Neural stem cell and application thereof in repairing spinal cord injury

Abstract

The invention discloses a fusion polypeptide TGNL and application of neural stem cells modified by the fusion polypeptide TGNL in spinal cord injury repair. The fusion polypeptide TGNL is composed of a TAT penetrating domain, a GSTP targeting domain, an NICDi inhibition domain, an MMP-2 activation domain and a flexible Linker connecting the three domains, and the full-length amino acid sequence is as shown in SEQ ID NO. 1. The invention also provides a preparation method of the polypeptide, a neural stem cell (hNSCs-TGNL) for expressing the polypeptide and a construction method of the neural stem cell (hNSCs-TGNL). Experiments show that the fusion polypeptide TGNL can efficiently penetrate nerve cells, specifically target glial scars, is selectively activated by MMP-2, and promotes neural stem cells to differentiate into neurons by inhibiting Notch signals; the hNSCs-TGNL has high survival rate and strong penetrability in an in-vitro glial scar model, and can significantly promote motor function recovery, reduce injury volume, increase axon density and reduce glial scars in a rat spinal cord contusion model. The invention provides a novel candidate drug and a cell treatment strategy for clinical treatment of spinal cord injury, and has remarkable clinical application value.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a neural stem cell and its application in repairing spinal cord injury. Background Art

[0002] Spinal cord injury (SCI) is a traumatic central nervous system disorder with a high disability rate, posing a serious threat to human health. Epidemiological studies indicate that traffic accidents account for 40%-50% of the causes of SCI, while falls from height, violent acts, and sports injuries account for 20%-30%, 10%-15%, and 5%-10%, respectively. According to the World Health Organization, the global annual incidence of SCI is approximately 40-80 per million people, and is increasing with the development of modern transportation and extreme sports. After SCI occurs, localized ischemia-reperfusion injury to the spinal cord triggers acute necrosis and delayed apoptosis of nerve cells, leading to interrupted neural pathways, limb paralysis, sensory loss, and autonomic dysfunction, placing a heavy burden on families and society.

[0003] Current clinical treatments for SCI have limited efficacy. Although surgery can stabilize the structure through spinal fixation and decompression, it cannot repair damaged nerves and carries risks such as postoperative infection and epidural hematoma. High-dose methylprednisolone has a neuroprotective effect only within 8 hours of injury, and long-term use can easily lead to adverse reactions such as gastrointestinal ulcers and immunosuppression. Biologics such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are affected by the blood-spinal cord barrier and enzymatic metabolism, making it difficult to form effective concentrations at the site of injury. Phase III clinical trials have shown that their neurological function improvement rate is less than 15%. Although rehabilitation therapy can improve motor function and self-care ability, it cannot achieve fundamental repair of neural structure.

[0004] The multidirectional differentiation potential of neural stem cells (NSCs) brings hope for the treatment of SCI, but simple transplantation faces many challenges. In the inhibitory microenvironment formed after SCI, macrophages and microglia release proinflammatory cytokines such as TNF-α and IL-6, which activate the Caspase-3 pathway to inhibit the survival of NSCs; overproliferating astrocytes and chondroitin sulfate proteoglycans form a physical barrier and secrete growth inhibitory factors, hindering the regeneration of nerve axons; at the same time, the lack of neurotrophic factors and insufficient angiogenesis in the injured area limit the survival, differentiation and integration of NSCs with host nerves. Meta-analysis shows that the cell survival rate after traditional NSC transplantation is less than 10%, and the neuronal differentiation rate is less than 5%. Therefore, there is an urgent need to develop new NSCs modification technologies to break through bottlenecks and promote the development of SCI treatment. Summary of the Invention

[0005] The core purpose of the present invention is to provide a specially modified neural stem cell that, by introducing a specific fusion polypeptide, has a stronger ability to survive, migrate, differentiate and promote neural repair in the spinal cord injury microenvironment. At the same time, a method for applying the neural stem cell in repairing spinal cord injury is provided to significantly improve the neurological function recovery of patients with spinal cord injury.

[0006] Therefore, on the one hand, the present invention discloses a fusion polypeptide TGNL. The fusion polypeptide TGNL consists of a TAT penetration domain, a GSTP targeting domain, a NICDi inhibitory domain, an MMP-2 activation domain and a flexible linker connecting them. The full-length amino acid sequence is shown in SEQ ID NO.1. Among them, the TAT domain is responsible for cell penetration, the GSTP domain targets the CHI3L1 protein highly expressed in glial scars, the NICDi domain antagonizes the Notch signal, and the MMP-2 sensitive linker (PLGLAG) ensures that the polypeptide is specifically activated at the local site of injury. The fusion polypeptide TGNL was synthesized by solid-phase chemical synthesis, purified by HPLC (purity ≥98%), and stored at -80°C for future use.

[0007] The present invention also discloses a neural stem cell expressing the fusion polypeptide TGNL and a method for constructing the same. First, a nucleic acid sequence encoding TGNL (SEQ ID NO. 2) is cloned into a pCDNA3.1 vector (containing a CMV promoter and a G418 resistance gene) to construct a recombinant vector, pCDNA3.1-TGNL. The recombinant vector is then transfected into human embryonic neural stem cells (hNSCs) using Lipofectamine 3000. Stable expression strains of hNSCs-TGNL are obtained by screening with G418 (400 μg / mL), and polypeptide expression is verified by Western blot.

[0008] The fusion polypeptide TGNL and modified neural stem cells hNSCs-TGNL of the present invention have significant beneficial effects: after optimization, the TAT domain increases the cell penetration rate by 24%, the GSTP domain achieves targeted enrichment of glial scars (fluorescence intensity is 5.2 times that of normal tissue), the MMP-2 sensitive linker ensures local activation of the injury (efficiency reaches 92%), and the NICDi domain effectively inhibits Notch signaling (Hes1 / Hey1 downregulation by 59.2%-68.5%), significantly promoting the differentiation of neural stem cells into neurons (differentiation rate 68.3%±5.1%); the hNSCs-TGNL has a survival rate of 92.3% and a scar penetration depth of 285 μm in an in vitro model. In vivo, it can improve the BBB score of rats with spinal cord injury to 17.2, reduce the injury volume by 68.4%, increase the density of new axons by 1.7 times, and reduce the thickness of glial scar by 67.1%. Multiple mechanisms synergistically break through the bottleneck of spinal cord injury repair.

[0009] This technology is expected to be developed into a new cell preparation or peptide drug for the treatment of spinal cord injury, achieving precise repair through local injection, and is particularly suitable for patients with acute and subacute spinal cord injury. At the same time, its modular design concept can be extended to the targeted treatment of other neurodegenerative diseases (such as Parkinson's disease and Alzheimer's disease), providing innovative solutions for the field of central nervous system injury and repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 MMP-2 activation specificity detection results (Western blot).

[0011] Figure 2 GFP results were observed under a fluorescence microscope.

[0012] Figure 3 Western blot detection results of hNSCs-TGNL. DETAILED DESCRIPTION

[0013] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0014] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0015] Example 1: Design, preparation and testing of fusion polypeptide TGNL

[0016] 1. Design and Preparation of Fusion Peptides

[0017] There are three key issues to be addressed in spinal cord injury repair: ① It is difficult for large molecules to penetrate the nerve cell membrane; ② The drug is poorly accumulated at the injury site; ③ Overactivation of Notch signaling inhibits nerve regeneration. Based on this, the present invention designs a multi-domain fusion polypeptide to achieve efficient repair through a three-level synergistic mechanism of "cell penetration-scar targeting-signal regulation". The design of each domain is shown in Table 1

[0018] Table 1 Design, sequence and results of each domain

[0019]

[0020] The polypeptide designed above is named fusion polypeptide TGNL, and its amino acid sequence is shown in SEQ ID NO. 1: YGRKKRRQRRRR-GGGGSGGGGSGGGGS-CRHSQMTVTSRL-GGGGSGGGGSGGGGS-PLGLAG-GGGGSGGGGSGGGGS-GWNNNDYEPQV.

[0021] The final obtained fusion polypeptide TGNL (SEQ ID NO. 1) was sent to Shengong Biotechnology (Shanghai) Co., Ltd. for synthesis, and after synthesis, the HPLC purity was ≥98%, and it was stored at -80°C for standby.

[0022] II. Test of the fusion polypeptide

[0023] (I) Cell penetration efficiency

[0024] Experimental procedure: Take primary cultured 7d rat spinal cord neurons (isolated from the spinal cord of newborn SD rats, and identified by β-III tubulin immunofluorescence with a purity of >90%), and inoculate 5x10 cells / well in a 24-well plate. After adhesion, divide into two groups: the experimental group is added with FITC-labeled fusion polypeptide TGNL (10 μM), and the control group is added with FITC-labeled unmodified TAT polypeptide (C-terminal without arginine, 10 μM), and incubate at 37°C, 5% CO for 1h; wash with PBS for 3 times (5min each time) to remove free polypeptide, collect cells after trypsin digestion, and detect the proportion of FITC positive cells by flow cytometry, with 3 independent repeated experiments for each group.

[0025] The experimental results are shown in Table 2: the cell penetration efficiency of the fusion polypeptide TGNL is significantly higher than that of the unmodified TAT group (P<0.01), and the introduction of C-terminal arginine prolongs the action time of the polypeptide by enhancing the anti-protease degradation ability, so that the penetration efficiency is relatively improved by 24%, which confirms its advantage in neural cell delivery and provides a premise for the subsequent function domain to play a role.

[0026] Table 2: Results of cell penetration efficiency detection

[0027] Group Positive cell rate (%) Standard deviation (±) Relative promotion rate (%) Fusion polypeptide TGNL group 89.3 3.2 24 Unmodified TAT group 72.1 2.8 -

[0028] (II) Glial scar targeting

[0029] Experimental procedures: SD rats (n=3) were anesthetized for 7 days after Allen's method modeling. Spinal cord tissue was obtained and 10 μm frozen sections were prepared. The tissues were fixed with 4% paraformaldehyde for 15 minutes, blocked with 5% BSA for 30 minutes, and incubated with FITC-labeled fusion peptide TGNL (5 μM) at 37°C for 1 hour. After washing with PBS, CHI3L1 primary antibody was added dropwise and incubated at 4°C overnight. Cy3-labeled secondary antibody was incubated at room temperature for 1 hour, and nuclei were stained with DAPI. Confocal microscopy was used for observation, and the FITC fluorescence intensity of CHI3L1-positive areas (glial scars) and normal spinal cord tissue was quantitatively analyzed using Image-ProPlus 6.0 software. Six fields of view were analyzed in each group.

[0030] The experimental results showed that the mean FITC fluorescence intensity in the glial scar region (CHI3L1-positive) was 285.6±21.3 (n=6) and 54.9±8.7 (n=6) in the normal spinal cord tissue region. One-way ANOVA showed F=189.2, P<0.001, indicating that the former was 5.2-fold±0.6-fold greater than the latter. Furthermore, the colocalization rate of FITC and Cy3 reached 89.7%±4.2%. Therefore, the fusion peptide TGNL was significantly enriched in the glial scar region with high CHI3L1 expression and highly colocalized with the target protein CHI3L1, confirming its specific targeting ability to spinal cord injury lesions and providing experimental evidence for reducing off-target effects and increasing local drug concentrations.

[0031] (III) MMP-2 activation specificity

[0032] Experimental procedures: The fusion peptide TGNL (10 μM) was divided into three groups: ① MMP-2 group (recombinant human MMP-2 added, 1 μg / mL), ② MMP-9 group (recombinant human MMP-9 added, 1 μg / mL), and ③ control group (no enzyme added). All groups were incubated in reaction buffer (50 mM Tris-HCl, 10 mM CaCl, pH 7.5) at 37°C for 2 h. The reaction products were detected and analyzed by special Western blot (primary antibody was anti-TGNL peptide-specific antibody, which was a polyclonal antibody prepared by multiple high-dose immunization of rabbits with peptide, 1:1000).

[0033] The experimental results show that ( Figure 1) : Two clear bands (molecular weight approximately 6.3kDa and 3.1kDa) were observed in the MMP-2 group, consistent with the expected cleavage product; only the full-length polypeptide band (approximately 9.4kDa) was observed in the MMP-9 group and the no-enzyme control group, with a cleavage rate of <5%. One-way ANOVA showed that the cleavage efficiency in the MMP-2 group was significantly higher than that in the other groups. Therefore, the fusion peptide TGNL can be specifically cleaved and activated by MMP-2, but not by the homologous protease MMP-9, confirming the enzyme specificity of its activation. This matches the pathological characteristics of high MMP-2 expression in localized spinal cord injury, supporting its design concept of "lesion-targeted activation" and reducing nonspecific effects in normal tissues.

[0034] (IV) Notch signaling inhibition

[0035] Experimental procedures: Third-generation neural stem cells (NSCs, purity >95% as determined by Nestin immunofluorescence) were seeded in 6-well plates at 2×10 cells / well. The experimental group was treated with the fusion peptide TGNL (5 μM), while the control group was treated with an equal amount of PBS. The cells were cultured at 37°C for 24 h. Total RNA was extracted using Trizol and reverse-transcribed into cDNA. The mRNA expression of Hes1, Hey1, and the internal control GAPDH was detected by qPCR, and the relative expression levels were calculated using the 2-ΔΔCt method. Each group was repeated three times.

[0036] The experimental results are shown in Table 3: After treatment with the fusion polypeptide TGNL, the mRNA expression of Notch signaling downstream target genes Hes1 and Hey1 was significantly downregulated (P < 0.001), confirming that the NICDi domain can competitively bind to the Notch1 receptor, effectively blocking the Notch signaling pathway, and providing molecular mechanism support for promoting the differentiation of NSCs into neurons and inhibiting the excessive proliferation of glial cells.

[0037] Table 3 Results of Hes1 and Hey1 mRNA content detection

[0038]

[0039]

[0040] 3. Summary

[0041] The fusion polypeptide TGNL prepared in this example has a correct structure and a purity of >98%, and has the functions of efficient cell penetration, specific targeting of glial scars, controllable activation of MMP-2, and Notch signal inhibition.

[0042] Example 2: Preparation of cells (hNSCs-TGNL) fused with the polypeptide TGNL

[0043] 1. Cell Culture (hNSCs Culture System for Expression of Adaptive Fusion Peptides)

[0044] Low-passage (P3-P5) human embryonic neural stem cells (hNSCs, purity >95% as determined by Nestin immunofluorescence) were seeded into T25 cell culture flasks coated with poly-lysine (10 μg / mL). The culture medium used was a complete medium specifically for neural stem cells: DMEM / F12 basal medium supplemented with 2% B27 (without vitamin A), 20 ng / mL EGF, 20 ng / mL bFGF, 1% N2 supplement, 100 U / mL penicillin, 100 μg / mL streptomycin, 2 mM L-glutamine (to maintain cell metabolism), and 10 ng / mL leukemia inhibitory factor (LIF, to inhibit differentiation). Culture in a cell culture incubator at 37°C, 5% CO, and 95% humidity. Change half the medium every two days. When the cell spheres reach a diameter of 150-200 μm or a confluency of 70%-80%, digest with Accutase (superior to trypsin-EDTA to minimize cell damage) at 37°C for 5 minutes. After centrifugation at 1000 rpm for 5 minutes, cells are passaged at a 1:3 ratio. During the passage process, cell viability is tested weekly by trypan blue staining (ensuring >90%), and Nestin positivity is tested by flow cytometry (ensuring >90%) to ensure that the cells are in an undifferentiated state.

[0045] 2. Construction of fusion polypeptide gene expression vector

[0046] 1. Target gene design and synthesis

[0047] Based on the fusion polypeptide TGNL sequence (SEQ ID NO.1), the codon-optimized nucleotide sequence is shown in SEQ ID NO.2; BamHI and XhoI restriction sites were introduced at both ends of the SEQ ID NO.2 sequence, and the product was chemically synthesized by GenScript.

[0048] 2. Vector Construction

[0049] The pCDNA3.1 vector (containing the CMV promoter and G418 resistance gene) was extracted and digested with the synthesized target gene fragment using BamHI and XhoI, respectively: the reaction system (20 μL) contained 1 μg of DNA, 2 μL of 10× FastDigest Buffer, 1 μL of BamHI, and 1 μL of XhoI, supplemented with ddHO, and incubated at 37°C for 1 hour. The digestion products were separated by 1% agarose gel electrophoresis (120V, 30 minutes), and the vector and target gene fragments were recovered. Ligation was performed at a molar ratio of 1:3 vector:target gene, incubated at 16°C overnight, and transformed into DH5α competent cells. The cells were plated on LB plates containing ampicillin (100 μg / mL) and incubated at 37°C for 16 hours. Three single colonies were selected, and the plasmids were extracted and verified by BamHI / XhoI double digestion and sequencing to confirm the correct sequence and reading frame. The recombinant vector was named pCDNA3.1-TGNL.

[0050] The pEGFP-N1 vector (containing the CMV promoter and EGFP tag for easy expression tracking) was extracted and digested with the synthesized target gene fragment using BamHI and XhoI, respectively. The reaction system (20 μL) contained 1 μg of DNA, 2 μL of 10× FastDigest Buffer, 1 μL of BamHI, and 1 μL of XhoI, supplemented with ddHO. The reaction was incubated at 37°C for 1 hour. The digestion products were separated by 1% agarose gel electrophoresis (120 V, 30 minutes) and purified using a DNA gel extraction kit. T4 DNA ligase (1 μL) and 10× T4 Ligase Buffer (2 μL) were added at a molar ratio of 1:3 vector:target gene, and ligation was carried out overnight at 16°C. The ligation product was transformed into DH5α competent cells, spread on LB plates containing ampicillin (100 μg / mL), and cultured at 37°C for 16 h. Three single colonies were picked, shaken, and the plasmid was extracted. The plasmid was verified by BamHI / XhoI double enzyme digestion and sent for sequencing. The recombinant vector with correct sequence and reading frame was named pEGFP-TGNL.

[0051] 3. Lipofectamine transfection (high-efficiency introduction into hNSCs)

[0052] One day before transfection, seed hNSCs at 1×10 cells / well in a poly-lysine-coated 6-well plate. Add 2 mL of complete medium and culture until the cells reach 70%-80% confluency. Prepare the transfection complex: add 2 μg of pCDNA3.1-TGNL plasmid to 250 μL of Opti-MEM medium and 3 μL of P3000 reagent, mix well. Separately, add 5 μL of Lipofectamine 3000 to 250 μL of Opti-MEM, mix well, and incubate at room temperature for 5 minutes. Combine the two solutions and incubate at room temperature for 20 minutes to form the liposome-DNA complex. Aspirate the old medium, add 1.5 mL of fresh complete medium to each well, add 500 μL of the complex dropwise, shake gently, and incubate at 37°C for 48-72 hours.

[0053] Transfection efficiency assessment: Because there is no fluorescent label, the parallel well control method is used - the same batch of cells are transfected with pEGFP-N1 (1μg / well), and the GFP positive rate is observed under a fluorescence microscope after 48 hours (the transfection efficiency can reach 65%-75%, Figure 2 ), which indirectly reflects the transfection efficiency of pCDNA3.1-TGNL.

[0054] IV. Screening of cell lines (obtaining stable expression lines)

[0055] A preliminary experiment determined the minimum lethal concentration of G418 for hNSCs: the cells were seeded in a 24-well plate, culture medium containing 100-800 μg / mL G418 was added, and the cells were observed for 10 days to determine that the minimum lethal concentration was approximately 400 μg / mL. 72 hours after transfection, the hNSCs transfected above were replaced with complete culture medium containing 400 μg / mL G418 for screening, and the screening culture medium was replaced every 3 days (while removing floating dead cells). After 14 days of screening, positive cell clones were formed, and single clones were picked with a cloning ring and transferred to a 24-well plate for expanded culture. After the cells were fully grown, they were gradually passaged to T25 flasks, and the expression of the fusion polypeptide was verified by Western blot (the primary antibody was an anti-TGNL polypeptide-specific antibody, a polyclonal antibody prepared by immunizing rabbits with polypeptides, 1:1000) Figure 3 For cryopreservation, cells were resuspended in freezing buffer containing 10% DMSO, 40% complete culture medium, and 50% fetal bovine serum to a concentration of 2 × 10 cells / mL. Aliquots were then placed in a programmed cooling box at -80°C overnight and transferred to liquid nitrogen for long-term storage. The cells were labeled hNSCs-TGNL.

[0056] Example 3: Application of hNSCs-TGNL

[0057] 1. In vitro functional verification

[0058] Experimental Procedure: 3rd-passage hNSCs-TGNL (experimental group), empty vector-transfected hNSCs (control group), and unmodified hNSCs (blank group) were seeded at 5×10 cells / well in 24-well plates pre-coated with TGF-β1 (10 ng / mL)-activated primary astrocytes (simulating a glial scar model). Co-cultured with complete neural stem cell medium was performed at 37°C and 5% CO for 7 days. Cell morphology was observed daily, and on day 7, the following assays were performed: ① Cell viability was assessed by CCK-8 assay; ② Neuronal differentiation was assessed by β-III tubulin / GFAP double-labeling (proportion of β-III tubulin-positive cells relative to total cells); ③ Notch signaling activity was assessed by Hes1 promoter-driven dual-luciferase reporter gene expression, expressed as relative fluorescence intensity; and ④ Confocal microscopy was used to measure the maximum depth of cell penetration into the glial scar using Z-stack scanning.

[0059] The experimental results are shown in Table 4. hNSCs-TGNL showed significant advantages in the glial scar model: the cell survival rate was increased by 21.1% compared with the control group, the neuronal differentiation rate increased by about 3 times, the Notch signaling activity was inhibited by 78%, and the scar penetration depth increased by nearly 2 times, confirming that the fusion polypeptide TGNL can enhance the survival, directional differentiation and penetration ability of hNSCs in the scar microenvironment, and its mechanism is related to the inhibition of Notch signaling.

[0060] Table 4 Comparison of experimental results

[0061]

[0062] 2. Rat Spinal Cord Contusion Model

[0063] Experimental procedures: A modified Allen's method was used to establish a T10 spinal cord contusion model in SD rats (200g×25mm impact force) [The rats were anesthetized with 10% chloral hydrate (300mg / kg) intraperitoneally and fixed on the operating table in a prone position. Routine disinfection and draping were performed, and the skin was incised along the midline of the back to separate the muscles and expose the 10th thoracic segmental vertebral lamina. Use bone rongeurs to carefully bite off the 10th thoracic segmental vertebral lamina to fully expose the spinal cord. Use a spinal cord striker, adjust the impact force to 200g×25mm, and strike the spinal cord vertically to cause spinal cord injury. After the impact, local contusion and bleeding of the spinal cord were visible. After confirming that the injury was successful, the muscles and skin were sutured layer by layer.], and the rats were randomly divided into 3 groups (n=10): Group A (hNSCs-TGNL transplantation), Group B (ordinary hNSCs transplantation), and Group C (normal saline control). Twenty-four hours after injury, a cell suspension (1×10 cells / μL, 2 μL per site) or an equal volume of saline was injected into four points surrounding the central area of ​​injury using a stereotaxic apparatus. BBB scores were performed 1, 2, 4, and 8 weeks after surgery (double-blind, three times per week). Eight weeks after surgery, the rats were sacrificed, and spinal cord tissue from the injured segment was obtained. Lesion volume was calculated by HE staining (ImageJ software). β-III tubulin immunofluorescence was used to count new axonal densities, and GFAP immunofluorescence was used to measure average glial scar thickness.

[0064] The experimental results are shown in Table 5. Eight weeks after surgery, the BBB score of group A (hNSCs-TGNL) was significantly higher than that of groups B and C. The injury volume was reduced by 68.4%, the density of new axons increased by 1.7 times, and the thickness of glial scar decreased by 67.1%. These results indicate that hNSCs-TGNL can effectively promote motor function recovery after spinal cord injury, reduce the scope of injury, promote axon regeneration, and inhibit excessive glial scar formation. Its in vivo therapeutic effect is superior to that of ordinary hNSCs.

[0065] Table 5 Comparison of experimental results

[0066]

[0067] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A fusion polypeptide TGNL, characterized in that: The amino acid sequence of the fusion polypeptide TGNL is shown in SEQ ID NO.

1.

2. The fusion polypeptide TGNL according to claim 1, characterized in that The nucleotide sequence of the fusion polypeptide TGNL after codon optimization is shown in SEQ ID NO.

2.

3. An expression vector, characterized in that The expression vector comprises the nucleic acid molecule of claim 2.

4. The expression vector according to claim 3, characterized in that The vector is pCDNA3.

1.

5. A host cell, characterized in that The cell is transformed or transfected with the expression vector according to claim 3 or 4.

6. The host cell according to claim 5, characterized in that The cells are human embryonic neural stem cells, named hNSCs-TGNL.

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