CN109400711A - A kind of PDGFR β targeting tumor necrosin relative death inducing ligand variant and its preparation method and application - Google Patents
A kind of PDGFR β targeting tumor necrosin relative death inducing ligand variant and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of tumor necrosin relative death inducing ligand variants, it is tumor necrosin relative death inducing ligand and ZPDGFRβFusion protein, ZPDGFRβThe N-terminal or C-terminal of tumor necrosin relative death inducing ligand are connected to by connexon.The invention also discloses a kind of recombinant vector, recombinant bacteriums such as nucleotide sequence and including it, also disclose the preparation method and purposes of aforementioned variant.TRAIL variant protein Z-hTRAIL of the invention has good anti-tumor activity, and also has clear curative effect to hepatic fibrosis-renal tubular ectasia syndrome, and potential applicability in clinical practice is good.
Description
Technical field
The present invention relates to biotech drug fields, and in particular to PDGFR β targeting tumour promotees apoptosis induction ligand variation
Body and its preparation method and application.
Background technique
Tumor necrosis factor apoptosis correlation inducing ligand (TRAIL) belongs to tumor necrosis factor (TNF) family member, C
End 114-281 amino acids can be soluble extracellular fragment by protease hydrolytic, form homotrimer, have receptor
Binding ability.The membrane receptor of TRAIL includes death receptor (DR4 and DR5) and Decoy receptor (DcR1 and DcR2).Death receptor
Contain death domain in DR4 and DR5 molecule, in conjunction with TRAIL after can be induced cell apoptosis to transmitting dead signal intracellular.
On the contrary, Decoy receptor DcR1 molecule not death domain-containing, the death domain of DcR2 is imperfect, and the two can be tied with TRAIL
It closes, but dead signal cannot be transmitted, because without inducing cell apoptosis.Tumour cell and the high expression of some abnormal activation cells
Death receptor, TRAIL can promote these Apoptosis.Normal cell often high expression Decoy receptor and the wound from TRAIL
Evil.Therefore, TRAIL may be currently being developed to ideal curative drug.
The study found that tumor cell surface height expresses death receptor, thus superpower swell is shown under TRAIL conditions in vitro
Cytotoxic effect activity, it is considered to be potential anti-tumor drug.But the internal antitumous effect of TRAIL is external right with it
The killing activity of tumour cell mismatches.Possible reason include: 1) TRAIL molecular weight it is small, Half-life in vivo is short.It solves
Method extends half-life period including the use of the methods of polyethyleneglycol modified or serum albumin fusion/combination.2) due to Decoy receptor
Wide expression is largely consumed after entering in vivo by normal tissue in normal tissue, TRAIL, and it is few thus anti-to reach tumor locus amount
Tumor effect is bad.Improved method mainly utilizes guide molecule to deliver TRAIL, it is made to be enriched in tumor locus, Jin Erti
High antitumous effect.Existing research carries out passing for TRAIL using tumour cell or endothelial cells in tumor neogenetic blood vessels as target cell
It send.Pericyte is important cells of vascular wall, is distributed on rear side of vascular endothelial cell, and the formation and stabilization to blood vessel have weight
The regulating and controlling effect wanted.But it there is no the research for submitting TRAIL into tumor using pericyte as target cell.
Hepatic Stellate Cell Activation is the initiating link of liver fibrosis.The hepatic stellate cells for inhibiting or removing activation can delay liver
Fibrotic processes.The study found that can high expression death receptor DR4 and DR5 during Hepatic Stellate Cell Activation.Therefore, TRAIL
It is capable of the apoptosis on hepatic stellate cells of induced activation and shows anti-fibrosis effect.Long-actingization that existing research is modified with PEG
TRAIL alleviates rat liver fibrosis process.But since TRAIL lacks targeting to hepatic stellate cells, therapeutic effect is not
It is good.
Summary of the invention
To solve the above-mentioned problems, the present invention provides a kind of PDGFR β targeting tumours to promote apoptosis induction ligand variant
And its preparation method and application.
Tumor necrosin relative death inducing ligand variant of the present invention, it is tumor necrosin relative death inducing
Ligand and ZPDGFRβFusion protein, ZPDGFRβThe N of tumor necrosin relative death inducing ligand is connected to by connexon
End or C-terminal.
Wherein, the ZPDGFRβAmino acid sequence as shown in SEQ ID NO:1.Preferably, the ZPDGFRβBy SEQ ID
It is nucleotide sequence coded shown in NO:2.
Wherein, the amino acid sequence of the tumor necrosin relative death inducing ligand is as shown in SEQ ID NO:3.It is excellent
Selection of land, the tumor necrosin relative death inducing ligand are nucleotide sequence coded as shown in SEQ ID NO:4.
Wherein, the connexon is made of 2~20 amino acid.
Wherein, the connexon is (G4S)3Connexon, amino acid sequence is as shown in SEQ ID NO:5.Preferably,
The connexon is nucleotide sequence coded as shown in SEQ ID NO:6.
Wherein, the amino acid sequence of the variant is as shown in SEQ ID NO:7.Preferably, the variant is by SEQ
It is nucleotide sequence coded shown in ID NO:8.
The present invention also provides a kind of nucleotide sequences, it includes the coding of tumor necrosin relative death inducing ligand
Sequence and ZPDGFRβCoded sequence, connected by the coded sequence of connexon therebetween.
Wherein, the ZPDGFRβCoded sequence as shown in SEQ ID NO:2.
Wherein, the coded sequence of the tumor necrosin relative death inducing ligand is as shown in SEQ ID NO:4.
Wherein, the connexon is (G4S)3Connexon, nucleotide sequence is as shown in SEQ ID NO:6.
Wherein, as shown in SEQ ID NO:8.
The present invention also provides the recombinant vector of foregoing nucleotide sequence or recombinant bacteriums.
The present invention also provides a kind of method for preparing forgoing neoplasms necrosin relative death inducing ligand variant,
Be characterized in that: it is to be prepared using foregoing nucleotide sequence as target fragment using the method for genetic engineering.
The present invention also provides forgoing neoplasms necrosin relative death inducing ligand variants to increase in preparation treatment cell
Purposes in the drug of natural disposition disease.
Wherein, the drug of the treatment cell hyperplastic disease is the drug for treating tumour or autoimmune disease.Institute
Stating tumour is Colon and rectum gland cancer.
The present invention also provides a kind of anti-tumor drugs, it is with the change of aforementioned tumor necrosin relative death inducing ligand
Allosome is active constituent, in addition the preparation that pharmaceutically acceptable auxiliary material is prepared.
The present invention also provides forgoing neoplasms necrosin relative death inducing ligand variants to prepare treating organs fibre
Purposes in the drug of dimensionization disease.
Wherein, the drug is the drug for treating hepatic fibrosis-renal tubular ectasia syndrome.
The present invention also provides a kind of drugs for treating hepatic fibrosis-renal tubular ectasia syndrome, it is with aforementioned tumor necrosin relative death
Inducing ligand variant is active constituent, in addition the preparation that pharmaceutically acceptable auxiliary material is prepared.
The present invention utilizes the affine body Z of energy specific recognition PDGFR βPDGFRβFor guide molecule, by itself and source of people TRAIL
(hTRAIL) it merges, constructs fusion protein Z-hTRAIL, and find that its inside and outside is antitumor and effect of anti hepatic fibrosis ratio
HTRAIL is remarkably reinforced, and potential applicability in clinical practice is good.
Above content according to the present invention, according to the ordinary technical knowledge and customary means of relevant art, not
It is detached under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
Detailed description of the invention
The MOLECULE DESIGN of Fig. 1 variant Z-hTRAIL；
The SDS-PAGE electrophoresis of Fig. 2 Z-hTRAIL and hTRAIL；
Fig. 3 Z-hTRAIL and hTRAIL gel permeation chromatography；
Fig. 4 Z-hTRAIL and hTRAIL in conjunction with pericyte after killing to tumour cell；
The tumor-targeting of Fig. 5 Z-hTRAIL and hTRAIL compare；A: the somatoscopy B: Tissue distribution being enriched in tumor；
Cylinder therapeutic effect of Fig. 6 Z-hTRAIL and hTRAIL to LS174T tumour；
Cylinder therapeutic effect of Fig. 7 Z-hTRAIL and hTRAIL to COLO205 tumour；
Cylinder therapeutic effect of Fig. 8 Z-hTRAIL and hTRAIL to HCT116 tumour；
Combination (A) and killing (B) of Fig. 9 Z-hTRAIL and hTRAIL to hepatic stellate cells；
Mouse liver function index after Figure 10 Z-hTRAIL and hTRAIL treatment；
Murine liver tissue sirius red stains after Figure 11 Z-hTRAIL and hTRAIL treatment；
Murine liver tissue Fibrosis score (A) and hydroxyproline content (B) after Figure 12 Z-hTRAIL and hTRAIL treatment；
The cell in vitro killing (A) of Figure 13 reference protein Zf-TRAIL and TRAIL and internal antitumous effect (B, C) ratio
Compared with.
Specific embodiment
The specific embodiment of form by the following examples makees further specifically above content of the invention
It is bright.But this should not be interpreted as to the scope of the above subject matter of the present invention is limited to the following embodiments.It is all above-mentioned interior based on the present invention
Hold realized technology to all belong to the scope of the present invention.
The MOLECULE DESIGN and clone's building of embodiment 1Z-hTRAIL variant
1, the MOLECULE DESIGN of Z-hTRAIL variant
ZPDGFRβ(table 1) is formed by 58 amino acid.The amino acid that hTRAIL is human TNF related apoptosis-inducing ligand extracellular fragment 114-281 forms
Segment (is shown in Table 1).Pass through (G4S)3Linker is by ZPDGFRβIt is connected to the N-terminal of hTRAIL, construction of fusion protein ZPDGFRβ-
(G4S)3- hTRAIL (abbreviation Z-hTRAIL) (Fig. 1).
2, the building of Z-TRAIL variation body expression vector
According to the amino acid sequence of ZPDGFR β, its initial code gene is designed, then after foranalysis of nucleic acids software optimization, by
The synthesis of Nanjing Jin Sirui company progress gene order.It is restricted at the both ends of sequence EcoRI/BamHI to be added respectively when synthesis
Endonuclease digestion site (EcoRI:gaattc/BamHI ggatcc).By double digestion and connection function, by the base of ZPDGFR β
Because sequence is loaded into the expression matter of pQE30-hTRAIL (nucleotide sequence of laden Z-hTRAIL segment is as shown in table 1)
On grain.After connection product converts TOP10 E. coli competent, recombinant plasmid is obtained.After recombinant plasmid is sequenced
It was found that ZPDGFRβ segment is successfully loaded on expression plasmid carrier.Expression plasmid pQE30-Z-hTRAIL is transferred to M15 large intestine bar
In bacterium competence, successfully bacterial strain M15-pQE30-Z-hTRAIL is expressed in building.
The fusion protein amino acid of the present invention of table 1 and nucleotide sequence
It the protein expression of embodiment 2Z-TRAIL variant and isolates and purifies
The monoclonal of picking expression bacterial strain M15-pQE30-Z-hTRAIL (preparation of embodiment 1) is inoculated into Double (containing ammonia
100 μ g/ml of benzyl XiLin, 30 μ g/ml of kanamycins) in LB liquid medium, 37 DEG C of shaken cultivations, as bacterial concentration A600Extremely
When 0.8 or so, 0.05mM isopropyl-β-D-thiogalactoside (Isopropyl β-D-1- is added
Thiogalactopyranoside, IPTG), 26 DEG C oscillation Fiber differentiation 14-16 hours.(7000g, 10min) is centrifuged to collect
Thallus, with Lysis buffer (50mM phosphate buffer, pH8.0；300mM NaCl；20mM imidazoles；10mM β-sulfydryl second
Alcohol) it is resuspended, phenylmethylsulfonyl fluoride (Phenylmethanesulfonyl fluoride, PMSF) extremely final concentration of 1mM is added,
Carrying out ultrasonic bacteria breaking under condition of ice bath (power 300W, work 10s, is spaced 30s, total 40min).After the completion of broken bacterium, centrifugation (4 DEG C,
25000g, 10min), it is repeated 4 times, collects bacteria break supernatant.The supernatant of acquisition is pressed with Ni-NTA resin gel (being purchased from Qiagen)
It is mixed according to appropriate volume ratio (such as V/V=50:1), 4 DEG C of oscillations combine 2h.Gel filler after combination is poured into chromatographic column, to
Protein sample is crossed after the completion of column, with Wash buffer (50mM phosphate buffer, pH8.0；300mM NaCl；40mM imidazoles；
10mM beta -mercaptoethanol) it is more than 30 times of column volumes of detergent gel column.Then Elution buffer (50mM phosphate-buffered is used
Liquid, pH7.6； 300mM NaCl；300mM imidazoles；10mM beta -mercaptoethanol) it elutes and collects protein sample, SDS-PAGE electrophoresis is aobvious
It is shown as single band (Fig. 2).Gel permeation chromatography is shown as unimodal (Fig. 3).
The result shows that we obtain pure variant protein Z-hTRAIL, using phosphate buffer PBS (10mM
Na2HPO4,137 mM NaCl,2.68mM KCl,2mM KH2PO4, pH 7.4) and dialysed overnight is spare.
Tumour cell after embodiment 3Z-TRAIL variant combination pericyte around killing
After being incubated for using PDGFR β specific antibody and pericyte, then use flow cytometry.As a result as shown in Figure 4 A,
The high PDGF-B expression R β in pericyte surface.By the Z-hTRAIL of pericyte and FAM label, (Example 2 prepares Z-hTRAIL, FAM
Label) or hTRAIL be incubated for altogether, then use flow cytometry, discovery Z-hTRAIL can be in conjunction with pericyte, and this knot
Conjunction can be closed by PDGFR β specific antibody, illustrate to merge ZPDGFRβIt can allow hTRAIL in conjunction with pericyte.For detection and Z-
HTRAIL in conjunction with pericyte after whether also have the function of tumor cytotoxicity, first by pericyte and Z-hTRAIL (embodiment 2
Preparation) (1 μM) preincubate 1h, after PBS washing again with tumour cell (LS174T and HCT116,1.5*104；COLO205,2*
104) co-culture overnight, CCK-8 detects cell survival rate.
As a result as shown in Figure 4 B, the survival rate of tumour cell increasing and drop with the pericyte number being incubated for Z-hTRAIL
It is low.After co-culturing with the pericyte that hTRAIL is incubated for, how much the survival rate and pericyte number of tumour cell are without obvious relation.
This illustrates that Z-hTRAIL of the present invention can be in conjunction with pericyte and killing tumor cell.And hTRAIL cannot in conjunction with pericyte, because
This can not killing tumor cell.
The tumor-targeting of embodiment 4Z-TRAIL variant is analyzed
The pH of Z-hTRAIL (preparation of embodiment 2) of the present invention be adjusted to 8.0 after with fluorescent dye CF750 according to molar ratio 1:8
Carry out mixed mark.After reacting at room temperature 1h, free CF750 fluorescent dye is excluded with PBS buffer solution dialysis.Egg after label
It is white to be entered in LS174T model of nude mice bearing tumor by tail vein injection, utilize small animal living body imaging system SPECTRAL Lago
And Lago X Imaging Systems probes into the tumor-targeting of Z-hTRAIL.
The results show that 0.5h after administration, the tumor locus of Z-hTRAIL group have stronger fluorescence signal, intensity is obviously high
In hTRAIL group, two groups of tumor bearing nude mice renal tracts can detect very strong fluorescence signal.Subsequent time point (1,2,
4,6h), Z-hTRAIL group tumor locus still detects stronger fluorescence signal, and hTRAIL group fluorescence signal weakens rapidly
Until disappearing (Fig. 5 A).Mice with tumor is put to death after 6h, is scanned after removing its main organs and tumor tissues, the results show that Z-
Stronger fluorescence signal can be detected in hTRAIL group tumor tissues, signal strength is about 3 times (Fig. 5 B) of hTRAIL group.
These results indicate that Z-hTRAIL ratio hTRAIL of the present invention can be enriched in tumor locus, show preferably swollen
Tumor targeting.
The internal antitumous effect of embodiment 5Z-TRAIL variant
Using LS174T, HCT116 and COLO205 model of nude mice bearing tumor is further evaluated Z-hTRAIL of the present invention and (is implemented
Example 2 prepare) internal antitumous effect.
In LS174T tumour (Colon and rectum gland cancer) model, the variant of the hTRAIL of 10mg/kg and identical molal quantity
Z-hTRAIL is administered three times by tail vein, when putting to death nude mice at the 16th day, PBS group knurl mean size for 850 ±
150mm3, hTRAIL group is 390 ± 80mm3, and Z-hTRAIL group is then minimum, only 102 ± 75mm3；Knurl average weight is
PBS group 0.652 ± 0.2g, hTRAIL group 0.302 ± 0.608g, Z-hTRAIL group then only have 0.091 ± 0.039g (Fig. 6).
In COLO205 tumour (Colon and rectum gland cancer) model, the variant of the hTRAIL of 5mg/kg and identical molal quantity
Z-hTRAIL is administered twice by tail vein, when putting to death nude mice at the 20th day, PBS group knurl mean size for 670 ±
140mm3, hTRAIL group is 400 ± 80mm3, and Z-hTRAIL group is then minimum, only 70 ± 60mm3；Knurl average weight is
PBS group 0.266 ± 0.114g, hTRAIL group 0.23 ± 0.053g, Z-hTRAIL group then only have 0.038 ± 0.035g (Fig. 7).
In HCT116 tumour (Colon and rectum gland cancer) model, the variant of the hTRAIL of 10mg/kg and identical molal quantity
Z-hTRAIL is administered four times by tail vein, when putting to death nude mice at the 19th day, PBS group knurl mean size for 782 ±
106.53mm3, hTRAIL group is 368.4 ± 104.6mm3, and Z-hTRAIL group is then minimum, only 205.1 ± 74.6mm3；Tumor
Body average weight be PBS group 0.491 ± 0.032g, hTRAIL group 0.319 ± 0.06g, Z-hTRAIL group then only 0.11 ±
0.036g (Fig. 8).
These results indicate that Z-hTRAIL ratio hTRAIL of the present invention has stronger internal anti-tumor capacity, illustrate to merge
ZPDGFRβThe internal antitumous effect of hTRAIL can be significantly increased, it is especially clear to the curative effect of colorectal cancer.
The effect of anti hepatic fibrosis of embodiment 6Z-TRAIL variant
The hepatic stellate cells of activation is to promote the key cells of liver fibrosis.Activate the high PDGF-B expression R β of sternzellen and dead
Receptor is died, therefore, compared with hTRAIL, Z-hTRAIL may have more advantage in terms of the combination and killing of activation sternzellen.
The albumen of FAM label and activation sternzellen are incubated for, then found by flow cytometer detection, activation sternzellen is integrated to
Z-hTRAIL (preparation of embodiment 2) is more than hTRAIL.Activation sternzellen is handled with various concentration albumen, is used again within second day
CCK8 measures residual cells quantity.The cell survival rate handled with PBS calculates the cell killing rate of albumen for 100%.It has been shown that,
Cell survival rate after the Z-hTRAIL effect of 10 and 20nM is respectively 24.6 ± 4.5% and 14.2 ± 1.2%, and identical dose
Cell survival rate after measuring hTRAIL effect is respectively 81.4 ± 4.8% and 59.7 ± 0.25% (Fig. 9).This illustrates Z-
The ability of killing activation sternzellen is stronger under hTRAIL ratio hTRAIL conditions in vitro.
In order to compare the internal anti-fibrotic effects of Z-hTRAIL and hTRAIL, we establish hepatic fibrosis in mice mould
Type.Carbon tetrachloride is dissolved in olive oil, concentration 25%.Four week old female C57 mouse peritoneals are injected with the tetrachloro dissolved
Change carbon, frequency of injection is that twice a week, it is 5ml/kg that injection dosage is 2.5ml/kg for the first time later.It, will be real at modeling 4 weeks
It tests mouse and is divided into model group and treatment group.Model group injects PBS, Z-hTRAIL or hTRAIL albumen is given respectively by treatment group
(10mg/kg), twice a week.During treatment, three groups of mouse maintain carbon tetrachloride injection.Mice serum was collected at the 6th week, is examined
Survey serum glutamic oxalacetic transaminase, glutamic-pyruvic transaminase and total bilirubin.Liver tissue slices show that collagen is fine with sirius red stains
It ties up and is scored by ISHAK standard degree of fibrosis, while detecting hydroxyproline in liver with kit, reflect collagen
Fiber content.
Figure 10 shows, model group liver function index (serum glutamic oxalacetic transaminase, glutamic-pyruvic transaminase and total bilirubin) it is horizontal with
There were significant differences for normal group, illustrates that hepatocellular injury is serious, models successfully.
Three kinds of Serum Indexes levels of hTRAIL treatment group and model group no significant difference.But three kinds of blood of Z-hTRAIL treatment group
Clear index is below hTRAIL treatment group.Sirius red stains show that Collagen fiber deposition is less than in Z-hTRAIL treatment group liver
HTRAIL treatment group (Figure 11), Fibrosis score and hydroxyproline content are below hTRAIL treatment group (Figure 12).
These results suggest that Z-hTRAIL of the present invention has preferably treatment to mouse liver fibrosis relative to hTRAIL
Effect.
Comparative example 1 merges influence of other affinity bodies to TRAIL antitumous effect
In order to illustrate fusion ZPDGFRβTo the enhancing active importance of TRAIL, we have selected and ZPDGFRβSequence is similar, but
The different affinity body Z of identification specificityFcRnFor control.By ZFcRnIt is connect with hTRAIL, construction of fusion protein Zf-hTRAIL.Into
One step determines whether fusion control affinity body equally can be enhanced the antitumous effect of hTRAIL compared with hTRAIL.
1, the MOLECULE DESIGN and preparation of Zf-hTRAIL variant
According to embodiment 1, design ZFcRnIt is connected to hTRAIL N-terminal, construction of fusion protein Zf-hTRAIL.According to reality
Example 2 is applied, with condition identical with Z-hTRAIL, prepares fusion protein Zf-hTRAIL with escherichia expression system.
2, the Cytotoxicity in vitro and Anticancer effect in vivo to tumour cell of Zf-hTRAIL variant
In order to test Zf-hTRAIL to the killing activity of tumour cell, first by LS174T cell (1 × 104It is a) it is inoculated in
96 orifice plates, it is adherent overnight after be added various concentration albumen.Effect overnight after, then plus CCK-8 measurement residual cell quantity.It is right
According to the PBS that same volume is added in hole.With the cell survival rate of PBS processing group for 100%, it is thin to tumour to calculate Zf-hTRAIL
The killing-efficiency of born of the same parents.It is compared with hTRAIL, judges to merge ZFcRnIt is living to the killing of tumour cell whether hTRAIL is affected
Property.As a result as shown in FIG. 13A, Zf-hTRAIL is similar to hTRAIL to the killing-efficiency of LS174T cell, illustrates to merge ZFcRn
The activity of hTRAIL is had no significant effect.
According to the method that embodiment 5 describes, compare Zf-hTRAIL and TRAIL to the therapeutic effect of LS174T tumour.Naked
Mouse inoculates after LS174 cell 7 days, tail vein injection Zf-hTRAIL, hTRAIL or PBS.Then tumour body is measured daily
Product draws growth curve.Observation terminates, and puts to death mouse, strips knurl weighing.As shown in Figure 13 B, Zf-hTRAIL and hTRAIL
Processing group tumor growth rate is slower than PBS processing group, and tumor weight is also lighter than PBS group.But at Zf-hTRAIL and hTRAIL
The tumour of the reason either speed of growth or knurl weight illustrate to merge affinity body Z all without notable differenceFcRnIncrease there is no significant
The internal antitumous effect of strong hTRAIL.
2 reference protein amino acid of table and nucleotide sequence
To sum up, the present invention utilizes the affine body Z of energy specific recognition PDGFR βPDGFRβFor guide molecule, by itself and source of people
The fusion protein Z-hTRAIL that TRAIL (hTRAIL) is constructed, inside and outside is antitumor and effect of anti hepatic fibrosis ratio
HTRAIL is remarkably reinforced, and uses other recognition factors, e.g., ZFcRnThe fusion protein of building is then ineffective.
Claims (17)
1. a kind of tumor necrosin relative death inducing ligand variant, it is characterised in that: it is tumor necrosis factor correlation
Apoptosis induction ligand and ZPDGFRβFusion protein, ZPDGFRβTumor necrosin relative death inducing is connected to by connexon to match
The N-terminal or C-terminal of body.
2. tumor necrosin relative death inducing ligand variant according to claim 1, it is characterised in that: described
ZPDGFRβAmino acid sequence as shown in SEQ ID NO:1；
And/or the amino acid sequence of the tumor necrosin relative death inducing ligand is as shown in SEQ ID NO:3；
And/or the connexon is made of 2~20 amino acid, the preferably described connexon is (G4S)3Connexon, amino acid
Sequence is as shown in SEQ ID NO:5.
3. tumor necrosin relative death inducing ligand variant according to claim 23, it is characterised in that: described
ZPDGFRβIt is nucleotide sequence coded as shown in SEQ ID NO:2；And/or the tumor necrosin relative death inducing ligand
It is nucleotide sequence coded as shown in SEQ ID NO:4；And/or connexon nucleotides sequence as shown in SEQ ID NO:6
Column coding.
4. tumor necrosin relative death inducing ligand variant according to any one of claims 1 to 3, feature
Be: its amino acid sequence is as shown in SEQ ID NO:7.
5. tumor necrosin relative death inducing ligand variant according to claim 4, it is characterised in that: its by
It is nucleotide sequence coded shown in SEQ ID NO:8.
6. a kind of nucleotide sequence, it is characterised in that: it includes the coded sequence of tumor necrosin relative death inducing ligand
With ZPDGFRβCoded sequence, connected by the coded sequence of connexon therebetween.
7. coded sequence according to claim 6, it is characterised in that: the ZPDGFRβCoded sequence such as SEQ ID NO:2
It is shown；And/or the coded sequence of the tumor necrosin relative death inducing ligand is as shown in SEQ ID NO:4；And/or
The connexon is (G4S)3Connexon, nucleotide sequence is as shown in SEQ ID NO:6.
8. nucleotide sequence according to claim 7, it is characterised in that: it is as shown in SEQ ID NO:8.
9. recombinant vector or recombinant bacterium comprising nucleotide sequence described in claim 6~8 any one.
10. a kind of side for preparing tumor necrosin relative death inducing ligand variant described in Claims 1 to 5 any one
Method, it is characterised in that: it is to be arranged with nucleotides sequence described in claim 6~8 any one for target fragment, using genetic engineering
Method be prepared.
11. tumor necrosin relative death inducing ligand variant described in Claims 1 to 5 any one is thin in preparation treatment
Purposes in the drug of born of the same parents' proliferative disease.
12. purposes according to claim 11, it is characterised in that: the drug of the treatment cell hyperplastic disease is treatment
The drug of tumour or autoimmune disease.
13. purposes according to claim 12, it is characterised in that: the tumour is Colon and rectum gland cancer.
14. a kind of anti-tumor drug, it is characterised in that: it is with tumor necrosis factor phase described in Claims 1 to 5 any one
Pass apoptosis induction ligand variant is active constituent, in addition the preparation that pharmaceutically acceptable auxiliary material is prepared.
15. tumor necrosin relative death inducing ligand variant described in Claims 1 to 5 any one is preparing therapeutic equipment
Purposes in the drug of official's fibrotic disease.
16. purposes according to claim 15, it is characterised in that: the drug is the drug for treating hepatic fibrosis-renal tubular ectasia syndrome.
17. a kind of drug for treating hepatic fibrosis-renal tubular ectasia syndrome, it is characterised in that: it is with tumour described in Claims 1 to 5 any one
Necrosin relative death inducing ligand variant is active constituent, in addition the system that pharmaceutically acceptable auxiliary material is prepared
Agent.
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| CN113444182A (en) * | 2021-06-22 | 2021-09-28 | 四川大学华西医院 | Fusion protein carrier for targeted delivery of IgG antibody and application thereof |
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| WO2018219301A1 (en) | 2018-12-06 |
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