Anti-TRAIL nano antibody and application thereof

Abstract

The invention belongs to the field of biological medicine, and relates to a nano antibody. The amino acid sequence of the anti-TRAIL nano antibody is as shown in SEQ ID No. 1. The anti-TRAIL nano-antibody can also be a series antibody consisting of two or more than two repeated anti-TRAIL nano-antibodies. The anti-TRAIL nano antibody has the advantages of small molecular weight, strong tissue penetrability and high affinity, the anti-TRAIL nano antibody has good binding affinity with human TRAIL protein, and the anti-TRAIL nano antibody has good TRAIL-DR5 pathway blocking activity, can significantly alleviate cerebral ischemia reperfusion injury after cerebral infarction, and has wide clinical application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to nano antibodies. Background Art

[0002] Cerebral infarction is mainly a disease caused by impaired blood supply to the brain, which leads to ischemia, hypoxic necrosis of brain tissue and neurological dysfunction. The incidence, disability and mortality of cerebral infarction are extremely high. There are about 12 million patients with cerebral infarction in my country, and nearly 2 million people die from cerebral infarction each year. It is the "number one killer" threatening healthy life. The best treatment for this disease is to restore cerebral blood perfusion as soon as possible through intravenous thrombolysis and mechanical thrombectomy, but reperfusion will cause rapid cascade damage to neurons, induce apoptosis or necrosis, and further aggravate the pathological damage of ischemic tissue, which is called cerebral ischemia / reperfusion injury (IRI). Researching how to reduce brain IRI and developing appropriate therapeutic drugs is an urgent problem to be solved in clinical practice.

[0003] The pathological mechanism of brain IRI is very complex, mainly involving energy metabolism disorders, excitotoxicity, Ca 2+ Overload, oxidative stress, cell apoptosis, inflammatory response and blood-brain barrier damage. TRAIL protein is an important apoptosis-inducing molecule in the human body. In addition to inducing cell apoptosis, TRAIL protein binds to DR5 receptor and directly participates in the process of inflammatory response, such as the short TRAIL antibody and method of use disclosed in the application with publication number CN110494152A. Studies have found that TRAIL protein is not expressed in normal brain tissue, but TRAIL protein expression is upregulated in astrocytes and activated microglia during cerebral ischemia-reperfusion in mice. At the same time, DR5 is upregulated in neurons and has a good correlation with the distribution of apoptotic cells. Using soluble DR5 protein, anti-TRAIL monoclonal antibody CD253 or sDR5-Fc fusion protein to block the interaction between TRAIL and DR5 through the intraventricular pathway can reduce neuronal damage. Therefore, blocking the TRAIL-DR5 pathway in the ischemic penumbra of the brain after cerebral infarction may be an ideal strategy for treating brain IRI.

[0004] The blood-brain barrier (BBB) ​​is a dynamic interface between the central nervous system and the blood circulation system. It is mainly composed of brain microvascular endothelial cells, pericytes, astrocytes and basement membranes that are tightly connected. It strictly regulates the transport of substances between the blood and the brain and provides a stable internal environment for the realization of neural function. Studies have shown that the permeability of the BBB is significantly increased in animal models of cerebral ischemia and clinical stroke patients. However, the reported anti-TRAIL monoclonal antibody CD253, soluble DR5 protein and sDR5-Fc fusion protein prepared in our laboratory in the early stage cannot penetrate the blood-brain barrier and still need intracranial injection, which limits their clinical application. Summary of the invention

[0005] The present invention provides an anti-TRAIL nano antibody and a preparation method thereof, which solves the problem that conventional monoclonal antibodies cannot penetrate the blood-brain barrier to alleviate ischemia-reperfusion injury after cerebral infarction.

[0006] The technical solution of the present invention is achieved in this way: The primary object of the present invention is to provide a nanobody against human tumor necrosis factor-related apoptosis inducing ligand (TRAIL), the amino acid sequence of the anti-TRAIL nanobody being selected from any one of the following: as shown in SEQ ID No.1, as shown in SEQ ID No.2, as shown in SEQ ID No.3, as shown in SEQ ID No.4, as shown in SEQ ID No.5, as shown in SEQ ID No.6, as shown in SEQ ID No.7, as shown in SEQ ID No.8, as shown in SEQ ID No.9 and as shown in SEQ ID No.10.

[0007] The second object of the present invention is to provide an antibody, which comprises one or more anti-TRAIL nanoantibodies, and the antibody comprises two or more nanoantibody units repeated in series, and the amino acid sequence of the nanoantibody unit is shown in any one of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQIDNo.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9 and SEQ ID No.10.

[0008] The third object of the present invention is to provide a gene fragment encoding the anti-TRAIL nanobody, the nucleotide sequence of the gene fragment being as shown in any one of SEQ ID No.11, SEQ ID No.12, SEQ ID No.13, SEQID No.14, SEQ IDNo.15, SEQ ID No.16, SEQ ID No.17, SEQ ID No.18, SEQ ID No.19 and SEQID No.20.

[0009] The fourth object of the present invention is to provide an expression vector containing the gene fragment.

[0010] The fifth object of the present invention is to provide a host cell, wherein the host cell contains the expression vector, or the gene fragment is integrated into its genome.

[0011] The sixth object of the present invention is to provide an immunoconjugate, wherein the immunoconjugate contains: (a) the above-mentioned nanobody or antibody; and / or (b) a conjugated portion selected from the following: any one of a detectable marker, a drug, a toxin, a cytokine, a radionuclide, an enzyme, a gold nanoparticle, a nanorod, a nanomagnetic particle and a viral coat protein.

[0012] The seventh object of the present invention is to provide a pharmaceutical composition, comprising: (i) the above-mentioned anti-TRAIL nanobody, or the above-mentioned antibody, or the above-mentioned immunoconjugate; and (ii) a pharmaceutically acceptable carrier.

[0013] The eighth object of the present invention is to provide a method for preparing an anti-TRAIL nanobody, wherein the expression vector containing the encoding gene is transfected into competent cells, and the cells are cultured to obtain the anti-TRAIL nanobody; and the host cells are cultured to obtain the anti-TRAIL nanobody.

[0014] The ninth objective of the present invention is to provide the use of the nanobody or the antibody in developing drugs for treating acute cerebral infarction.

[0015] The present invention has the following beneficial effects: The present invention provides an anti-TRAIL nanobody, the amino acid sequence of the anti-TRAIL nanobody is shown in any one of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9 and SEQ ID No.10, the nanobody of the present application has a small molecular weight of about 15kDa, high affinity, strong tissue penetration, and has the function of blocking the binding of human TRAIL protein to death receptor 5 (Deathreceptor 5, DR5). Intravenous injection of the nanobody can reduce cerebral ischemia-reperfusion injury after cerebral infarction, and has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 The electrophoresis diagram is the construction of phagemid vector; A is the gel run diagram of total RNA extracted from peripheral blood lymphocytes separated from alpacas after five immunizations; B is the electrophoresis diagram of the first round of nested PCR; C is the electrophoresis diagram of the second round of nested PCR, lanes 1 and 2 are IgG2c and IgG2b subtype antibody gene fragments, respectively; D is the electrophoresis diagram of VHH gene before and after restriction digestion, lanes 1 and 3 are IgG2c and IgG2b subtype antibody gene fragments, respectively, and lanes 2 and 4 are IgG2c and IgG2b subtype antibody gene fragments after restriction digestion, respectively; E is the electrophoresis diagram of pComb3XSS vector, lanes 1, 2 and 3 are pComb3XSS vector after restriction digestion, pComb3XSS vector after ligation and pComb3XSS original vector, respectively.

[0018] Figure 2 The figure shows the diversity and capacity of the library after electroporation; A is the plate for determining the number of transformants in the library; B is the PCR electrophoresis diagram of the bacterial solution to determine the insertion rate of the target gene in the library; and C is the comparison result of the amino acid sequence of the monoclonal bacterial solution VHH.

[0019] Figure 3 The results of phage clone ELISA.

[0020] Figure 4 This is the SDS-PAGE run of different TRAIL nanobodies expressed in prokaryotes.

[0021] Figure 5 These are the results of Jurkat cell viability detection after treatment with TRAIL protein and TRAIL nanoantibody.

[0022] Figure 6 This is the plasmid map of pComb3XSS-Nb19.

[0023] Figure 7 The protective effect of TRAIL nanoantibody Nb19 in the mouse cerebral ischemia-reperfusion model; A is TTC staining of mouse brain; B is the comparison result of mouse brain tissue infarction rate; C is the comparison result of mouse mNSS. DETAILED DESCRIPTION

[0024] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] Unless otherwise specified, the experimental methods used in the following experimental examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0026] In the technical scheme claimed for protection in the present invention, the amino acid sequence of TRAIL nanobody is as shown in any one of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8 and SEQ ID No.9, and the nucleotide sequence encoding the antibody amino acid is as shown in any one of SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.13, SEQ ID No.14, SEQ ID No.15, SEQ ID No.16, SEQ ID No.17 and SEQ ID No.18.

[0027] In the following examples, 1 L of 2YT medium contained 16 g of peptone, 10 g of yeast extract, and 5 g of sodium chloride.

[0028] Example 1: Construction of TRAIL Nanobody Library (1) 0.2 mg of human TRAIL antigen (purchased from Suzhou Jinan Protein Technology Co., Ltd.) was mixed with an equal volume of Freund's complete adjuvant (primary immunization) / incomplete adjuvant (purchased from Sigma, USA), stirred and emulsified overnight at 4°C, and injected subcutaneously into the neck of two-year-old male alpacas (purchased from Shenghong Breeding Farm in Jiaxiang County, Shandong Province) with an interval of 15 days for a total of 5 immunizations to stimulate B cells to express antigen-specific nanobodies; (2) One week after the fifth immunization, a booster immunization was performed. Three days later, 100 mL of blood was collected from the jugular vein of the alpaca, and peripheral blood lymphocytes were separated and total RNA was extracted and reverse transcribed into cDNA. (3) Nested PCR was used to amplify the variable domain of heavy chain of heavy-chain antibody (VHH) gene of alpaca heavy chain antibody; (4) 10 μg of pComb3XSS phage display vector (purchased from Biovector) and 4 μg of VHH were digested with restriction endonuclease Sfi I (purchased from Beijing NEB Company) and ligated with T4 ligase (purchased from Beijing NEB Company) to construct phagemid; (5) The ligation product was electroporated into TG1 competent cells (purchased from Lucigen, USA), and then plated after gradient dilution. The reservoir size was measured to be 3.28×10 9 pfu, and 48 clones were randomly selected for colony PCR detection.

[0029] The heavy chain variable region gene of the alpaca heavy chain antibody is as follows Figure 1 As shown, the nanobody gene insertion is as follows Figure 2 As shown, it can be seen that the insertion rate of the nano antibody gene is 100%. The bacterial library was infected with helper phage and shaken overnight, and the phage-displayed TRAIL nano antibody library was obtained after PEG precipitation.

[0030] Example 2: Screening and identification of TRAIL nanobodies Antibody Screening: (1) Coat 4 wells of an ELISA plate (purchased from Corning, USA) with 10 μg / mL human TRAIL antigen, 100 μL per well, and incubate at 4°C overnight. (2) Wash 4 times with PBST (PBS + 0.05% Tween-20), add 300 μL of 5% skim milk powder, and block at 37°C for 1 h; (3) After washing, add 100 μL of phage library (2×10 11 cfu), shaking and binding at room temperature for 2 h; (4) Wash the plate 10 times with PBST to remove non-specifically bound phages; (5) Add 100 μL of 10 μg / mL sDR5-Fc antibody fusion protein (made in our laboratory) to the ELISA well and shake for 2 h. Collect the supernatant and set it as the output of this round. Infect the output with TG1 Escherichia coli (purchased from Beijing Huayueyang Biological Company), shake and culture overnight after auxiliary phage infection, and set the PEG-precipitated phage as the input of the next round. The same screening process was repeated for a total of 4 rounds, during which the screening pressure was continuously increased. The antigen coating concentrations in the second, third and fourth rounds were 5, 2 and 1 μg / mL, respectively.

[0031] Identification of positive phage clones: (1) A total of 192 monoclonal colonies were picked from the four output titer plates and inoculated into 1 mL of 2YT medium containing 100 μg / mL ampicillin. The culture was shaken at 37°C and 250 rpm until the OD 600 The value is about 0.6. After the helper phage is statically infected, kanamycin is added and shaken overnight. The next day, centrifuge at 10,000 rpm for 15 min at 4°C, and take the supernatant for phage enzyme-linked immunosorbent assay detection; (2) Coat 5 μg / mL human TRAIL protein and BSA protein at 4°C overnight, add 50 μL of sample diluent and 5 μg / mL sDR5-Fc antibody fusion protein to two TRAIL protein coated wells, add 50 μL of sample diluent to one BSA protein coated well, and then add 50 μL of monoclonal phage supernatant to three wells, incubate at 37°C for 30 min. After washing the plate, add 100 μL of phage secondary antibody (purchased from Chengdu Apak Company) and react at 37°C for 30 min. After washing the plate, add 100 μL of TMB colorimetric solution (purchased from Solebao Biotechnology Co., Ltd.), color at room temperature for 10 min, add 50 μL of 1 M HCl to stop, and read at 450 nm; (3) When the OD value of the PBS well is higher than that of the sDR5-Fc well and more than 3 times greater than that of the BSA well, the monoclonal colony is determined to be a positive clone. All positive clones were sequenced and analyzed, and a total of 10 different nanobody amino acid sequences were obtained, numbered 16, 18, 19, 76, 119, 146, 147, 151, 155 and 175. The results are as follows Figure 3 shown.

[0032] Example 3: Expression and purification of TRAIL nanobodies (1) Spread the positive clones on a 2YT culture plate containing 100 μg / mL ampicillin and culture them inverted at 37°C overnight; (2) Select a single colony and inoculate it into 3 mL of 2YT culture medium containing 100 μg / mL ampicillin and 0.05% glucose, and incubate at 37°C, 250 rpm, and shake overnight. (3) Inoculate 1 mL of overnight culture into 500 mL of 2YT medium containing 100 μg / mL ampicillin and 0.05% glucose and culture until OD 600 When the value reached 0.6-0.8, IPTG (purchased from Solebow Biotechnology Co., Ltd.) was added and the culture was shaken at 30°C and 220 rpm overnight; (4) Collect the bacteria by centrifugation and use ultrasonic method to break the bacteria to obtain the crude antibody extract; (5) The nanoantibodies Nb16 (amino acid sequence as shown in SEQ ID No.1, nucleotide sequence as shown in SEQ ID No.11), Nb18 (amino acid sequence as shown in SEQ ID No.2, nucleotide sequence as shown in SEQ ID No.12), Nb19 (amino acid sequence as shown in SEQ ID No.3, nucleotide sequence as shown in SEQ ID No.13), Nb76 (amino acid sequence as shown in SEQ ID No.4, nucleotide sequence as shown in SEQ ID No.14), Nb119 (amino acid sequence as shown in SEQ ID No.5, nucleotide sequence as shown in SEQ ID No.15), Nb146 (amino acid sequence as shown in SEQ ID No.6, nucleotide sequence as shown in SEQ ID No.16), Nb147 (amino acid sequence as shown in SEQ ID No.7, nucleotide sequence as shown in SEQ ID No.17), Nb151 (amino acid sequence as shown in SEQ ID No.8, nucleotide sequence as shown in SEQ ID No.18), Nb155 (amino acid sequence as shown in SEQ ID No.19), Nb206 (amino acid sequence as shown in SEQ ID No.20, nucleotide sequence as shown in SEQ ID No.21), Nb207 (amino acid sequence as shown in SEQ ID No.22, nucleotide sequence as shown in SEQ ID No.23), Nb208 (amino acid sequence as shown in SEQ ID No.24, nucleotide sequence as shown in SEQ ID No.25), Nb210 (amino acid sequence as shown in SEQ ID No.25, nucleotide sequence as shown in SEQ ID No.26), Nb211 (amino acid sequence as shown in SEQ ID No.26, nucleotide sequence as shown in SEQ ID No.27), Nb212 (amino acid sequence as shown in SEQ ID No.27, nucleotide sequence as shown in SEQ ID No.28), Nb213 (amino acid sequence as shown in SEQ ID No.29 No.9, nucleotide sequence as SEQ ID No.19) and Nb175 (amino acid sequence as SEQ ID No.10, nucleotide sequence as SEQ ID No.20), the purification results are shown in Figure 4 shown.

[0033] Example 4: Cell viability assay (MTS) to detect the blocking function of TRAIL nanobodies (1) Add 20 μL of 1 μg / mL human TRAIL protein into a 96-well cell culture plate, and then add an equal volume of cell culture medium or different concentrations of TRAIL nanoantibodies. The control group directly adds 40 μL of cell culture medium. (2) 1.25×10 cells were plated in each cell culture well. 5Jurkat cells (purchased from ATCC), 60 μL / well. The plated cells were placed in a 37°C, 5% CO2 incubator and cultured for 24 hours; (3) Add 10 μL of MTS to each well and continue culturing for 3 hours. Then measure the absorbance at 490 nm. The cell viability is calculated as follows: Cell viability (%) = (OD 对照组 -OD 实验组 ) / OD 对照组 ×100%.

[0034] The results of cell viability assays were Figure 5 As shown, all 10 TRAIL nanobodies can block the TRAIL-DR5 pathway to inhibit cell apoptosis, among which Nb19 has the best effect. 5 μg / mL Nb19 can increase the cell viability from 16% to 96% after TRAIL protein treatment alone. The TRAIL-DR5 pathway blocking effect is the best, so Nb19 is used for the following examples.

[0035] The plasmid map of pComb3XSS-Nb19 is as follows Figure 6 shown.

[0036] Example 5: TRAIL Nanobody Affinity Determination Using the Sartorius Octet® R4 system to detect: (1) The HIS1K sensor (purchased from Sartorius, Germany) was pre-wetted in PBST buffer for 10 min, and then cured with Nb19 for 180 s at a curing concentration of 10 ug / ml; (2) Human TRAIL protein and mouse TRAIL protein (purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.) were diluted to 500 nM, 250 nM, 125 nM, 62.5 nM and 0 nM respectively and added to the sample wells. The binding and dissociation time was set to 180 s. (3) When measuring different concentrations, rinse with 10 mM Glycine (purchased from Sigma, USA) to regenerate the sensor; (4) Calculate the equilibrium dissociation constant (K) by real-time monitoring of the biofilm thickness D ), K D The smaller the value, the stronger the affinity. The calculation formula is: K D =Kd / Ka.

[0037] The measurement results are shown in Table 1: Table 1 Affinity determination results of Nb19 with mouse and human TRAIL proteins As shown in Table 1, the binding affinity of nanobody Nb19 to human TRAIL protein is higher than that to mouse TRAIL protein, which is 2.4×10 -9 M and 2.425×10 -7 M.

[0038] Example 6: Study on the protective effect of TRAIL nanobody on cerebral ischemia-reperfusion injury (1) The mouse model of middle cerebral artery occlusion / reperfusion (MCAO / R) was established. C57BL / 6N mice weighing about 23 g, aged 6-8 weeks, were selected (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) and anesthetized with intraperitoneal injection of sodium pentobarbital. The body temperature of the mice was monitored and maintained at 36.5-37.5℃ using a feedback heating pad. The left common carotid artery, internal carotid artery, and external carotid artery of the mice were dissected and exposed. The distal end of the external carotid artery and the proximal end of the common carotid artery were ligated, and then the external carotid artery was opened obliquely and a nylon suture was inserted into the anterior cerebral artery to block the blood flow of the middle cerebral artery. After 2 hours of occlusion of the middle cerebral artery, the nylon suture was removed and reperfusion was performed. PBS, 30 mg / kg Nb19, or 90 mg / kg Nb19 were injected into the mice through the tail vein 5 minutes before reperfusion. Each mouse was injected with 200 μL, and there were 5 mice in each group.

[0039] (2) TTC staining. The mouse brain was removed 24 hours after reperfusion, quickly frozen and cut into thin slices. The slices were placed in a preheated 2% 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) solution for staining for 30 min. After washing with PBS, the slices were placed face up and photographed. Finally, the infarction rate was calculated using Image J software.

[0040] (3) mNSS. Motor, sensory, reflex, and balance abilities were assessed 24 h after reperfusion. The maximum deficit score was 18. The higher the score, the more severe the neurological dysfunction.

[0041] like Figure 7 As shown in the figure, 30 and 90 mg / kg Nb19 reduced the brain infarction rate of MCAO / R model mice by 63% and 82%, respectively, and the mNSS values ​​were significantly reduced. The experimental results show that Nb19 plays a protective role against cerebral ischemia-reperfusion injury.

[0042] In summary, the present invention provides an anti-TRAIL nanobody, which has a small molecular weight and strong tissue penetrability. The anti-TRAIL nanobody has good binding affinity with human TRAIL protein, and the anti-TRAIL nanobody has good TRAIL-DR5 pathway blocking activity, can significantly reduce ischemia-reperfusion injury after cerebral infarction, and has broad clinical application prospects.

[0043] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An anti-TRAIL nanobody, characterized in that The amino acid sequence of the anti-TRAIL nanobody is selected from any one of the following: as shown in SEQ ID No.1, as shown in SEQ ID No.2, as shown in SEQ ID No.3, as shown in SEQ ID No.4, as shown in SEQ ID No.5, as shown in SEQ ID No.6, as shown in SEQ ID No.7, as shown in SEQ ID No.8, as shown in SEQ ID No.9 and as shown in SEQ ID No.

10.

2. An antibody, characterized in that: The antibody comprises two or more nano antibody units repeated in series, and the amino acid sequence of the nano antibody unit is shown in any one of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9 and SEQ ID No.

10.

3. The gene fragment encoding the anti-TRAIL nanobody according to claim 1, characterized in that: The nucleotide sequence of the gene fragment is shown in any one of SEQ ID No.11, SEQ ID No.12, SEQ ID No.13, SEQ ID No.14, SEQ ID No.15, SEQ ID No.16, SEQ ID No.17, SEQ ID No.18, SEQ ID No.19 and SEQ ID No.

20.

4. An expression vector, characterized in that: The expression vector contains at least one gene fragment according to claim 3.

5. A host cell, characterized in that: The host cell contains the expression vector according to claim 4 or the gene fragment according to claim 3 is integrated into its genome.

6. An immunoconjugate, characterized in that: The immunoconjugate contains an antibody portion and a conjugated portion; wherein the antibody portion is selected from the anti-TRAIL nanobody described in claim 1 or the antibody described in claim 2.

7. The immunoconjugate according to claim 6, characterized in that: The coupling part is one or more of a detectable marker, a drug, a toxin, a cytokine, a radionuclide, an enzyme, a gold nanoparticle, a nanorod, a nanomagnetic particle and a virus coat protein.

8. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises: (1) one or more of the anti-TRAIL nanobody according to claim 1, the antibody according to claim 2, or the immunoconjugate according to claim 6 or 7; (2) or / and a pharmaceutically acceptable carrier for the antibody or immunoconjugate described in (1).

9. A method for preparing an anti-TRAIL nanobody, characterized in that: The host cell according to claim 5 is cultured and the expression product is purified to obtain the anti-TRAIL nanobody.

10. Use of the pharmaceutical composition according to claim 8 in developing drugs for treating acute cerebral infarction.

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