A neural stem cell and its application in repairing spinal cord injury

Abstract

The application discloses a kind of fusion polypeptide TGNL and its modified neural stem cell in the application of spinal cord injury repair.The fusion polypeptide TGNL is composed of TAT penetration domain, GSTP targeting domain, NICDi inhibiting domain, MMP-2 activation domain and flexible Linker connecting them, and full-length amino acid sequence is shown as SEQ ID NO.1.The application also provides the preparation method of the polypeptide, neural stem cell (hNSCs-TGNL) expressing the polypeptide and its construction method.Experiments show that the fusion polypeptide TGNL can efficiently penetrate nerve cells, specifically target glial scar, be selectively activated by MMP-2, and promote neural stem cell differentiation into neuron by inhibiting Notch signal;hNSCs-TGNL has high survival rate and strong penetration ability in in-vitro glial scar model, can significantly promote motor function recovery, reduce injury volume, increase axon density and reduce glial scar in rat spinal cord contusion model.The application provides a new candidate drug and cell treatment strategy for clinical treatment of spinal cord injury, and has significant clinical application value.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a neural stem cell and its application in repairing spinal cord injuries. Background Technology

[0002] Spinal cord injury (SCI) is a highly disabling traumatic disease of the central nervous system, seriously threatening human health. Epidemiological studies show that traffic accidents account for 40%-50% of SCI cases, while falls from heights, violent behavior, and sports injuries account for 20%-30%, 10%-15%, and 5%-10%, respectively. World Health Organization data shows that the global annual incidence of SCI is approximately (40-80) per million people, and it is on the rise with the development of modern transportation and extreme sports. After SCI occurs, local ischemia-reperfusion injury of the spinal cord triggers acute necrosis and delayed apoptosis of nerve cells, leading to the interruption of nerve conduction pathways, causing limb paralysis, sensory loss, and autonomic dysfunction, placing a heavy burden on families and society.

[0003] Current clinical treatments for spinal cord injury (SCI) have limited efficacy. While 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 only provides neuroprotection within 8 hours of injury, and long-term use can easily lead to adverse reactions such as gastrointestinal ulcers and immunosuppression. Biological agents such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are difficult to achieve effective concentrations at the injury site due to blood-spinal cord barrier barriers and enzymatic metabolism; phase III clinical trials showed that their neurological function improvement rate was less than 15%. Rehabilitation therapy can improve motor function and self-care ability, but it cannot achieve fundamental repair of the neural structure.

[0004] The multi-directional differentiation potential of neural stem cells (NSCs) offers hope for the treatment of spinal cord injury (SCI), but simple transplantation faces numerous challenges. In the inhibitory microenvironment formed after SCI, macrophages and microglia release pro-inflammatory factors such as TNF-α and IL-6, activating the Caspase-3 pathway and inhibiting NSC survival. Overproliferating astrocytes and chondroitin sulfate proteoglycans form a physical barrier and secrete growth-inhibiting factors, hindering axonal regeneration. Simultaneously, the lack of neurotrophic factors and insufficient angiogenesis at the injury site limits NSC survival, differentiation, and integration with the host nervous system. 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 novel NSC modification technologies to overcome these bottlenecks and advance the treatment of SCI. Summary of the Invention

[0005] The core objective of this invention is to provide a specially modified neural stem cell that, by introducing specific fusion peptides, possesses a stronger ability to survive, migrate, differentiate, and promote neural repair in the microenvironment of spinal cord injury. Simultaneously, this invention provides a method for applying this neural stem cell in repairing spinal cord injury, thereby significantly improving the neurological function recovery of patients with spinal cord injury.

[0006] Therefore, this invention discloses a fusion peptide TGNL. The fusion peptide TGNL consists of a TAT penetration domain, a GSTP targeting domain, a NICDi inhibition domain, an MMP-2 activation domain, and a flexible linker connecting them, with its full-length amino acid sequence shown in SEQ ID NO.1. Specifically, the TAT domain is responsible for cell penetration, the GSTP domain targets the highly expressed CHI3L1 protein in glial scars, the NICDi domain antagonizes Notch signaling, and the MMP-2 sensitive linker (PLGLAG) ensures specific activation of the peptide at the site of injury. The fusion peptide TGNL was synthesized using a solid-phase chemical synthesis method, purified by HPLC (purity ≥98%), and stored at -80℃ for later use.

[0007] This invention also discloses a neural stem cell expressing the fusion polypeptide TGNL and its construction method: First, the nucleic acid sequence encoding TGNL (SEQ ID NO.2) is cloned into the pCDNA3.1 vector (containing the CMV promoter and G418 resistance gene) to construct the recombinant vector pCDNA3.1-TGNL. Human embryonic neural stem cells (hNSCs) are transfected with the recombinant vector using Lipofectamine 3000. A stable expression line hNSCs-TGNL is obtained through selection with G418 (400 μg / mL), and polypeptide expression is verified by Western blot.

[0008] The fusion peptide TGNL and the modified neural stem cells hNSCs-TGNL of this invention have significant beneficial effects: the TAT domain, after optimization, increases cell penetration 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 at the injury site (efficiency up to 92%); the NICDi domain effectively inhibits Notch signaling (Hes1 / Hey1 downregulated by 59.2%-68.5%), and significantly promotes the differentiation of neural stem cells into neurons (differentiation rate 68.3% ± 5.1%); 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 increase the BBB score of spinal cord injury rats 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 scars by 67.1%, breaking through the bottleneck of spinal cord injury repair through multiple mechanisms.

[0009] This technology holds promise for development into novel cell preparations or peptide drugs for the treatment of spinal cord injuries, enabling precise repair through local injection. It is particularly suitable for patients with acute and subacute spinal cord injuries. Furthermore, its modular design concept can be extended to targeted therapy for 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. Attached Figure Description

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

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

[0012] Figure 3 Western blot results of hNSCs-TGNL. Detailed Implementation

[0013] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the 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 this invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

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

[0016] I. Design and Preparation of Fusion Peptides

[0017] Three key issues need to be addressed in spinal cord injury repair: ① large molecules have difficulty penetrating nerve cell membranes; ② drugs accumulate inefficiently at the injury site; and ③ excessive activation of Notch signaling inhibits nerve regeneration. Based on these issues, this invention designs a multi-domain fusion peptide that achieves efficient repair through a three-tiered 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 peptide designed above is named the fusion peptide TGNL, and its amino acid sequence is shown in SEQ ID NO.1: YGRKKRRQRRRR-GGGGSGGGGSGGGGS-CRHSQMTVTSRL-GGGGSGGGGSGGGGS-PLGLAG-GGGGSGGGGSGGGGS-GWNNNDYEPQV.

[0021] The final fusion peptide TGNL (SEQ ID NO.1) was sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis. After synthesis, the HPLC purity was ≥98%, and it was stored at -80℃ for later use.

[0022] II. Detection of Fusion Peptides

[0023] (a) Cell penetration efficiency

[0024] Experimental procedure: Primary cultured rat spinal cord neurons (isolated from the spinal cord of newborn SD rats, with a purity >90% as determined by β-IIItubulin immunofluorescence) were seeded at 5 × 10⁶ cells / well in 24-well plates. After adhesion, the cells were divided into two groups: the experimental group was treated with FITC-labeled fusion peptide TGNL (10 μM), and the control group was treated with FITC-labeled unmodified TAT peptide (without arginine added at the C-terminus, 10 μM). The cells were incubated at 37°C and 5% CO₂ for 1 h. The cells were washed three times with PBS (5 min each time) to remove free peptides. After digestion with 0.25% trypsin, the cells were collected, and the proportion of FITC-positive cells was detected by flow cytometry. Each group was tested three times independently.

[0025] The experimental results are shown in Table 2: The cell penetration efficiency of the fusion peptide TGNL was significantly higher than that of the unmodified TAT group (P<0.01). The introduction of C-terminal arginine prolonged the peptide action time by enhancing the anti-protease degradation ability, which increased the penetration efficiency by 24%, confirming its advantage in nerve cell delivery and providing a prerequisite for the subsequent functional domains to play their role.

[0026] Table 2 Results of cell penetration efficiency detection

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

[0028] (II) Targeted treatment of glial scars

[0029] Experimental procedure: SD rats (n=3) with Allen's method model 7 days old were anesthetized, and spinal cord tissue was harvested and 10μm frozen sections were prepared. The sections were fixed with 4% paraformaldehyde for 15 min, blocked with 5% BSA for 30 min, and incubated with FITC-labeled fusion peptide TGNL (5μM) at 37℃ for 1 h. After washing with PBS, CHI3L1 primary antibody was added and incubated overnight at 4℃. Cy3-labeled secondary antibody was added and incubated at room temperature for 1 h. The nuclei were stained with DAPI. The sections were observed under a confocal microscope, 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 for each group.

[0030] Experimental results showed that the average fluorescence intensity of FITC in the glial scar area (CHI3L1 positive) was 285.6±21.3 (n=6), while that in the normal spinal cord tissue area was 54.9±8.7 (n=6). One-way ANOVA showed F=189.2, P<0.001, with the former being 5.2 times ±0.6 times that of the latter, and the co-localization rate of FITC and Cy3 reached 89.7%±4.2%. Therefore, the fusion peptide TGNL was significantly enriched in the glial scar area with high CHI3L1 expression and highly co-localized with the target protein CHI3L1, confirming its specific targeting ability for spinal cord injury lesions and providing experimental evidence for reducing off-target effects and increasing local drug concentration.

[0031] (III) MMP-2 activation specificity

[0032] Experimental procedure: The fusion peptide TGNL (10 μM) was divided into 3 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 a specially prepared Western blot (the primary antibody was a TGNL peptide-specific antibody, which was a polyclonal antibody prepared by immunizing rabbits with multiple high doses of the peptide, 1:1000).

[0033] Experimental results show that ( Figure 1Two clear bands (approximately 6.3 kDa and 3.1 kDa) were observed in the MMP-2 group, consistent with the expected cleavage product. Only the full-length polypeptide band (approximately 9.4 kDa) was observed in the MMP-9 group and the enzyme-free control group, with a cleavage rate <5%. One-way ANOVA showed that the cleavage efficiency of the MMP-2 group was significantly higher than that of the other groups. Therefore, the fusion polypeptide TGNL can be specifically cleaved and activated by MMP-2, but not by its homologous protease MMP-9, confirming its enzyme specificity. This matches the pathological characteristic of high MMP-2 expression in the spinal cord injury site, supporting its "lesion-targeted activation" design concept and reducing non-specific effects in normal tissues.

[0034] (iv) Notch signal suppression

[0035] Experimental procedure: Third-generation neural stem cells (NSCs, purity >95% as determined by Nestin immunofluorescence) were seeded at 2 × 10⁶ cells / well in 6-well plates. The experimental group was treated with the fusion peptide TGNL (5 μM), while the control group was treated with an equal volume of PBS. The cells were cultured at 37°C for 24 h. Total RNA was extracted using the Trizol method, and cDNA was synthesized by reverse transcription. The mRNA expression of Hes1, Hey1, and the internal reference GAPDH was detected by qPCR. The relative expression levels were calculated using the 2-ΔΔCt method. Each group was repeated in triplicate.

[0036] The experimental results are shown in Table 3: After treatment with the fusion peptide TGNL, the mRNA expression of downstream target genes Hes1 and Hey1 of Notch signaling was significantly downregulated (P < 0.001), confirming that the NICDi domain can competitively bind to the Notch1 receptor and effectively block the Notch signaling pathway, 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] III. Summary

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

[0042] Example 2: Preparation of cells (hNSCs-TGNL) with fusion peptide TGNL

[0043] I. Cell Culture (hNSCs Culture System Adapted for Fusion Peptide Expression)

[0044] Low-passage (P3-P5) human embryonic neural stem cells (hNSCs, purity >95% as determined by Nestin immunofluorescence) were seeded in T25 cell culture flasks coated with poly-L-lysine (10 μg / mL). The culture medium used was complete neural stem cell culture medium: 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, and 100 μg / mL streptomycin, with an additional 2 mM L-glutamine (to maintain cell metabolism) and 10 ng / mL leukemia inhibitory factor (LIF, to inhibit differentiation). The cells were cultured in a cell culture incubator at 37℃, 5% CO2, and 95% humidity. Half of the culture medium was replaced every two days. When the cell clones reached a diameter of 150-200 μm or a confluence of 70%-80%, they were digested with Accutase digestion solution (superior to trypsin-EDTA, reducing cell damage) at 37℃ for 5 min, centrifuged at 1000 rpm for 5 min, and then passaged at a 1:3 ratio. During passage, cell viability was assessed weekly using trypan blue staining (ensuring >90%), and Nestin positivity was assessed by flow cytometry (ensuring >90%) to ensure the cells remained in an undifferentiated state.

[0045] II. Construction of Fusion Peptide Gene Expression Vector

[0046] 1. Design and synthesis of target genes

[0047] Based on the fusion polypeptide TGNL sequence (SEQ ID NO.1), the nucleotide sequence optimized by this codon 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 then chemically synthesized by GenScript.

[0048] 2. Carrier Construction

[0049] The pCDNA3.1 vector (containing the CMV promoter and G418 resistance gene) was extracted and digested with BamHI and XhoI, respectively, along with the synthesized target gene fragment. The reaction system (20 μL) contained 1 μg DNA, 2 μL 10×FastDigest Buffer, 1 μL BamHI, 1 μL XhoI, and ddHO to make up the volume. The reaction was carried out at 37℃ for 1 h. The digestion products were separated by 1% agarose gel electrophoresis (120V, 30 min), and the vector fragment and target gene fragment were recovered. Ligation was performed at a vector:target gene ratio of 1:3, incubated overnight at 16℃, and transformed into DH5α competent cells. The cells were plated on LB agar plates containing ampicillin (100 μg / mL) and cultured at 37℃ for 16 h. Three single colonies were picked, and the plasmid was extracted, verified by BamHI / XhoI double digestion, and sequenced. The sequence was confirmed to be correct and the reading frame was correct. 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 double-digested with BamHI and XhoI, along with the synthesized target gene fragment. The reaction system (20 μL) contained 1 μg DNA, 2 μL 10×FastDigest Buffer, 1 μL BamHI, 1 μL XhoI, and ddHO to make up the volume. The reaction was carried out at 37℃ for 1 h. The digestion products were separated by 1% agarose gel electrophoresis (120V, 30 min) and purified using a DNA gel extraction kit. At a vector:target gene ratio of 1:3 (molar ratio), T4 DNA ligase (1 μL) and 10×T4 Ligase Buffer (2 μL) were added, and ligation was carried out overnight at 16℃. The ligation product was transformed into DH5α competent cells, plated on LB agar plates containing ampicillin (100 μg / mL), and incubated at 37°C for 16 h. Three single colonies were picked, and plasmids were extracted after shaking. The plasmids were verified by double digestion with BamHI / XhoI and sent for sequencing. The recombinant vector with correct sequence and reading frame was named pEGFP-TGNL.

[0051] III. Liposome Transfection (Efficient Introduction of hNSCs)

[0052] One day before transfection, hNSCs were seeded at 1×10⁶ cells / well in 6-well plates coated with poly-L-lysine, and 2 mL of complete culture medium was added. Cells were cultured until confluence reached 70%-80%. To prepare the transfection complex: 2 μg of pCDNA3.1-TGNL plasmid was added to 250 μL of Opti-MEM medium, along with 3 μL of P3000 reagent, and mixed well. Separately, 5 μL of Lipofectamine 3000 was added to 250 μL of Opti-MEM, mixed well, and incubated at room temperature for 5 min. The two solutions were then mixed and incubated at room temperature for 20 min to form the liposome-DNA complex. The old culture medium was removed, and 1.5 mL of fresh complete culture medium was added to each well. 500 μL of the complex was added dropwise, gently shaken, and incubated at 37°C for 48-72 h.

[0053] Transfection efficiency assessment: Due to the lack of fluorescent tags, a parallel-well control method was used—cells from the same batch were transfected with pEGFP-N1 (1 μg / well), and the GFP positivity rate was observed under a fluorescence microscope after 48 hours (transfection efficiency reached 65%-75%). Figure 2 This indirectly reflects the transfection efficiency of pCDNA3.1-TGNL.

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

[0055] Preliminary experiments determined the minimum lethal concentration of G418 for hNSCs: Cells were seeded in 24-well plates, and medium containing 100-800 μg / mL G418 was added. After 10 days of observation, the minimum lethal concentration was determined to be approximately 400 μg / mL. 72 hours after transfection, the transfected hNSCs were replaced with complete medium containing 400 μg / mL G418 for selection. The selection medium was changed every 3 days (while removing floating dead cells). After 14 days of selection, positive cell clones were observed to form. Single clones were picked using a cloning loop and transferred to 24-well plates for expansion culture. Once the cells reached confluence, they were progressively passaged to T25 flasks. Western blot analysis (primary antibody was an anti-TGNL peptide-specific antibody, a polyclonal antibody prepared by immunizing rabbits with the peptide, 1:1000) was used to verify the expression of the fusion peptide. Figure 3 The expected band size is approximately 9.4 kDa. During cryopreservation, cells were resuspended in cryopreservation solution containing 10% DMSO, 40% complete culture medium, and 50% fetal bovine serum, and the concentration was adjusted to 2 × 10 cells / mL. After aliquoting, cells were incubated overnight at -80°C in a programmed cooling box and then transferred to liquid nitrogen for long-term storage, labeled as hNSCs-TGNL.

[0056] Example 3: Application of hNSCs-TGNL

[0057] I. In vitro functional verification

[0058] Experimental Procedure: Third-generation hNSCs-TGNL (experimental group), hNSCs transfected with empty vector (control group), and unmodified hNSCs (blank group) were seeded at 5 × 10⁶ cells / well in 24-well plates containing primary astrocytes (simulating a glial scar model) pre-coated with TGF-β1 (10 ng / mL) for activation. Cells were co-cultured in complete neural stem cell culture medium at 37°C and 5% CO₂ for 7 days. Cell morphology was observed daily. On day 7, the following methods were used to assess cell viability: ① CCK-8 assay for cell viability; ② β-III tubulin / GFAP immunofluorescence dual-labeling counting of neuronal differentiation rate (proportion of β-III tubulin+ cells); ③ Notch signaling activity (detected using a Hes1 promoter-driven dual-luciferase reporter gene assay, expressed as relative fluorescence intensity); ④ Z-stack scanning using confocal microscopy to measure the maximum depth of cell penetration into the glial scar.

[0059] The experimental results are shown in Table 4. hNSCs-TGNL showed significant advantages in the glial scar model: cell survival rate increased by 21.1% compared with the control group, neuronal differentiation rate increased by about 3 times, Notch signaling activity was inhibited by 78%, and scar penetration depth increased by nearly 2 times. This confirms that the fusion peptide 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] II. Rat spinal cord contusion model

[0063] Experimental Procedure: A T10 spinal cord contusion model in SD rats was established using the modified Allen's method (200g × 25mm impact force). [Rats were anesthetized with 10% chloral hydrate (300mg / kg) via intraperitoneal injection and fixed in a prone position on the operating table. After routine disinfection and draping, the skin was incised along the midline of the back, muscles were separated, and the T10 vertebral lamina was exposed. The T10 vertebral lamina was carefully removed using bone forceps to fully expose the spinal cord. A spinal cord impactor was used, with the impact force adjusted to 200g × 25mm, to vertically strike the spinal cord, causing spinal cord injury. Local contusions and hemorrhages were observed after the impact. After confirming successful injury, the muscles and skin were sutured layer by layer.] Rats were randomly divided into 3 groups (n=10): Group A (hNSCs-TGNL transplantation), Group B (ordinary hNSCs transplantation), and Group C (saline control). Twenty-four hours post-injury, cell suspension (1×10 cells / μL, 2μL per point) or an equal volume of saline was injected at four points around the injury center using a stereotaxic instrument. BBB scores were assessed at 1, 2, 4, and 8 weeks post-surgery (double-blind method, three times weekly). At 8 weeks post-surgery, rats were sacrificed, and spinal cord tissue from the injured segment was harvested. Injury volume was calculated using HE staining (ImageJ software), new axon density was counted using β-III tubulin immunofluorescence, and the average thickness of the glial scar was measured using GFAP immunofluorescence.

[0064] The experimental results are shown in Table 5. At 8 weeks post-operation, the BBB score of group A (hNSCs-TGNL) was significantly higher than that of groups B and C. The lesion volume was reduced by 68.4%, the density of new axons increased by 1.7 times, and the thickness of glial scars was reduced by 67.1%. This indicates that hNSCs-TGNL can effectively promote the recovery of motor function after spinal cord injury, reduce the lesion range, promote axon regeneration, and inhibit excessive glial scar formation. Its in vivo treatment effect is better than that of ordinary hNSCs.

[0065] Table 5 Comparison of Experimental Results

[0066]

[0067] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

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

1.

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

2.

3. An expression vector, characterized by, The expression vector comprises the nucleotide sequence shown as SEQ ID NO. 2 according to claim 2.

4. The expression vector of claim 3, wherein, 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 of claim 5, wherein, The cell is human embryonic neural stem cell, named hNSCs-TGNL.

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