CN109400711B - PDGFR beta targeting tumor necrosis factor related apoptosis inducing ligand variant and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a tumor necrosis factor related apoptosis inducing ligand variant, which is tumor necrosis factor related apoptosis inducing ligand variantDeath inducing ligand and ZPDGFRβThe fusion protein of, ZPDGFRβIs connected with the N terminal or the C terminal of the TRAIL through a linker. The invention also discloses a nucleotide sequence, a recombinant vector and a recombinant bacterium comprising the nucleotide sequence, and a preparation method and application of the variant. The TRAIL variant protein Z-hTRAIL has good tumor killing activity, and also has definite curative effect on liver fibrosis, and has good clinical application prospect.
Description
Technical Field
The invention relates to the field of biotechnology drugs, in particular to a PDGFR beta targeting tumor apoptosis-promoting inducing ligand variant and a preparation method and application thereof.
Background
The tumor necrosis factor apoptosis-related inducing ligand (TRAIL) belongs to a Tumor Necrosis Factor (TNF) family member, and the C-terminal 114-281-bit amino acid of the TRAIL can be hydrolyzed into a soluble extracellular segment by protease to form a homotrimer, and has receptor binding capacity. Membrane receptors for TRAIL include death receptors (DR4 and DR5) and decoy receptors (DcR1 and DcR 2). The death receptors DR4 and DR5 contain death structural domain in molecule, and can transmit death signal to cell after being combined with TRAIL to induce cell death. In contrast, the decoy receptor DcR1 molecule does not contain a death domain, and the death domain of DcR2 is incomplete, both of which are capable of binding to TRAIL but are unable to transmit a death signal and therefore do not induce apoptosis. Tumor cells and some abnormally activated cells all highly express death receptors, and TRAIL can promote apoptosis of the cells. Normal cells often express decoy receptors at high levels to protect them from TRAIL. Thus, TRAIL may be developed as an ideal therapeutic drug.
Researches show that the surface of tumor cells highly expresses death receptors, so that the TRAIL shows super tumor cell killing activity under the in vitro condition, and is considered as a potential anti-tumor drug. However, the in vivo antitumor effect of TRAIL is not matched to its in vitro killing activity against tumor cells. Possible reasons include: 1) TRAIL has small molecular weight and short half-life in vivo. The solution includes using polyethylene glycol modification or serum albumin fusion/combination to prolong half-life. 2) Because the decoy receptor is widely expressed in normal tissues, TRAIL is greatly consumed by the normal tissues after entering the body, and the quantity of TRAIL reaching tumor sites is small, so the anti-tumor effect is poor. The improved method is mainly to utilize guide molecules to deliver TRAIL, so that the TRAIL is enriched at tumor parts, and the anti-tumor effect is further improved. The delivery of TRAIL has been studied using tumor cells or tumor neovascular endothelial cells as target cells. Pericytes are important vascular wall cells, are distributed behind vascular endothelial cells, and have important regulation and control functions on the formation and the stability of blood vessels. However, there has been no study on the delivery of TRAIL into tumors using pericytes as target cells.
Hepatic stellate cell activation is the initiating link in hepatic fibrosis. Inhibition or elimination of activated hepatic stellate cells can delay the process of hepatic fibrosis. The research shows that death receptors DR4 and DR5 are highly expressed during the activation process of the hepatic stellate cells. Thus, TRAIL is able to induce apoptosis of activated hepatic stellate cells to show anti-fibrotic effects. Long-acting TRAIL modified by PEG has been studied to alleviate hepatic fibrosis progression in rats. However, TRAIL lacks targeting to hepatic stellate cells, and thus has poor therapeutic effect.
Disclosure of Invention
In order to solve the problems, the invention provides a PDGFR beta targeting tumor apoptosis-inducing ligand variant and a preparation method and application thereof.
The tumor necrosis factor related apoptosis inducing ligand variant of the present invention is tumor necrosis factor related apoptosis inducing ligandBody and ZPDGFRβThe fusion protein of, ZPDGFRβIs connected with the N terminal or the C terminal of the TRAIL through a linker.
Wherein, Z isPDGFRβThe amino acid sequence of (a) is as shown in SEQ ID NO: 1 is shown. Preferably, Z isPDGFRβConsisting of SEQ ID NO: 2 in sequence listing.
Wherein, the amino acid sequence of the TRAIL is shown as SEQ ID NO: 3, respectively. Preferably, the tnf-related apoptosis-inducing ligand consists of SEQ ID NO: 4.
Wherein the linker consists of 2-20 amino acids.
Wherein the linker is (G4S)3A linker having the amino acid sequence set forth in SEQ ID NO: 5, respectively. Preferably, the linker consists of SEQ ID NO: 6.
Wherein the amino acid sequence of the variant is shown as SEQ ID NO: shown at 7. Preferably, the variant consists of SEQ ID NO: 8 in sequence listing.
The invention also provides a nucleotide sequence which comprises the coding sequence of the TRAIL and ZPDGFRβThe coding sequence of (1), which are linked by the coding sequence of a linker.
Wherein, Z isPDGFRβThe coding sequence of (A) is shown in SEQ ID NO: 2, respectively.
Wherein, the coding sequence of the TRAIL is shown as SEQ ID NO: 4, respectively.
Wherein the linker is (G4S)3A linker having the nucleotide sequence set forth in SEQ ID NO: and 6.
Wherein, it is shown as SEQ ID NO: shown in fig. 8.
The invention also provides a recombinant vector or a recombinant bacterium of the nucleotide sequence.
The invention also provides a method for preparing the TRAIL variant, which is characterized by comprising the following steps: the gene is prepared by taking the nucleotide sequence as a target fragment and adopting a genetic engineering method.
The invention also provides the application of the TRAIL variant in preparing medicines for treating cell proliferative diseases.
Wherein, the medicament for treating the cell proliferative diseases is a medicament for treating tumors or autoimmune diseases. The tumor is colorectal adenocarcinoma.
The invention also provides an anti-tumor medicament which is a preparation prepared by taking the tumor necrosis factor-related apoptosis-inducing ligand variant as an active ingredient and adding pharmaceutically acceptable auxiliary materials.
The invention also provides the application of the TRAIL variant in preparing medicines for treating organ fibrosis diseases.
Wherein the medicament is a medicament for treating liver fibrosis.
The invention also provides a medicament for treating liver fibrosis, which is a preparation prepared by taking the tumor necrosis factor-related apoptosis-inducing ligand variant as an active ingredient and adding pharmaceutically acceptable auxiliary materials.
The invention utilizes the affinity Z capable of specifically recognizing PDGFR betaPDGFRβAs a guide molecule, the fusion protein Z-hTRAIL is constructed by fusing the targeting molecule with human TRAIL (hTRAIL), and the in vivo and in vitro anti-tumor and anti-hepatic fibrosis effects of the targeting molecule Z-hTRAIL are found to be obviously enhanced compared with hTRAIL, so that the targeting molecule has good clinical application prospect.
According to the above-mentioned aspects of the present invention, many other modifications, substitutions, or alterations can be made without departing from the basic technical concept of the present invention as defined by the general technical knowledge and common practice in the field relevant to the present invention.
Drawings
FIG. 1 molecular design of variant Z-hTRAIL;
FIG. 2 SDS-PAGE electrophoresis of Z-hTRAIL and hTRAIL;
FIG. 3Z-hTRAIL and hTRAIL gel filtration chromatography;
FIG. 4Z-killing of tumor cells following binding of hTRAIL and hTRAIL to pericytes;
FIG. 5 tumor targeting comparison of Z-hTRAIL and hTRAIL; a, in-tumor enrichment and in-vivo observation B, tissue distribution;
FIG. 6 in vivo therapeutic effect of Z-hTRAIL and hTRAIL on LS174T tumor;
FIG. 7 in vivo therapeutic effect of Z-hTRAIL and hTRAIL on COLO205 tumors;
FIG. 8 in vivo therapeutic efficacy of Z-hTRAIL and hTRAIL on HCT116 tumors;
FIG. 9Z-binding of hTRAIL and hTRAIL to hepatic stellate cells (A) and killing (B);
FIG. 10Z-hTRAIL and mouse liver function indices following hTRAIL treatment;
FIG. 11Z-hTRAIL and hTRAIL treatment following sirius red staining of mouse liver tissue;
FIG. 12Z-hTRAIL and mouse liver tissue fibrosis score (A) and hydroxyproline content (B) after hTRAIL treatment;
FIG. 13 comparison of the cell killing in vitro (A) and the anti-tumor effect in vivo (B, C) of the control proteins Zf-TRAIL and TRAIL.
Detailed Description
The present invention will be described in further detail with reference to the following examples. It should not be understood that the scope of the above-described subject matter of the present invention is limited to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Example 1 molecular design and cloning construction of Z-hTRAIL variants
1. Molecular design of Z-hTRAIL variants
ZPDGFRβConsisting of 58 amino acids (table 1). hTRAIL is a fragment consisting of the amino acids of the extracellular segment 114-281 of human TRAIL (see Table 1). By (G4S)3Linker will ZPDGFRβIs connected to the N terminal of hTRAIL to construct fusion protein ZPDGFRβ-(G4S)3hTRAIL (Z-hTRAIL for short) (FIG. 1).
2. Construction of Z-TRAIL variant expression vector
Based on the amino acid sequence of ZPDGFR beta, the initial coding gene is designed and then the soft coding gene is analyzed by nucleic acidAfter the optimization, the gene sequence was synthesized by Nanjing Kinshire. EcoRI/BamHI restriction endonuclease sites (EcoRI: gattc/BamHI ggatcc) were added to both ends of the sequence during synthesis. By double digestion and ligation, Z isPDGFRThe gene sequence of β was loaded onto the expression plasmid of pQE30-hTRAIL (the nucleotide sequence of the loaded Z-hTRAIL fragment is shown in Table 1). After the ligation product was transformed into TOP10 E.coli competence, a recombinant plasmid was obtained. Discovery of Z by sequencing of recombinant plasmidsPDGFRThe beta fragment was successfully loaded into an expression plasmid vector. The expression plasmid pQE30-Z-hTRAIL is transferred into M15 escherichia coli competence, and an expression strain M15-pQE30-Z-hTRAIL is successfully constructed.
TABLE 1 amino acid and nucleotide sequences of fusion proteins to which the invention relates
EXAMPLE 2 protein expression and isolation and purification of Z-TRAIL variant
The expression strain M15-pQE30-Z-hTRAIL (prepared in example 1) was picked up and inoculated into double-resistant (ampicillin-containing 100. mu.g/ml, kanamycin 30. mu.g/ml) LB liquid medium, and cultured with shaking at 37 ℃ until the concentration of A is reached600When the temperature is about 0.8, 0.05mM Isopropyl-beta-D-thiogalactoside (Isopropyl beta-D-1-thiogalactopyranoside, IPTG) is added, and shaking induction culture is carried out at 26 ℃ for 14-16 hours. The cells were collected by centrifugation (7000g, 10min), resuspended in lysine buffer (50mM phosphate buffer, pH 8.0; 300mM NaCl; 20mM imidazole; 10 mM. beta. -mercaptoethanol), phenylmethylsulfonyl fluoride (PMSF) was added to a final concentration of 1mM, and disrupted by sonication in ice bath (power 300W, working for 10s, interval 30s, total 40 min). After the completion of the disruption, centrifugation (4 ℃, 25000g, 10min) was performed for 4 times, and the disrupted supernatant was collected. The supernatant obtained was mixed with Ni-NTA resin gel (purchased from Qiagen) in appropriate volumesMix at a ratio (e.g., V/V-50: 1) and bind with shaking at 4 ℃ for 2 h. The combined gel filler was poured into a chromatographic column, and after the protein sample was passed through the column, the gel column was washed with Wash buffer (50mM phosphate buffer, pH 8.0; 300mM NaCl; 40mM imidazole; 10 mM. beta. -mercaptoethanol) over 30 column volumes. The protein samples were then collected by Elution with an Elution buffer (50mM phosphate buffer, pH 7.6; 300mM NaCl; 300mM imidazole; 10 mM. beta. -mercaptoethanol) and shown as a single band by SDS-PAGE (FIG. 2). Gel filtration chromatography showed a single peak (fig. 3).
The results show that we obtained the pure variant protein Z-hTRAIL using phosphate buffered saline PBS (10mM Na)2HPO4,137 mM NaCl,2.68mM KCl,2mM KH2PO4pH 7.4) was dialyzed overnight for further use.
Example 3 killing of peripheral tumor cells after binding of Z-TRAIL variant to peripheral cells
After incubation with pericytes using antibodies specific for PDGFR β, they were analyzed by flow cytometry. The results are shown in a of fig. 4, where PDGFR β is highly expressed on the pericyte surface. Pericytes were co-incubated with FAM-labeled Z-hTRAIL (prepared as described in example 2, FAM-labeled) or hTRAIL, and flow cytometry analysis revealed that Z-hTRAIL could bind to pericytes and that this binding could be blocked by PDGFR beta-specific antibodies, indicating fusion of Z-hTRAILPDGFRβAllowing hTRAIL to bind to the pericytes. To determine whether Z-hTRAIL bound to pericytes could also kill tumor cells, pericytes were preincubated for 1h with Z-hTRAIL (prepared in example 2) (1. mu.M), washed with PBS, and tumor cells (LS174T and HCT116, 1.5X 10)4；COLO205，2*104) The cells were co-cultured overnight and the cell viability was measured by CCK-8.
As a result, as shown in B of FIG. 4, the survival rate of tumor cells decreased with the increase in the number of pericytes incubated with Z-hTRAIL. After coculture with pericytes incubated with hTRAIL, the survival of tumor cells was not significantly related to the number of pericytes. This demonstrates that Z-hTRAIL of the present invention can bind to pericytes and kill tumor cells. hTRAIL, however, does not bind to pericytes and therefore cannot kill tumor cells.
EXAMPLE 4 tumor-Targeted analysis of Z-TRAIL variants
The Z-hTRAIL (prepared in example 2) of the present invention was mixed-labeled with the fluorescent dye CF750 at a molar ratio of 1:8 after adjusting its pH to 8.0. After 1h reaction at room temperature, free CF750 fluorochrome was removed by dialysis against PBS buffer. The marked protein is injected into a LS174T tumor-bearing nude mouse model through tail vein, and the tumor targeting of Z-hTRAIL is explored by using a small animal living body Imaging system SPECTRAL Lago and Lago X Imaging Systems.
The result shows that 0.5h after administration, the tumor part of the Z-hTRAIL group has stronger fluorescence signal, the intensity of the fluorescence signal is obviously higher than that of the hTRAIL group, and the kidney parts of two groups of tumor-bearing nude mice can detect the stronger fluorescence signal. At the subsequent time points (1,2,4,6h), strong fluorescence signals were still detected at the tumor sites in the Z-hTRAIL group, whereas the hTRAIL group fluorescence signals rapidly decreased until they disappeared (FIG. 5A). After 6h, the tumor-bearing mice were sacrificed and the main organs and tumor tissues thereof were removed and then scanned, and the results showed that strong fluorescence signals could be detected in the tumor tissues of the Z-hTRAIL group, and the signal intensity was about 3 times that of the hTRAIL group (fig. 5B).
These results indicate that Z-hTRAIL of the present invention can be enriched at tumor sites compared to hTRAIL, showing better tumor targeting.
EXAMPLE 5 in vivo anti-tumor Effect of Z-TRAIL variant
The Z-hTRAIL (prepared in example 2) of the present invention was further evaluated for its anti-tumor effect in vivo using LS174T, HCT116 and COLO205 tumor-bearing nude mouse models.
In LS174T tumor (colorectal adenocarcinoma) model, hTRAIL 10mg/kg and the same number of moles of variant Z-hTRAIL were administered three times via the tail vein, and at the time of sacrifice of nude mice at day 16, the PBS group had an average tumor size of 850. + -. 150mm3The hTRAIL group is 390 plus or minus 80mm3While the Z-hTRAIL group is the smallest and only 102 + -75 mm3(ii) a The mean tumor weights were 0.652. + -. 0.2g for PBS, 0.302. + -. 0.608g for hTRAIL, and only 0.091. + -. 0.039g for Z-hTRAIL (FIG. 6).
In COLO205 tumor (colorectal adenocarcinoma) model, 5mg/kg of hTRAIL and the same number of moles of variant Z-hTRAIL were administered twice via the tail vein, and at the time of sacrifice of nude mice at day 20, PBS groupThe average size of tumor body is 670 +/-140 mm3The hTRAIL group is 400 +/-80 mm3While the Z-hTRAIL group is the smallest and only 70 + -60 mm3(ii) a The average tumor weights were 0.266. + -. 0.114g in PBS, 0.23. + -. 0.053g in hTRAIL, and only 0.038. + -. 0.035g in Z-hTRAIL (FIG. 7).
In the HCT116 tumor (colorectal adenocarcinoma) model, hTRAIL at 10mg/kg and the same number of moles of the variant Z-hTRAIL were administered four times via the tail vein, and at the time of sacrifice of nude mice at day 19, the PBS group had an average tumor size of 782. + -. 106.53mm3The hTRAIL group is 368.4 +/-104.6 mm3While the Z-hTRAIL group is the smallest and only 205.1 + -74.6 mm3(ii) a The average tumor body weight was 0.491. + -. 0.032g in PBS group, 0.319. + -. 0.06g in hTRAIL group, and only 0.11. + -. 0.036g in Z-hTRAIL group (FIG. 8).
These results indicate that Z-hTRAIL of the present invention has a stronger in vivo anti-tumor ability than hTRAIL, indicating that fusion ZPDGFRβCan obviously enhance the in vivo anti-tumor effect of hTRAIL, and particularly has definite curative effect on colorectal cancer.
Example 6 anti-hepatic fibrosis Effect of Z-TRAIL variant
Activated hepatic stellate cells are key cells to promote liver fibrosis. Activated stellate cells highly express PDGFR β and death receptors, and therefore Z-hTRAIL may be more advantageous in binding and killing activated stellate cells than hTRAIL. FAM-labeled proteins were incubated with activated stellate cells and then found by flow assay to bind more Z-hTRAIL (prepared in example 2) to activated stellate cells than hTRAIL. The stellate cells were activated by treatment with different concentrations of protein and the number of residual cells was determined the next day with CCK 8. The cell killing rate of the protein was calculated with the cell survival rate of PBS treatment as 100%. It was revealed that the cell survival rates after the action of Z-hTRAIL at 10 and 20nM were 24.6. + -. 4.5% and 14.2. + -. 1.2%, respectively, while the cell survival rates after the action of hTRAIL at the same dose were 81.4. + -. 4.8% and 59.7. + -. 0.25%, respectively (FIG. 9). This indicates that Z-hTRAIL has a stronger ability to kill activated stellate cells than hTRAIL under in vitro conditions.
To compare the in vivo anti-fibrotic effects of Z-hTRAIL and hTRAIL, we established a mouse liver fibrosis model. Carbon tetrachloride was dissolved in olive oil at a concentration of 25%. Four-week-old female C57 mice were injected intraperitoneally with dissolved carbon tetrachloride twice a week at an injection dose of 2.5ml/kg for the first time, and then 5ml/kg for the subsequent time. At 4 weeks of modeling, the experimental mice were divided into a model group and a treatment group. Model groups were injected with PBS and treatment groups were given Z-hTRAIL or hTRAIL protein (10mg/kg), respectively, twice weekly. During treatment, three groups of mice maintained carbon tetrachloride injections. Serum from mice was collected on the sixth week and tested for serum glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase, and total bilirubin. The liver tissue is sliced, the collagen fiber is displayed by using sirius red staining, the fibrosis degree is scored according to the ISHAK standard, and meanwhile, the hydroxyproline in the liver is detected by using a kit to reflect the content of the collagen fiber.
Fig. 10 shows that the liver function indexes (serum glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase and total bilirubin) in the model group are significantly different from those in the normal group, which indicates that the liver cells are seriously damaged and the modeling is successful.
The levels of the three serum indexes of the hTRAIL treatment group have no obvious difference with those of the model group. However, the three serum indexes of the Z-hTRAIL treatment group are lower than those of the hTRAIL treatment group. Sirius red staining showed less intrahepatic collagen deposition in the Z-hTRAIL treated group than in the hTRAIL treated group (fig. 11), and lower fibrosis scores and hydroxyproline levels than in the hTRAIL treated group (fig. 12).
The above results indicate that Z-hTRAIL of the present invention has a better therapeutic effect on mouse liver fibrosis than hTRAIL.
Comparative example 1 Effect of fusion of other aptamers on TRAIL antitumor Effect
To illustrate the fusion ZPDGFRβOf importance for enhancing TRAIL Activity, we selected ZPDGFRβThe affinity entities Z having similar sequences but different specificitiesFcRnAs a control. Will ZFcRnConnecting with hTRAIL to construct fusion protein Zf-hTRAIL. Further comparison with hTRAIL determines whether the fusion control affibody can also enhance the antitumor effect of hTRAIL.
1. Molecular design and preparation of Zf-hTRAIL variant
According to example 1, Z isFcRnLigation to the N-terminus of hTRAIL to constructFusion protein Zf-hTRAIL. The fusion protein Zf-hTRAIL was prepared according to example 2 using the E.coli expression system under the same conditions as for Z-hTRAIL.
2. In vitro killing and in vivo anti-tumor effects of Zf-hTRAIL variant on tumor cells
To test the killing activity of Zf-hTRAIL on tumor cells, LS174T cells (1X 10) were first tested4One) were seeded in 96-well plates and different concentrations of protein were added after overnight adherence. After overnight exposure, the number of residual cells was determined by adding CCK-8. The same volume of PBS was added to the control wells. The killing efficiency of Zf-hTRAIL on tumor cells was calculated with the cell survival rate of PBS-treated group as 100%. Comparing with hTRAIL to judge fusion ZFcRnWhether the killing activity of hTRAIL on tumor cells is influenced. The results are shown in FIG. 13A, where Zf-hTRAIL kills LS174T cells with a similar efficiency as hTRAIL, indicating that fusion Z is fusedFcRnHas no significant effect on the activity of hTRAIL.
Zf-hTRAIL was compared with TRAIL for LS174T tumor treatment effect according to the method described in example 5. 7 days after the nude mice were inoculated with LS174 cells subcutaneously, Zf-hTRAIL, hTRAIL or PBS was injected into the tail vein. Tumor volumes were then measured daily and growth curves were plotted. At the end of the observation, the mice were sacrificed and the tumor bodies were stripped and weighed. As shown in FIG. 13B, Zf-hTRAIL and hTRAIL treated groups showed slower tumor growth rate and lighter tumor body weight than PBS treated group. However, there was no significant difference between Zf-hTRAIL and hTRAIL-treated tumors, neither growth rate nor tumor weight, indicating that fusion affibody Z wasFcRnThe in vivo anti-tumor effect of hTRAIL was not significantly enhanced. TABLE 2 control protein amino acid and nucleotide sequences
In conclusion, the present invention utilizes the affinity Z capable of specifically recognizing PDGFR betaPDGFRβTo be guideThe fusion protein Z-hTRAIL constructed by fusing it with human TRAIL (hTRAIL) has obviously higher in vivo and in vitro anti-tumor and anti-hepatic fibrosis effects than hTRAIL, and adopts other recognition factors, such as ZFcRnThe constructed fusion protein has poor effect.
Claims (12)
1. Tumor necrosis factor related apoptosis inducing ligandA variant characterized by: it is a TRAIL and ZPDGFRβThe fusion protein of, ZPDGFRβConnecting to the N-terminal of TRAIL through a linker; z isPDGFRβThe amino acid sequence of (a) is as shown in SEQ ID NO: 1 is shown in the specification; the amino acid sequence of the TRAIL is shown as SEQ ID NO: 3 is shown in the specification; the linker is (G4S)3A linker having the amino acid sequence set forth in SEQ ID NO: 5, respectively.
2. The TRAIL variant according to claim 1, wherein: z isPDGFRβConsisting of SEQ ID NO: 2; and/or, the TRAIL is composed of SEQ ID NO: 4; and/or, the linker consists of SEQ ID NO: 6.
3. The TRAIL variant according to claim 1 or 2, wherein: the amino acid sequence is shown as SEQ ID NO: shown at 7.
4. The TRAIL variant according to claim 3, wherein: it consists of SEQ ID NO: 8 in sequence listing.
5. A nucleotide, characterized in that: it includes the coding sequence of TRAIL and ZPDGFRβThe coding sequences of (a) and (b) are connected by a coding sequence of a linker; zPDGFRβConnecting to the N-terminal of TRAIL through a linker; z isPDGFRβThe coding sequence of (A) is shown in SEQ ID NO: 2 is shown in the specification; the coding sequence of the TRAIL is shown as SEQ ID NO: 4 is shown in the specification; the linker is (G4S)3A linker having the nucleotide sequence set forth in SEQ ID NO: and 6.
6. The nucleotide of claim 5, wherein: the nucleotide sequence is shown as SEQ ID NO: shown in fig. 8.
7. A recombinant vector or recombinant bacterium comprising the nucleotide of claim 5 or 6.
8. A method for preparing TRAIL variant is characterized in that: it is prepared by taking the nucleotide as the target fragment according to claim 5 or 6 and adopting a genetic engineering method.
9. Use of the TRAIL variant of any one of claims 1-4 in the preparation of a medicament for treating tumor or liver fibrosis.
10. Use according to claim 9, characterized in that: the tumor is colorectal adenocarcinoma.
11. An antitumor agent characterized by: the TRAIL variant of any one of claims 1 to 4 is used as an active ingredient, and pharmaceutically acceptable excipients are added to prepare the medicine.
12. A medicament for treating liver fibrosis, which is characterized in that: the TRAIL variant of any one of claims 1 to 4 is used as an active ingredient, and pharmaceutically acceptable excipients are added to prepare the medicine.
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