNA and add 2μL 2× RNA loading buffer, mix well. Perform electrophoresis test. (If three clear bands appear, it proves that the RNA integrity is good)

[0075] 12. RNA purity test: Detect OD values ​​at 260nm and 280nm and calculate the A260 / A280 ratio. The ratio of pure RNA should be around 2.0.

[0076] 2.2.2 RNA reverse transcription

[0077] The reverse transcription system was added with reactants according to Table 3, and the reverse transcription procedure was according to Table 4.

[0078] Table 3 Reverse transcription reaction system

[0079]

[0080] Table 4 Reverse transcription procedure

[0081] temperature time 25℃ 5min 55℃ 15min 85℃ 5min

[0082] 2.2.3 cDNA amplification

[0083] The amplification primer sequences used were

[13] :

[0084] CALL001 Primer:GTCCTGGCTGCTCTTCTACAAGG;

[0085] CALL002 Primer:GGTACGTGCTGTTGAACTGTTCC.

[0086] The cDNA amplification system was added with reactants according to Table 5, and the cDNA amplification program was according to Table 6.

[0087] Table 5 cDNA amplification system

[0088]

[0089] Table 6 cDNA amplification procedure

[0090]

[0091] 2.2.4PCR-Step 2

[0092] The cDNA amplification product was recovered by gel recovery according to the instructions of the gel recovery kit, and the recovered product was used for PCR-Step 2. The primer sequences used were: VHH-Back:

[0093] GATGTGCAGCTGCAGGAGTCTGGRGGAGG; VHH-For:CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT.

[0094] The PCR-Step 2 system was added with reactants according to Table 7, and the PCR-Step 2 program was according to Table 8.

[0095] Table 7 PCR-Step 2 System

[0096]

[0097]

[0098] Table 8 PCR-Step 2 Program

[0099]

[0100] 2.2.5 Phage plasmid construction

[0101] The PCR-Step 2 amplification product was recovered by gel according to the instructions of the gel recovery kit, and the recovered product, namely the VHH fragment and the pMES4 vector, was subjected to double enzyme digestion.

[0102] Add the reactants according to Table 9 for the double enzyme digestion system of VHH fragments and according to Table 10 for the double enzyme digestion system of pMES4 vectors

[0103] Table 9 VHH fragment double enzyme digestion system

[0104]

[0105] Table 10 pMES4 vector double restriction enzyme digestion system

[0106]

[0107]

[0108] After digestion at 37°C for 1 h, digestion was continued at 4°C overnight.

[0109] The double-digested VHH fragment was ligated to the pMES4 vector at a molar ratio of 3:1. The pMES4-VHH ligation system was added with reactants according to Table 11.

[0110] Table 11 pMES4-VHH ligation system

[0111] Reactants volume pMES4 (600ng) 23μL VHH fragment (200ng) 7μL T4 Ligase 5μL 10×T4 DNA Ligase Buffer 5μL <![CDATA[ddH2O]]> 10μL Total volume 50μL

[0112] Connect overnight at 16℃. The connection product was purified using the SanPrep column PCR product purification kit according to the instructions. 30μL sterile water was used for elution, and mixed with TG1 competent cells for electroporation. The electroporation conditions were: GeminniX2 electroporator, 1cm electroporation cup, voltage 1.8KV, 25μFD and 200 ohms. After electroporation, the cells were added with 1mL SOC culture medium, shaken at 37℃ for 1h, diluted 1000 times, and coated with LB plates containing 100μg / mL ampicillin and 2% glucose, and cultured at 37℃ overnight.

[0113] 15 single clones were randomly selected and colony PCR was performed to verify their insertion rate, which should be above 75% in theory. The insertion rate of the present invention reached 93.3%. The primer sequences used for PCR verification were: MP57: TTATGCTTCCGGCTCGTATG; GIII: CCACAGACAGCCCTCATAG.

[0114] The colony PCR system contains reactants according to Table 12, and the colony PCR program is according to Table 13.

[0115] Table 12 Colony PCR system

[0116]

[0117]

[0118] Table 13 Colony PCR program

[0119]

[0120] The constructed pMES4-VHHs plasmid was verified to be qualified by the above insertion rate detection, and then multiple rounds of electroporation were performed to construct a phage display library.

[0121] In the first round of electroporation, 2 μL of plasmid (14.91 ng / μL) and 25 μL of competent cells were used. After 1000-fold dilution, only 81 clones grew, and the calculated transformation efficiency was about 2.7×10 6 cfu / μg.

[0122] In the second round of electroporation, the electroporation conditions were the same as the first round. After dilution 1000 times, 114 clones grew, and the calculated transformation efficiency was about 3.82×10 6 cfu / μg.

[0123] In the third round of electroporation, the electroporation conditions were the same as above. After dilution 1000 times, 247 clones grew, and the calculated transformation efficiency was about 8.28×10 6cfu / μg.

[0124] The library was built through three rounds of electroporation. According to the minimum requirements in the literature, at least 10 7 A separate transformant library meets the requirements for library construction.

[0125] Example 3 Expansion and screening of SV2C-L4 nanobody library

[0126] 3.1 Materials

[0127] Bovine serum albumin V (Beijing Solebow Technology Co., Ltd., Catalog No. A8020), high-efficiency RIPA tissue / cell rapid lysis buffer (Beijing Solebow Technology Co., Ltd., Catalog No. R0010), TMB single-component colorimetric solution (Beijing Solebow Technology Co., Ltd., Catalog No. PR1200), 1kD dialysis bag (Beijing Solebow Technology Co., Ltd., Catalog No. YA1047), Anti-His6 TagRabbit pAb (Sevier Biotechnology Co., Ltd., Catalog No. GB111251), HRP-Goat Anti Rabbit IgG (H+L) (Immuno Way, Catalog No. RS0002), Bovine Thrombin (Shanghai Yishen Biotechnology Co., Ltd., Catalog No. 20402ES03).

[0128] 3.2 Experimental procedures

[0129] The total number of E. coli cells in the library constructed by the present invention was counted to be 4.8×10 9 cells.

[0130] 3.2.1 Amplification of phage library:

[0131] Inoculation: Take 50 mL of 2×YT to inoculate 100 μL of the total library, add 100 μg / mL ampicillin and 2% glucose solution, and culture at 37°C, 200 rpm until the OD600 value is about 0.5.

[0132] Add 20 mL of the above bacterial solution to a 50 mL centrifuge tube and add 50 μL of 4 × 10 10 pfu helper phage VCSM13, stand at 37℃ for 30min. Centrifuge at 2800g, 20℃ for 10min. Discard the supernatant, add 100mL 2×YT culture medium to resuspend, add ampicillin at a final concentration of 100μg / mL, 25μL kanamycin, and culture at 37℃, 200rpm overnight.

[0133] 3.2.2 Enrichment of phage library:

[0134] 1. Centrifuge the overnight culture at 5000g, 4°C for 15 min.

[0135] 2. Take the supernatant to a new centrifuge tube, add 10 mL of pre-cooled PEG6000, turn it upside down 5 times, mix well, and place it in an ice bath for 30 minutes.

[0136] 3. Centrifuge at 3200g, 4℃ for 10min, discard the supernatant, resuspend each tube with 1mL of ice-cold 1×PBS, transfer to a 1.5mL centrifuge tube, and centrifuge at 20000g, 4℃ for 1min.

[0137] 4. Transfer the supernatant to a new 1.5 mL centrifuge tube, add 250 μL PEG6000, invert and mix 10 times, and place on ice for 10 minutes.

[0138] Centrifuge at 5.4°C, 20,000 g for 15 min, discard the supernatant, aspirate clean, and resuspend in 1 mL PBS.

[0139] Centrifuge at 6.4°C, 20,000 g for 1 min and transfer the supernatant to a 1.5 mL centrifuge tube to obtain phage concentrate.

[0140] 3.2.3 Phage concentrate titer determination:

[0141] TG1 was inoculated into LB medium and cultured until the OD600 value was 0.5.

[0142] Prepare 15 200 μL tubes, add 90 μL 1× PBS, add 10 μL of the prepared phage concentrate to the first tube, mix well, take out 10 μL, add to the next tube, and dilute in sequence. Take samples from the last 8 tubes.

[0143] Prepare 8 200μL tubes, add 90μL TG1, and then add the samples of the last 8 tubes diluted in the previous step into these 8 tubes one by one, incubate at 37℃ for 15min, then divide 9 areas on 100μg / mL ampicillin and 2% glucose LB plates, add 5μL to each area, and culture at 37℃ overnight.

[0144] The titer of the phage concentrate was 10 12 And above meet the screening standards.

[0145] 3.2.4SV2C-L4 protein coating

[0146] 50 U of thrombin was added to each 1 mL of the GST-SV2C-L4 antigen protein purified previously, and the protein was digested at 25°C for 4 h. The GST tag was removed by GSH affinity chromatography to obtain the untagged SV2C-L4 protein in the penetration peak.

[0147] The SV2C-L4 protein with the GST tag removed was diluted to 6 μg / μL with PBS, 100 μL of 1×PBS was added to the control wells, and 100 μL of the diluted protein was added to the experimental wells. The enzyme labeling strip was covered and stored at 4°C overnight.

[0148] 3.2.5 Screening

[0149] The coated plate was washed three times with 250 μL of PBST per well, and then 250 μL of 5% BSA was added to each well at 700 rpm for 2 h for blocking.

[0150] Wash 5 times with 250μL PBST, then add 100μL of incubated phage to each of the control well and the experimental well. Shake at 700rpm for 2h, and wash 15 times with 250μL PBST. Phage incubation: 50μL phage concentrate, 50μL 5% BSA, 400μL PBS, shake at 700rpm for 0.5h.

[0151] 0.25% trypsin was diluted 10 times before use, 100 μL was added to each well, and shaken at 700 rpm for 0.5 h.

[0152] Prepare four 200 μL tubes, pre-add 5 μL AEBSF to each, transfer the samples from the control wells and experimental wells to the prepared tubes and mark them clearly.

[0153] The titer of the phage obtained after trypsin digestion was determined. The titer of the phage in the experimental group and the control group was determined according to the above-mentioned method for determining the titer of the phage concentrate.

[0154] While measuring the titer, prepare three 50mL centrifuge tubes, add 3mL of TG1 with an OD600 value of 0.5 to each tube, take 50μL of each of the three experimental group samples, add them to the corresponding tubes, and let them stand at 37℃ for 30min. Make up to 10mL with LB, add 10μL of 100μg / mL ampicillin and 2% glucose, culture at 200rpm, 37℃ overnight, and add 20% glycerol to store the strains obtained in the first round of screening at -80℃.

[0155] The strains screened in the previous round of experimental group were used for the second round of screening, and so on, until the screening results reached that the experimental group should be 2 orders of magnitude higher than the control group. After three rounds of screening, the phage sub-library with strong affinity to SV2C-L4 was obtained from 1# to 3#.

[0156] Example 4 Nanobody Binding Ability Screening

[0157] 4.1 Preparation of nanobodies

[0158] 1. Coating board

[0159] Take 10 μL of the phage sub-library obtained above, add 90 μL of LB culture medium, and dilute them to 10 -4 and 10 -5 , spread on LB plates containing 100 μg / mL ampicillin and 2% glucose, and culture at 37°C overnight.

[0160] 2. Make motherboard

[0161] Take a 96-well plate, add 100 μL of culture medium (2×YT, 100 μg / mL ampicillin, 2% glucose, 10% glycerol) to each well, pick a single clone from the above plate, add it to each well in turn, and keep 4 wells as controls to detect contamination. Seal the multi-well plate with sealing film and culture overnight at 37°C, 200 rpm.

[0162] 3. Induction

[0163] Add 1 mL of culture medium (2×YT, 100 μg / mL ampicillin, 2% glucose) to a 1.5 mL EP tube, add 10 μL of bacterial solution from the corresponding well on the mother plate to each tube, and culture at 37° C., 200 rpm for 6 h.

[0164] Add 1 μL 1M IPTG to each tube and culture overnight at 37°C, 200 rpm.

[0165] Centrifuge at 3200g, 20℃ for 10 min, discard the supernatant, add 250μL RIPA lysis buffer (100μL RIPA + 1μL PMSF) to each tube, shake to mix, place on ice, and shake for 1h.

[0166] Centrifuge at 10000 rpm, 4°C for 5 min.

[0167] Take 200 μL of supernatant and place it in a new 1.5 mL EP tube to obtain the nanobodies.

[0168] 4.2 ELISA test:

[0169] 1. Coat with SV2C-L4 protein at a concentration of 0.5 μg / mL (diluted with PBS) at 4°C overnight.

[0170] 2. Wash each well with 250 μL PBST for 5 times, add 250 μL 5% BSA, 700 rpm, and block for 2 hours;

[0171] 3. After blocking, add 250 μL PBST to each well and wash 5 times. Add 50 μL buffer (5% BSA: PBST = 1:20) to each well, add 50 μL corresponding nanobody to each well of the experimental well, and add 50 μL RIPA buffer (PMSF: RIPA = 1:100) to the control well. Shake at 700 rpm for 1 hour.

[0172] 4. Wash each well 5 times with 250 μL PBST, add 100 μL buffer (5% BSA:PBST=1:20) to the control wells without primary antibody, and add 100 μL Anti-His6 Tag Rabbit pAb primary antibody to the remaining wells. The primary antibody is diluted 1:2000 with buffer, and shake at 700 rpm for 1 hour.

[0173] 5. Wash each well 5 times with 250 μL PBST, add 100 μL buffer (5% BSA:PBST=1:20) to the control wells without secondary antibody, and add 100 μL HRP-Goat Anti Rabbit IgG (H+L) secondary antibody to the remaining wells. The secondary antibody was diluted 1:10000 with buffer, and shake at 700 rpm for 1 hour.

[0174] 6. Wash each well 10 times with 250 μL PBST, wipe the bottom of the ELISA plate clean and place it on tin foil.

[0175] 7. Add 100 μL TMB color development solution to each well, cover with tin foil, and let stand for 30 minutes away from light.

[0176] 8. Before the test, add 50 μL 2M H2SO4 to each well to terminate the reaction.

[0177] 9. Measure the absorbance at 450nm with an enzyme-labeled instrument and record the sample number with the highest absorbance value.

[0178] After all samples in the master plate were screened, 33 samples with high absorbance values ​​were selected for PCR verification and sent for testing. The bacterial solution PCR system was added with reactants according to Table 14, and the bacterial solution PCR program was according to Table 15.

[0179] Table 14 Bacterial liquid PCR system

[0180]

[0181]

[0182] Table 15 Bacterial liquid PCR program

[0183]

[0184] After sequencing, 33 samples were sequenced and 12 nanobody protein genes with different sequences were obtained, numbered as: 1-A4, 1-A5, 1-A10, 1-C6, 1-F8, 1-H9, 2-A2, 2-A5, 2-C4, 2-F1, 2-G11, 2-H4. The OD450 results of the affinity test of the 12 nanobody proteins are shown in Figure 3The bacterial solution containing the 12 nanoantibody plasmids was outsourced to Jinweizhi for sequencing. The computer-readable nucleic acid sequences are shown in Seq ID No: 2-13, and the protein sequence comparison is shown in Figure 4 The computer readable sequence is Seq ID No: 15-26. Example 5 SV2C-L4 Nanobody Purification

[0185] 5.1 Materials

[0186] MEM (containing NEAA) basal medium (Pnosai, catalog number 22E1061), FBS (gibco, catalog number 10099-141C), DMEM / HIGH GLUCOSE (cytiva, catalog number SH30022.01), Trypsin-EDTA solution (Beijing Solebow Technology Co., Ltd., catalog number T1320), penicillin-streptomycin mixture (100X) (Beijing Solebow Technology Co., Ltd., catalog number P1400), DMSO (Beijing Solebow Technology Co., Ltd., catalog number D8371).

[0187] 5.2 Inducible expression of SV2C-L4 nanobody

[0188] The 12 SV2C-L4 nanoantibody strains obtained by screening were inoculated into LB medium at 1%, cultured at 37°C, 200rpm until the OD600 value was about 0.6, and the uninduced sample was taken. IPTG was added to a final concentration of 0.5mmol / L, induced at 20°C for 16h, and the induced sample was taken. Centrifuged at 8000rpm for 5min to collect the bacteria.

[0189] The uninduced samples and induced samples were centrifuged and then subjected to SDS-PAGE electrophoresis. The results showed that both samples successfully induced the expression of SV2C-L4 nanoantibody.

[0190] 5.3 Purification of SV2C-L4 Nanobody

[0191] Add 20 times the weight of Binding Buffer to the collected cells, use a homogenizer at 800 bar to break the cells until they are clear, and centrifuge at 10000 rpm for 10 min. Take the supernatant and purify it by nickel ion affinity chromatography (20 column volumes of Binding Buffer balance, Binding Buffer: 20mM PB, pH 7.4, 20mM NaCl, 100mM imidazole; Elution Buffer: 20mM PB, pH7.4, 100mM NaCl, 500mM imidazole.) The target protein is present in the elution sample Elution, and the SDS-PAGE results are shown in Figure 5 .

[0192] Example 6 SF-680SE labeled SV2C-L4 nanobody

[0193] (1) Protein dissolution: Use a concentrator to replace the buffer of the protein solution with 0.1 M NaHCO3.

[0194] (2) Dye dissolution: After the activated Super Flour SE dye solid powder is placed in a desiccator from a refrigerator and slowly warmed to room temperature, the dye is prepared with anhydrous DMSO to a concentration of 1 mg / mL.

[0195] (3) Labeling: Slowly add an appropriate amount of dye solution to 1 mL of protein solution (protein: dye molar ratio = 1:20-50; the mass ratio can be optimized within the following range: 10-200 μg Super Flour SE per mg IgG antibody). Stir slowly for 1 hour at room temperature in the dark. Alternatively, the protein solution can be directly added to the reagent bottle of the solid powder of the dye, and stir slowly for 1 hour at room temperature in the dark.

[0196] (4) Purification: Purify the product using Sephadex G25.

[0197] 8 mL of dextran Sephadex G25 filler was loaded, and PBS solution was used for balance first. Then, the labeled SV2C nanobody protein solution was purified through Sephadex G25 column to wash away the excess fluorescent dye not labeled on the SV2C nanobody. The labeled protein solution was eluted with PBS solution, and the eluted components were collected in sections according to the reading of the UV detector. After SDS-PAGE detection, the purified component elution sample with high concentration was collected for later use.

[0198] Example 7 Experiment of SV2C-L4 nanobody targeting N2a cells after labeling

[0199] Take out the frozen Neura-2a cell line from liquid nitrogen, and thaw it as quickly as possible by shaking rapidly in a constant temperature water bath at 37°C, following the correct principle of "slow freezing and fast thawing" for cell recovery. Transfer the cell freezing solution (about 1mL) in the cryopreservation tube to a 5mL centrifuge tube, add 2mL of conventional cell culture medium, mix by blowing, and centrifuge at a low speed of 900r / min for 3min. After centrifugation, remove the supernatant, add 1mL of conventional cell culture medium, blow Neura-2a cells, mix, and use the pipette to absorb the cell suspension into a 10cm diameter culture dish containing 12mL of cell culture medium. After gently shaking left and right, the suspension can be carefully cultured in a cell culture incubator with a concentration of 5% CO2, 90% humidity, and a constant temperature of 37°C.

[0200] When the cells grow to about 80% fusion, they can be passaged. Use a pipette to suck the old culture medium into the waste bottle. After washing the cultured cells 2-3 times with 2 mL of sterile PBS solution, add 1 mL of 0.25% pancreatin to digest the cells. Note that you can hit the pancreatin on the wall of the culture dish to let it flow down to avoid damaging the cells. Carefully shake the culture dish to disperse the pancreatin evenly on the surface of the cells to be digested. Then place the digesting culture dish in the cell culture incubator for 1-2 minutes. Use a pipette to inject 1 mL of culture medium containing fetal bovine serum to neutralize the digestion effect of pancreatin. Use a pipette to blow the cells carefully. After digestion is complete, it can be blown down easily. Use a pipette to suck the cell digestion solution into a 10 mL centrifuge tube and centrifuge the cell suspension at 900 r / min for 3 minutes. After centrifugation, the supernatant was removed by aspiration, and the cells were resuspended in 1 mL of culture medium. After careful pipetting and mixing of the cells, the cell suspension was transferred to a 10 cm diameter culture dish containing 12 mL of cell culture medium at a ratio of 1:4.

[0201] At the same time as cell passage, Neura-2a cells were plated on 24-well plates at 10 cells per well. 5 Each well was added with labeled SV2C-L4 nanoantibodies to make the final concentration of the protein 2μM, and then incubated overnight. The binding of Neura-2a cells and labeled SV2C-L4 nanoantibodies was observed under a laser confocal microscope. Most of the labeled proteins bind to the receptors on the Neura-2a cell membrane, so red fluorescence can be observed on the cell membrane, while the negative control Hela cells have no receptors bound by nanoantibodies, so no fluorescence can be observed ( Figure 6 ).

[0202] The SV2 extracellular soluble portion nanobody provided by the present invention can be used as a fluorescent probe molecule to become a research tool for studying neuroendocrine tumors. Generally, according to research needs, the SV2 extracellular soluble portion nanobody can be coupled with any other fluorescent probe according to the method provided by the dye supplier. In addition, the SV2 extracellular soluble portion nanobody can also be coupled with HRP, alkaline phosphatase, etc. using conventional methods, which can be understood by those skilled in the art.

[0203] Obviously, 89 Zr, 68 Ga, 99 Tc, 111 In, 90 Nuclides such as Y are used to label the extracellular soluble part of SV2 with nanobodies through bifunctional chelators such as DOPA, DTPA, and HYNIC; 18 F by first synthesizing 18F precursor molecules are used to label the extracellular soluble part of SV2 with nanobodies; 131 I was used to label the SV2 extracellular soluble part of the nanobody by the chloramine T method. The SV2 extracellular soluble part of the nanobody was labeled with a nuclide, and the SV2 extracellular soluble part of the nanobody labeled with a nuclide can become a nuclear imaging probe molecule or a nuclear medicine molecule with a therapeutic effect, which can be understood by technicians in the same field.

[0204] The toxin molecules are coupled to the SV2 extracellular soluble part nanobody by conventional chemical methods. The SV2 extracellular soluble part nanobody coupled to the toxin small molecule can become a nuclear medicine molecule with therapeutic effect, which can be understood by technicians in the same field. Such toxin small molecules include: microtubule generation inhibitors such as auristatin, maytansine and its analogs; DNA damaging factors such as calicheamicin, duocarmycins, anthracyclines, pyrrolobenzodiazepine dimers; DNA transcription inhibitors such as amatoxin and quinoline alkaloid (SN-38).

[0205] The nucleotide sequence involved in the present invention is:

[0206] Seq ID No:1

[0207] SV2C-L4:

[0208] CATATGTTTTCCGGATGTGATTAAACCGCTGCAGAGCGATGAATATGCGCTGCTGACCCGCAACGTGGAACGCGATAAATATGCGAACTTTACCATTAACTTTACCATGGAAAATCAGATTCATACCGGCATGGAATATGATAACGGCCGCTTTATTGGCGTGAAATTTAAAAGCGTGACCTTTAAAGATAGCGTGT TTAAAAGCTGCACCTTTGAAGATGTGACGAGCGTGAACACCTATTTTAAAACTGCACCTTTATTGATACCGTGTTTGATAACACCGATTTTGAACCGTATAAATTTATTGATAGCGAATTTAAAAATTGCAGCTTTTTTCATAACAAAACCGGCTGTCAGATTACCTTTGATGACGATTATAGCGCGTAAGAATTC

[0209] Seq ID No:2

[0210] 1-A4:

[0211] CTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGGTCTCTAGGACTCTCCTGTGCAGCCTCTGGACGCACCATCAGTAGTTATGCCATGGCCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAACTTGTCGCAGTTATCAATTGGAGTGGTGACAGAACATACTATGGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACACGGTGTATCTGCGAATGAACAGCCTGAAACCTGAGGACACGGCCGTCTATTACTGTAATGTCCTAAGGTCATTTAATTCGCGCTCTGATCCCCTTTACAACTGGGGCCAGGGGACCCAGGTCACC

[0212] Seq ID No:3

[0213] 1-A5:

[0214] CTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAATCAGAATCAGTAACTATGCCATGGGCTGGTATCGCCAGGCTCCAGGAAAACAGCGCGAGTTGGTCGCACGTATTAATAGTGTTGGAGATTTAGACTATGCAGACTCCCTCAAGGGCCGATTCACCATCTCCAGAGACAGCGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCGGAGGACACGGCCCTCTATTACTGTAATACAGCCATGGGGGCCTGGAAACTCCCGGGTGTCAAGGAGCCCTGGGGCCAGGGGACCCAGGTCACC

[0215] Seq ID No:4

[0216] 1-A10:

[0217] CTGCAGGAGTCTGGAGGAGGCTTGGTGCATGGTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTCCGTCCATCTTCAGTGGCTTGTCCATGGCCTGGTACCGCCAGGCTCCAGGGGAGGAGCGCGAATTGGTCGCACGAATTAGTCGTGATGGAAGTACAAACTATGCAGACACCGTACAGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACACGGTGTATCTCCAAATGAACAGCCTGAAACCAGAGGACGCGGGCGTCTATTTCTGTAATAGAGGAATAGGTGCTAGAACCTTTTGGGGCCGGGGGACCCAGGTCACC

[0218] Seq ID No:5

[0219] 1-C6:

[0220] CTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAATCATAATCAGTAACTATGCCATGGGCTGGTATCGCCAGGCTCCAGGAAAACAGCGCGAGTTGGTCGCACGTATTAATAGTGTTGGAGATTTAGACTATGCAGACTCCCTCAAGGGCCGATTCACCATCTCCAGAGACAGCGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCGGAGGACACGGCCCTCTATTACTGTAATACAGCCATGGGGGCCTGGAAACTCCCGGGTGTCAAGGAGCCCTGGGGCCAGGGGACCCAGGTCACC

[0221] Seq ID No:6

[0222] 1-F8:

[0223] CTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGCTCTCTGAGTCTCTCCTGTACAGCCTCTACAATCAGAATCAGTAACTATGCCATGGGCTGGTATCGCCAGGCTCCAGGAAAACAGCGCGACTTGGTCGCTCGGATTAGTAGTGATGGTAGGTTAGACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAGCGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCCTCTATTACTGTAATACAGCCATGGGGGCCTGGAAACTCCCGAGTGCAAAGGAATCCTGGGGCCAGGGGACCCGGGTCACC

[0224] Seq ID No:7

[0225] 1-H9:

[0226] CTGCAGGAGTCTGGAGGAGAATTGGTGCAGTTTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTATAATCAGGATAAGTAGCTATTCCATGGGCTGGTATCGCCAGGCTCCAGGAAAACAGCGCGAGTTGGTCGCACGTATTAGTAGTGATGGCAGCACACGCTATTCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAGCGCCACGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCCTCTATTACTGTAATACAGCCATGGGGGCCTGGCAACTCCCAAGTGCCAAGGAACCCTGGGGCCCGGGGACCCAGGTCACC

[0227] Seq ID No:8

[0228] 2-A2:

[0229] CTGCAGGAGTCTGGAGGAGGCCAGGTGCATGCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTCCGTCCATCTTCAGTGGCTTGTCCATGGCCTGGTACCGCCAGGCTCCAGGGGAGGAGCGCGAATACGTCGCACGAATTAGTCGTGATGGAAGTACAGCCTATGCAGACACCGTACAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTCCAAATGAACAGCCTGAAACCAGAGGACACGGGCGTCTATTTCTGTAATAGAGGAGTAGGTGCTAGAACCTTTTGGGGCCGGGGGACCCAGGTCACC

[0230] Seq ID No:9

[0231] 2-A5:

[0232] CTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCGTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTCCGTCCATCTTCAGTGGCTTGTCCATGGCCTGGTACCGCCAGGCTCCAGGGGAGGAGCGCGAATTGGTCGCACGAATTAGTCGTGATGGAAGTACAAAGTATGCAGACACCGTACAGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACACGGTGTATCTCCAAATGAACAGCCTGAAACCAGAGGACGCGGGCGTCTATTTCTGTAATAGAGGAATAGGTGCTAGAACCTTTTGGGGCCAGGGGACCCAGGTCACC

[0233] Seq ID No:10

[0234] 2-C4:

[0235] CTGCAGGAGTCTGGGGGAGAATTGGTGCAGCCTGGGGGGTCTCTGACACTCTCCTGTGCTGCCTCTGGAATCAGAATCAGTAACTATGCCATGGGCTGGTATCGCCAGGCTCCAGGAAAACAGCGCGACTTGGTCGCACGTATTAGTAGTGACGGTAGTACACGCTATGCAGACTCCGTGAACGGCCGATTCACCATCTCCAGAGACAGCGCCAAGAACACGGTGTTTCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCCTCTATTACTGTAATACAGCCATGGGGGCCTGGAAACTCCCGACTACCAAGGAATCCTGGGGCCAGGGGACCCAGGTCACC

[0236] Seq ID No:11

[0237] 2-F1:

[0238] CTGCAGGAGTCTGGAGGAGGCCAGGTGCATGCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTCCGTCCATCTTCAGTGGCTTGTCCATGGCCTGGTACCGCCAGGCTCCAGGGGAGGAGCGCGAATACGTCGCACGAATTAGTCGTGATGGAAATACAGCCTATGCAGACACCGTACAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTCCAAATGAACAGCCTGAAACCAGAGGACACGGGCGTCTATTTCTGTAATAGAGGAGTAGGTGCTAGAACCTTTTGGGGCCGGGGGACCCAGGTCACC

[0239] Seq ID No:12

[0240] 2-G11:

[0241] CTGCAGGAGTCTGGGGGAGAATTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAATCAGAATCAGTAACTATGCCATGGGCTGGTATCGCCAGGCTCCAGGAAAACAGCGCGACTTGGTCGCACGTATTAGTAGTAGTGGTGATACACGCTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAGCGCCAAGAACACGGTGTATCTGCAAATGAATAGCCTGAAACCTGAGGACACGGCCCTCTATTACTGTAATACAGCCATGGGGGCCTGGAAAGTCCCGAGTGCCCAGGAATCCTGGGGCCAGGGGACCCAGGTCACC

[0242] Seq ID No:13

[0243] 2-H4:

[0244] CTGCAGGAGTCTGGAGGAGGCCAGGTGCATGCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTCCGTCCATCTTCAGTGGCTTGTCCATGGCCTGGTACCGCCAGGCTCCAGGGGAGGAGCGCGAATACGTCGCACGAATTAATCGTGATGGAAATACAGCCTATGCAGACACCGTACAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTCCAAATGAACAGCCTGAAACCAGAGGACACGGGCGTCTATTTCTGTAATAGAGGAGTAGGTGCTAGAACCTTTTGGGGCCGGGGGACCCAGGTCACC

[0245] The protein sequences involved in the present invention:

[0246] Seq ID No:14

[0247] SV2C-L4:

[0248] <h2 style=";text-align:left;direction:ltr">HMFPDVIKPLQSDEYALLTRNVERDKYANFTINFTMENQIHTGMEYDNGRFIGVKFKSVTFKDSVFKSCTFEDVTSVNTYFKNCTFIDTVFDNTDFEPYKFIDSEFKNCSFFHNKTGCQITFDDDYSA*EF<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0249] <h2 style=";text-align:left;direction:ltr"> Seq ID No:15<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0250] <h2 style=";text-align:left;direction:ltr"> 1-A4:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0251] <h2 style=";text-align:left;direction:ltr"> LQESGGGLVQAGGSLGLSCAASGRTISSYAMAWFRQAPGKERELVAVINWSGDRTYYGDSVKGRFTISRDDAKNTVYLRMNSLKPEDTAVYYCNVLRSFNSRSDPLYNWGQGTQVT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0252] <h2 style=";text-align:left;direction:ltr"> Seq ID No:16<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0253] <h2 style=";text-align:left;direction:ltr"> 1-A5:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0254] <h2 style=";text-align:left;direction:ltr"> LQESGGGLVQPGGSLRLSCAASGIRISNYAMGWYRQAPGKQRELVARINSVGDLDYADSLKGRFTISRDSAKNTVYLQMNSLKPEDTALYYCNTAMGAWKLPGVKEPWGQGTQVT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0255] <h2 style=";text-align:left;direction:ltr"> Seq ID No:17<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0256] <h2 style=";text-align:left;direction:ltr"> 1-A10:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0257] <h2 style=";text-align:left;direction:ltr"> LQESGGGLVHGGGSLRLSCAASPSIFSGLSMAWYRQAPGEERELVARISRDGSTNYADTVQGRFTISRDDAKNTVYLQMNSLKPEDAGVYFCNRGIGARTFWGRGTQVT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0258] <h2 style=";text-align:left;direction:ltr"> Seq ID No:18<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0259] <h2 style=";text-align:left;direction:ltr"> 1-C6:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0260] <h2 style=";text-align:left;direction:ltr"> LQESGGGLVQPGGSLRLSCAASGIIISNYAMGWYRQAPGKQRELVARINSVGDLDYADSLKGRFTISRDSAKNTVYLQMNSLKPEDTALYYCNTAMGAWKLPGVKEPWGQGTQVT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0261] <h2 style=";text-align:left;direction:ltr">Seq ID No:19<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0262] <h2 style=";text-align:left;direction:ltr"> 1-F8:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0263] <h2 style=";text-align:left;direction:ltr"> LQESGGGLVQPGGSLSLSCTASTIRISNYAMGWYRQAPGKQRDLVARISSDGRLDYADSVKGRFTISRDSAKNTVYLQMNSLKPEDTALYYCNTAMGAWKLPSAKESWGQGTRVT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0264] <h2 style=";text-align:left;direction:ltr"> Seq ID No:20<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0265] <h2 style=";text-align:left;direction:ltr"> 1-H9:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0266] <h2 style=";text-align:left;direction:ltr"> LQESGGELVQFGGSLRLSCAASIIRISSYSMGWYRQAPGKQRELVARISSDGSTRYSDSVKGRFTISRDSATNTVYLQMNSLKPEDTALYYCNTAMGAWQLPSAKEPWGPGTQVT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0267] <h2 style=";text-align:left;direction:ltr"> Seq ID No:21<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0268] <h2 style=";text-align:left;direction:ltr"> 2-A2:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0269] <h2 style=";text-align:left;direction:ltr"> LQESGGGQVHAGGSLRLSCAASPSIFSGLSMAWYRQAPGEEREYVARISRDGSTAYADTVQGRFTISRDNAKNTVYLQMNSLKPEDTGVYFCNRGVGARTFWGRGTQVT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0270] <h2 style=";text-align:left;direction:ltr"> Seq ID No:22<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0271] <h2 style=";text-align:left;direction:ltr"> 2-A5:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0272] <h2 style=";text-align:left;direction:ltr"> LQESGGGLVQRGGSLRLSCAASPSIFSGLSMAWYRQAPGEERELVARISRDGSTKYADTVQGRFTISRDDAKNTVYLQMNSLKPEDAGVYFCNRGIGARTFWGQGTQVT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0273] <h2 style=";text-align:left;direction:ltr"> Seq ID No:23<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0274] <h2 style=";text-align:left;direction:ltr"> 2-C4:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0275] LQESGGELVQPGGSLTLSCAASGIRISNYAMGWYRQAPGKQRDLVARISSDGSTRYADSVNGRFTISRDSAKNTVFLQMNSLKPEDTALYYCNTAMGAWKLPTTKESWGQGTQVT

[0276] Seq ID No:24

[0277] 2-F1:

[0278] LQESGGGQVHAGGSLRLSCAASPSIFSGLSMAWYRQAPGEEREYVARISRDGNTAYADTVQGRFTISRDNAKNTVYLQMNSLKPEDTGVYFCNRGVGARTFWGRGTQVT

[0279] Seq ID No:25

[0280] 2-G11:

[0281] LQESGGELVQPGGSLRLSCAASGIRISNYAMGWYRQAPGKQRDLVARISSSGDTRYADSVKGRFTISRDSAKNTVYLQMNSLKPEDTALYYCNTAMGAWKVPSAQESWGQGTQVT

[0282] Seq ID No:26

[0283] 2-H4:

[0284] LQESGGGQVHAGGSLRLSCAASPSIFSGLSMAWYRQAPGEEREYVARINRDGNTAYADTVQGRFTISRDNAKNTVYLQMNSLKPEDTGVYFCNRGVGARTFWGRGTQVT

Claims

1. A SV2 extracellular soluble part nanobody, characterized in that The protein sequence of the probe molecule contains any one of Seq ID No: 14 to Seq ID No:

26.

2. The SV2 extracellular soluble portion nanobody according to claim 1, characterized in that The nanobody is coupled to any fluorescent probe molecule using conventional methods.

3. A SV2 extracellular soluble portion nanobody according to any one of claims 1 to 2, characterized in that The nano antibody is coupled to any enzyme-labeled probe molecule using a conventional method.

4. A SV2 extracellular soluble portion nanobody according to any one of claims 1 to 3, characterized in that The nanobody is coupled to any nuclide molecule using conventional methods, and the nuclide molecule is selected from any one of the following: 68 Ga, 99 Tc, 131 I, 89 Zr, 111 In, 90 Y, 18 F.

5. A SV2 extracellular soluble portion nanobody according to any one of claims 1 to 4, characterized in that The nano antibody is coupled with any chemical toxin molecule by conventional methods, and the chemical toxin molecule is selected from any one of the following: auristatin, maytansine and its analogs, calicheamicin, duocarmycins, anthracyclines, pyrrolobenzodiazepine dimers, amatoxin, and quinoline alkaloid (SN-38).

6. A nano antibody to the extracellular soluble part of SV2, which can be used in the preparation of nuclear imaging probe molecules for neuroendocrine tumors.

7. A nano-antibody of the extracellular soluble part of SV2, which can be used in the preparation of radioactive drugs targeting neuroendocrine tumors.

8. A nanobody of the extracellular soluble part of SV2, which can be used in the preparation of nanobody chemical toxin-coupled drugs targeting neuroendocrine tumors.

9. A nanobody of the extracellular soluble part of SV2, which can be used in the preparation of fluorescent-labeled probe molecular tools targeting neuroendocrine tumors.

10. A nano antibody against the extracellular soluble part of SV2, which can be used in the preparation of enzyme-labeled probe molecular tools targeting neuroendocrine tumors.

Images