CN105461801B - The mutant of high activity tumor necrosin relative death inducing ligand - Google Patents

The mutant of high activity tumor necrosin relative death inducing ligand Download PDF

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CN105461801B
CN105461801B CN201510906764.1A CN201510906764A CN105461801B CN 105461801 B CN105461801 B CN 105461801B CN 201510906764 A CN201510906764 A CN 201510906764A CN 105461801 B CN105461801 B CN 105461801B
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trail
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apoptosis
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CN105461801A (en
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谭树华
付剑
黄传龙
古丽丽
孙玥盈
朱娟娟
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China Pharmaceutical University
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Abstract

The invention belongs to field of biological pharmacy, and in particular to a kind of tumor necrosin relative death inducing ligand (TRAIL) mutant and its pharmaceutical applications., and can be with low-down affinity combination soluble recepter OPG since wild type TRAIL can be in combination with tumor cell membrane surface death receptors DR4 and DR5 and Decoy receptor DcRl and DcR2, but it could inducing apoptosis of tumour cell only in conjunction with DR4 and DR5.In order to enhance the anti-tumor activity of TRAIL, the present invention designs and constructs the TRAIL variant that a kind of couple of DR5 has high-affinity, it is characterized in that containing E263G mutation (i.e. the 263rd Glu is mutated into Gly).Variant solubility expression and after purification in E.coli, through SEC-HPLC analysis shows that it exists with active trimeric form.The affinity of the variant and DR5 receptor significantly improves, and its anti-tumor activity is significantly enhanced.

Description

The mutant of high activity tumor necrosin relative death inducing ligand
Technical field
The invention belongs to field of biological medicine, and in particular to a kind of tumor necrosin relative death inducing ligand (TRAIL) mutant, the mutant have the death receptor DR5 of tumor cell surface compared with wild type TRAIL higher Affinity, while showing anti-tumor activity more higher than wild type TRAIL.
Background technique
Tumor necrosin relative death inducing ligand (TNF-related apoptosis-inducing ligand, TRAIL) be also known as apo 2 ligand (Apoptosis Ligand, APO-2L) belong to tumor necrosis factor (TNF) family at Member, it belongs to II type transmembrane protein as the other members of TNF superfamily.Since TRAIL can specificity induction tumour cell Apoptosis, and to normal cell almost without cytotoxicity, therefore have broad application prospects in terms of the immunization therapy of tumour (Cancer Lett 2013,332:156-162)。
Human TNF related apoptosis-inducing ligand gene encodes 281 amino acid, relative molecular weight 32.5KD altogether, and isoelectric point 7.63 is II type Transmembrane protein, by cytoplasmic domain (14 amino acid), transmembrane region (26 amino acid) and after birth outskirt (241 amino acid) composition. The intracellular space (cytoplasmic domain) of TRAIL does not have signal peptide sequence, can form solubility after the extracellular Partial digestion of TRAIL TRAIL(sTRAIL).STRAIL lacks transmembrane region and intracellular region, N-terminal no signal peptide structure, soluble type molecule N-terminal amino acid Missing do not influence its bioactivity.Two kinds of most-often used sTRAIL are TRAIL (95-281) and TRAIL under the prior art (114-281), research shows that constructing soluble TRAIL (sTRAIL) clone, expression since 95 or 114 amino acids residues Albumen out is active, and can be in conjunction with specific receptor.Film mating type TRAIL (overall length TRAIL) and extracellular soluble outskirt TRAIL, that is, TRAIL (95-281) or TRAIL (114-281) can be in conjunction with the specific receptor of tumor cell surface, selectivity Ground inducing apoptosis of tumour cell, and to normal cell almost without toxicity.
The receptor of TRAIL is divided into three classes, and one kind is death receptor (death receptor, DR), including TRAIL-R1 (DR4) and TRAIL-R2 (DR5), they contain death domain intracellular and rely on the identification of the apoptotic signal of Caspase approach Structural domain.TRAIL in conjunction with DR after the dead information of TRAIL is transmitted into the cell, to activate Caspase system, finally Cause Apoptosis.Another kind of is Decoy receptor (decoy receptor, DcR), including TRAIL-R3 (DcR1) and TRAIL- R4 (DcR2), the apoptosis that they cannot mediate TRAIL to induce due to lacking function Death Domain intracellular, therefore referred to as Decoy receptor. Decoy receptor contestable inhibits the combination of TRAIL and DR4 and DR5, rises and promotees apoptosis antagonism.Third class is soluble recepter OPG (osteoprotegrin) is tied under normal physiological conditions although it in conjunction with TRAIL and can inhibit its activity with TRAIL Resultant force very low (Curr Opin Pharmacol 2008,8:433-439).
TRAIL can induce apoptosis of tumor in conjunction with the death receptor DR4 and DR5 of tumour cell film surface, but simultaneously TRAIL can also be combined with Decoy receptor DcRl, DcR2 and OPG, and this combination is unable to inducing apoptosis of tumour cell.Therefore, Design and building have the TRAIL mutant of more high-affinity to death receptor DR, it is possible to reduce with Decoy receptor DcR and OPG Combination, while improving the activity of its inducing apoptosis of tumour cell.
The present invention constructs the TRAIL mutant for having more high affinity to tumor cell surface death receptor DR5, this is prominent Variant shows anti-tumor activity more higher than wild type TRAIL.
Summary of the invention
Since the extracellular soluble outskirt, that is, TRAIL (114-281) or TRAIL (95-281) and overall length TRAIL of TRAIL have The activity of identical inducing apoptosis of tumour cell, it is an object of the invention to construct to have more high-affinity to death receptor DR5 TRAIL extracellular soluble outskirt mutant, to improve its anti-tumor activity.
Specific technical solution of the present invention is as follows:
A kind of mutant of apoptosis induction ligand related to human tumor necrosis factor is mutated, i.e., with human TNF related apoptosis-inducing ligand containing E263G Full length amino acid sequence positioning, the site of mutation are located at the 263rd of human TNF related apoptosis-inducing ligand full length amino acid sequence, amino acid by Glu sports Gly.Due to the work of the extracellular soluble outskirt and overall length TRAIL inducing apoptosis of tumour cell having the same of TRAIL Property, therefore heretofore described mutant can be the TRAIL (overall length) of wild type, derivative type or the film of recombination mating type or contain There is the TRAIL active fragment of extracellular soluble outskirt.
The above-mentioned TRAIL active fragment containing extracellular soluble outskirt, preferably TRAIL (114-281)-E263G, amino acid sequence Column are as shown in SEQ ID No:1.Or preferably TRAIL (95-281)-E263G, amino acid sequence is as shown in SEQ ID No:2.
Another object of the present invention is to provide above-mentioned mutant to treat or prevent apoptosis related disease medicine in preparation Purposes in object.The dead information of TRAIL by being transmitted into the cell after in conjunction with DR5 by the mutant, to activate Caspase system, finally causes Apoptosis.The mutant enhances the binding force with DR5, and improves its activation Caspase system and the ability induced cell apoptosis.
Further, the drug can be described as anti-tumor drug.The drug contains mutant conduct of the present invention Active pharmaceutical ingredient and pharmaceutically acceptable carrier.
Further, the drug also contains other anti-tumor drugs and/or anti-tumor drug sensitizer.
Mutant of the present invention can prepare the volume of the mutant by artificial synthesized or gene cloning method Code gene, and expressed to obtain in corresponding host cell.The host cell includes Escherichia coli, yeast, insect Cell or mammalian cell.
In one embodiment of the invention, it is obtained using Computer-Aided Drug Design and method for screening active ingredients to dead Mutant TRAIL (114-281)-E263G (i.e. the 263rd Glu of original is mutated into Gly) that receptor DR5 has more high-affinity is died, The mutant shows higher anti-tumor activity.
By the method for molecular cloning, the encoding gene of TRAIL (114-281)-E263G mutant is constructed, and is cloned To the EcoR I of the prokaryotic expression carrier pTASH (Protein Expr Purif 2002,25:430-436) containing Tac promoter Between III restriction enzyme site of Hind, recombinant plasmid transformed is then subjected to intracellular expression into host strain E.coli JM109.Work Bacterium mud is collected by centrifugation after journey bacterium fermented and cultured, centrifugation supernatant is through cation exchange resin SP-Sepharose Fast after broken wall Flow column chromatographic isolation and purification obtains purity up to 95% or more sample, and through SEC-HPLC, analysis shows, expression product is with trimerization Body form exists.
Interaction of biomacromolecules instrument analysis shows that, mutant TRAIL (114-281)-E263G and death receptor DR5 Affinity (KD=7.73 ± 0.5x10-9M) be wild type TRAIL (114-281) Yu death receptor DR5 affinity (KD= 1.96±0.2x10-8M) 2.54 times.
MTT antineoplastic activity analysis shows that, to colon cancer cell COLO 205, mutant TRAIL (114-281)-E263G (IC50=0.44nM) anti-tumor activity be wild type TRAIL (114-281) (IC50=2.4nM) 5.5 times.
Flow cytometry analysis shows that under the conditions of same molar ratio, mutant TRAIL (114-281)-E263G promotees The apoptosis rate of colon cancer cell COLO 205 is significantly higher than wild type TRAIL (114-281).
Detailed description of the invention
Fig. 1: mutant TRAIL (114-281)-E263G prokaryotic expression carrier pTA-TRAIL-E263G schematic diagram. (Ptac:Tac promoter；AP: ampicillin resistance；TRAIL-E263G:TRAIL (114-281)-E263G encoding gene；Ori: PUC plasmid replication starting point；RrnBT1T2: transcription terminator).
Fig. 2: mutant TRAIL (114-281)-E263G protein expression situation.(arrow meaning is in place for purpose albumen institute It sets.M: low molecular weight protein Marker；Lane 1: wild type TRAIL (114-281) bacterial protein；Lane 2:TRAIL (114-281)-E263G bacterial protein；Lane 3:TRAIL (114-281) bacterial cell disruption supernatant；Lane 4:TRAIL (114- 281)-E263G bacterial cell disruption supernatant；Lane 5:TRAIL (114-281) bacterial cell disruption precipitating；Lane 6:TRAIL (114- 281)-E263G bacterial cell disruption precipitates).
Fig. 3: 10% non-reduced SDS-PAGE electrophoretic analysis mutant TRAIL (114-281)-E263G purified product.(M: Low molecular weight protein Marker；Lane 1: the wild type TRAIL (114-281) of purifying；Lane 2: the TRAIL (114- of purifying 281)-E263G)。
Fig. 4: SEC-HPLC four kinds of chromatography standard protein.(chromatographic column: Shodex PROTEIN KW-802.5, SHOWA DENKO K.K., Japan) analysis four kinds of standard proteins: BSA (MW=67kDa), chicken egg white (MW=43kDa), Chymotrypsinogen (MW=25kDa) and lysozyme (MW=14.4kDa).Their retention time is 11.1min respectively, 11.9min, 13.2min, 14.1min, corresponding retention volume are respectively 7.8ml, 8.3ml, 9.2ml, 9.9ml.
The wild type TRAIL (114-281) of Fig. 5: SEC-HPLC chromatography purifying.(chromatographic column: Shodex PROTEIN KW-802.5, SHOWA DENKO K.K., Japan) the wild type TRAIL (114-281) purified is analyzed, retention time is 11.618, retention volume 8.1ml, purity 96.53%).
TRAIL (114-281)-E263G mutant of Fig. 6: SEC-HPLC analysis purifying.(chromatographic column: Shodex PROTEIN KW-802.5, SHOWA DENKO K.K., Japan) TRAIL (the 114-281)-E263G mutant purified is analyzed, Retention time is 11.619, retention volume 8.1ml, purity 95.91%.
Fig. 7: mtt assay analyzes wild type TRAIL (114-281) and TRAIL (114-281)-E263G mutant to people's colon The pro-apoptosis bioactivity of cancer cell COLO 205.(the IC of wild type TRAIL (114-281)50=2.4nM；Mutant TRAIL (114- 281) IC of-E263G50=0.44nM).
Fig. 8: human colon cancer cell COLO 205 various concentration wild type TRAIL (114-281) and TRAIL (114- 281) the apoptosis rate difference after-E263G mutant effect 4h.(0.5nM mutant TRAIL (114-281)-E263G administration group Vs.0.5 wild type TRAIL (114-281) administration group, P < 0.05 *；1nM mutant TRAIL (114-281)-E263G administration group Vs.1nM wild type TRAIL (114-281) administration group, P < 0.01 * *；5nM mutant TRAIL (114-281)-E263G administration group Vs.5nM wild type TRAIL (114-281) administration group, P < 0.01 * *).
Fig. 9: wild type TRAIL (114-281) and mutant TRAIL (114-281)-E263G and death receptor DR5 is dissociated Constant KDMeasurement.(A: wild type TRAIL (114-281)；B: mutant TRAIL (114-281)-E263G).
Specific embodiment
The present invention is further illustrated by the following examples, but not as limitation of the present invention.
Design of the embodiment 1 to DR5 high-affinity TRAIL mutant
The building of TRAIL-R complex model: with TRAIL-DR5 compound (PDB ID:1D4V) for template, which is The monomer complex of TRAIL and DR5 extracellular domain, resolution ratio areWhat wherein DR5 was included is 69-184 bit amino Acid, the structures of TRAIL-DR5 trimer compositions using the website EBI PDBePISA server (http: // Pqs.ebi.ac.uk it) generates.The TARIL-DR5 trimer compositions structure obtained in this way certainly exists some unreasonable energy Amount is collided, therefore utilization MOE (Molecular Operating Environment, Chemical Computing Group, Montreal) software is energy-optimised to carrying out to it: adding hydrogen, power-up lotus to system with Protonate 3D first, then exists Under the field of force OPLS-AA, setting Gradient is 0.05, carries out energy minimum.Structure after optimizing in this way is as subsequent FoldX The initial configuration that (http://foldxsuite.crg.eu/, Center for Genomic Regulation) is calculated.
The selection in the mutational site TRAIL: it is known as the area 50s loop (51-65 bit amino there are two region in the structure of DR5 Acid) and the area 90s loop (91-104 amino acids), it is main portions (Mol Cell 1,999 4 of the DR5 in conjunction with TRAIL (4):563-571).These sites are selected in MOE, then use the Selection-Extend-Near of MOEFunction, The selected amino acid with 50s loop and 90s loop neighbouring TRAIL, and guarantee that selected mutational site is not TNF superfamily Conserved positions, it is final to determine that following site is mutated: T127, G128, T129, R130, K145, E155, S157, R158, S159、G160、H161、R191、Q193、S215、P217、D218、K251、T261、E263、H264、H270。
Energy balane: using the tripolymer TRAIL-DR5 of energy-optimised mistake as initial configuration, for as above selected 21 to Mutational site is successively virtually mutated it with FoldX4, then successively calculates the Conjugated free energy value of mutation front and back, foundation Δ Δ G < 0.5Kcal/mol screens alternative mutant.
Δ Δ G=Δ G (mutant)-Δ G (wild-type), that is, the combination after being mutated can subtract the combination energy before mutation. Δ Δ G < 0, affinity improves after indicating mutation.0.5Kcal/mol is the limits of error of FoldX, and only mutation front and back combination can become It is just considered believable for changing the result of the absolute value greater than 0.5Kcal/mol of (Δ Δ G).
According to the above method and screening principle (Δ Δ G < 0.5Kcal/mol), the result of FoldX is screened, is excluded, Available following mutant:
TRAIL (114-281)-G131N: combination can be -20.81Kcal/mol before mutation, be -23.38Kcal/ after mutation Mol, Δ Δ G=-23.38-(- 20.81)=- 2.57Kcal/mol；
TRAIL (114-281)-S159E: combination can be -20.90Kcal/mol before mutation, be -24.81Kcal/ after mutation Mol, Δ Δ G=-24.81-(- 20.90)=- 3.91Kcal/mol)；
TRAIL (114-281)-E263G: combination can be -20.87Kcal/mol before mutation, be -23.24Kcal/ after mutation Mol, Δ Δ G=-23.24-(- 20.87)=- 2.37Kcal/mol.
Subsequent experimental is done to above 3 select mutant, and active compared with the TRAIL of wild type, with verifying The result of design.
The building of 2 TRAIL of embodiment (114-281) and its mutant protein engineering bacteria
The building of TRAIL (114-281) expression bacterial strain.Design and synthesize following primer: P1 (5-CCGAATTCATGGTGA GAGAAAGAGGTCCTCAGAGAG-3, SEQ ID NO:6, underscore are the EcoR I restriction enzyme site introduced) and reverse primer P2 (5-ACAAGCTTAGCCAACTAAAAAGGCCCCGAAAAAACTG-3, SEQ ID NO:7, underscore are the Hind III introduced Restriction enzyme site).It is respectively upstream and downstream primer with P1 and P2 using Human plactnta cDNA as template, using high-fidelity TaqDAN polymerase PCR amplification TRAIL (114-281) encoding gene.PCR condition are as follows: 94 DEG C of denaturation 5min；94 DEG C of 30s, 50 DEG C of 50s, 72 DEG C of 90s Carry out 30 circulations；72 DEG C of extension 10min.PCR product is cloned into pMD19-T carrier (Dalian treasured biotech firm product) after purification In and serve Hai Meiji Biotechnology Co., Ltd sequencing.Sequencing correctly uses EcoR I and Hind III after clone extracts plasmid Double digestion is carried out, obtained target gene is subcloned into expression vector pTASH (Expr Purif 2002,25:430-436.) again EcoRI/Hind III digestion site, the engineering of expression TRAIL (114-281) is obtained after Transformed E .coli JM109 host strain Bacterium pTA-TRAIL (114-281)/JM JM109.
Mutant TRAIL (114-281)-E263G, TRAIL (114-281)-S159E and TRAIL (114-281)-G131N The building of engineering bacteria.On the basis of the plasmid pTA-TRAIL (114-281) of the expression wild type TRAIL (114-281) constructed On, using it as template, using Overlap Extension PCR method (Gene 1989,77 (1): 51-59) to TRAIL The encoding gene of (114-281) carries out rite-directed mutagenesis, to obtain mutant TRAIL (114-281)-E263G, TRAIL (114- 281) encoding gene of-S159E and TRAIL (114-281)-G131N, sequence is successively as shown in SEQ ID NO:3-5.Then It is subcloned into expression vector pTASH (the Protein Expr Purif 2002,25:430- of the promoter containing Tac respectively 436.) in EcoRI/Hind III digestion site, recombinant expression carrier pTA-TRAIL-E263G (as shown in Figure 1) is obtained, PTA-TRAIL-S159E, pTA-TRAIL-G131N, its correct sequence of sequence verification.CaCl is used again2Method convert to E.coli JM109 host strain obtains recombinant expression genetic engineering bacterium.
The expression of 3 TRAIL of embodiment (114-281) and its mutant protein
Recombination engineering single bacterium is fallen within 50ml LB liquid medium (containing 100 μ g/ml ampicillins), 37 DEG C, 220rpm cultivates 12h.By 2% inoculum concentration be inoculated in 200ml fermentation medium (1% tryptone, 0.5% yeast powder, 4% Sodium glutamate, 1% malt flour, 0.67%KH2PO4, 0.75%Na2HPO4 .12H2O, 0.1mM ZnSO4, 100 μ g/ml ammonia benzyls blueness Mycin, pH 6.5), 37 DEG C, 220rpm, culture is for 24 hours.
Fermentation liquid is centrifuged (6000rpm, 10min) and collects bacterium mud (wet bacterium weight, 6g/l), is resuspended with the ratio of 10% (w/v) In broken bacterium buffer (20mM Tris-HCl, 0.05mM ZnSO4, 0.25mM DTT, 0.1%Triton, pH 7.6), use homogeneous Machine (AH100B, ATS Engineering Inc., Canada) carries out clasmatosis.Broken liquid centrifugation (13000rpm, 30min) Collect supernatant.10%SDS-PAGE electrophoretic analysis bacterial protein, broken liquid supernatant and broken liquid precipitate.For example, wild type TRAIL (114-281) and mutant TRAIL (114-281)-E263G molecular weight respectively with software (http: // Web.expasy.org/compute_pi/) prediction theory value 19.62kDa and 19.55kDa (including translation initiation methionine) Unanimously (result is as shown in Figure 2).
The purifying of 4 TRAIL of embodiment (114-281) and its mutant protein
It is illustrated by taking wild type TRAIL (114-281) and TRAIL (114-281)-E263G mutant as an example.Firstly, With equilibration buffer (20mM Tris-HCl, 0.05mM ZnSO4, 0.25mM DTT, pH 7.6) and balance SP sepharose Fast Flow (GE) cation exchange resin chromatographic column (1.0 × 20cm of φ).Then, by bacterial cell disruption liquid supernatant with 0.5ml/ The flow velocity loading of min washes away unbonded albumen with equilibration buffer after completion of the sample, then miscellaneous slow with washing with the flow velocity of 1ml/min Fliud flushing (20mM Tris-HCl, 0.05mM ZnSO4, 0.25mM DTT, 100mM NaCl, pH 7.6) and it washes away in conjunction with upper miscellaneous egg It is white, finally with the flow velocity of 1ml/min elution buffer (20mM Tris-HCl, 0.05mM ZnSO4, 0.25mM DTT, 150mM NaCl, pH 7.6) lower destination protein is washed, the elution collection liquid in different pipes is subjected to non-reduced SDS-PAGE electrophoresis point Analysis, the albumen collected in the pipe of purity is high carry out subsequent analysis (Fig. 3).
5 TRAIL of embodiment (114-281) and its measurement of mutant protein molecular size range and homogeneity analysis
It is illustrated by taking wild type TRAIL (114-281) and TRAIL (114-281)-E263G mutant as an example.Due to TRAIL is there is and play its anti-tumor activity in the form of tripolymer, therefore whether final purifying gained sample is with three Dimer form exists most important to experimental result.
Using molecular exclusion method (Size Exclusion Chromatography, SEC) in HPLC system (LC-2010A HT, SHIMADZU Corp., Japan) on analyzed, chromatographic column be Shodex PROTEIN KW-802.5 (SHOWA DENKO K.K., Japan), mobile phase is 0.2M phosphate buffer (pH 7.4), Na containing 0.1M2SO4(press chromatographic column specification It is required that addition), flow velocity 0.7ml/min, Detection wavelength 280nm.
Independent analysis and hybrid analysis are carried out to four kinds of standard proteins, the results showed that, four kinds of standard protein BSA (MW= 67kDa), chicken egg white (MW=43kDa), chymotrypsinogen (MW=25kDa) and lysozyme (MW=14.4kDa) Retention time is 11.2min, 11.9min, 13.2min respectively, and retention volume corresponding to 14.1min is respectively 7.8ml, 8.3ml, 9.2ml, 9.9ml (result is as shown in Figure 4).Standard song is made according to the molecular weight of each standard protein and retention volume Line, obtaining molecular weight calculation formula is lgMW=-0.313 × Ve+4.265.
The retention time of wild type TRAIL (114-281) is 11.618min (as shown in Figure 5), and corresponding retention volume is 8.1ml, calculating wild type TRAIL (114-281) molecular weight according to molecular weight calculation formula is 52.48kDa, with ExPASy net Stand (http://web.expasy.org/compute_pi/) prediction trimeric molecules amount (methionine containing translation initiation) 58.87kDa is close.
The retention time of mutant TRAIL (114-281)-E263G is 11.619min (as shown in Figure 6), corresponding reservation Volume is 8.1ml, according to molecular weight calculation formula calculate its molecular weight be 52.48kDa, with the website ExPASy (http: // Web.expasy.org/compute_pi/) prediction molecular weight (methionine containing translation initiation) 58.66kDa basic one It causes.
It follows that mutant TRAIL (the 114-281)-E263G and wild type TRAIL (114-281) that purifying obtains are equal Exist with trimeric form.In addition, wild type TRAIL (114-281) purity is 96.53% (as shown in Figure 5), mutant TRAIL-E263G purity is 95.91% (result is as shown in Figure 6).Referring to the method for embodiment 3-5, TRAIL can be prepared (114-281)-S159E and TRAIL (114-281)-G131N mutant.
Embodiment 6 is using mtt assay measurement TRAIL (114-281) and its anti-tumor activity of mutant
Colon cancer cell COLO 205 is long to convergence degree 80%, after 0.25% trypsin solution digestion, with 2 × 104A cells/well (RPMI-1640 culture medium of the 100 μ l containing 1%FBS) is seeded to 96 porocyte culture plates, in 37 DEG C, 5%CO2 Overnight incubation in incubator.Give respectively various dose wild type TRAIL (114-281) or mutant protein (0,0.1, 0.5,1,5,10 and 100nM), 5 multiple holes are arranged in each concentration, and after processing for 24 hours, 12 μ LMTT (5mg/ml) are added in every hole, in CO2Continue culture 4 hours in incubator, then 100 μ L, tri- liquid is added in every hole, vibrates 10min, enzyme linked immunosorbent detection after 12h Instrument measures absorbance value (OD) in 570nm, and calculates cell inhibitory rate as follows: inhibiting rate (%)=(1- (medicine group OD Mean value-blank group OD mean value)/(control group OD mean value-blank group OD mean value)) × 100%.Experimental result GraphPad 5.0 software of Prism (GraphPad, USA) is handled, data means standard deviationIt indicates, compares between two groups It is examined compared with t, three groups and three groups or more are used one-way analysis of variance.
MTT measurement result is shown, wild type TRAIL (114-281) and mutant TRAIL (114-281)-E263G, is mutated Body TRAIL (114-281)-S159E and mutant TRAIL (114-281) four kinds of drugs of-G131N are to colon cancer cell COLO205 has obvious inhibiting effect, and there are dose-dependence, IC50It is 2.4nM, 0.44nM, 2.55nM respectively, 3.07nM。
Therefore, the preferred TRAIL of the present invention (114-281)-E263G mutant inhibits colon cancer cell COLO's 205 Activity is 5.5 times (result is as shown in Figure 7) of wild type TRAIL (114-281).
Embodiment 7
Flow cytometry TRAIL (114-281) and mutant TRAIL (114-281)-E263G induces COLO 205 Apoptosis
It is detected using the bis- dye methods of Annexin V-PE/7-ADD.Colon cancer cell COLO 205 is long to convergence degree 80%, after 0.25% trypsin solution digestion, with 1 × 105A cells/well is seeded in 6 orifice plates, is incubated overnight.Add difference Concentration (0,0.5,1 and 5nM) wild type TRAIL (114-281) and TRAIL (114-281)-E263G mutant drug-treated 4h Afterwards, collected by trypsinisation cell (containing the cell in supernatant), PBS are cleaned 2 times, are resuspended with the PBS containing 2%FBS, and adjustment cell is close It spends to 2~10 × 105A cell/mL.The 100 above-mentioned cell suspensions of μ l are taken to be uniformly mixed with the Guava Nexin reagent of 100 μ l, After room temperature is protected from light dyeing 20min, in Guava easyCyteTMOn flow cytometer (Merck Millipore, Germany) into Row Apoptosis assay analyzes experimental result with 2.6 software of GuavaSoft.
Flow cytometry analysis result (such as table 1, shown in Fig. 8) display is made in 0.5nM TRAIL (114-281)-E263G Apoptosis rate under, under apoptosis rate 22.18%, with 0.5nM wild type TRAIL (114-281) effect (7.28%) it compares, there is significant difference (P < 0.05 *)；Under 1nM TRAIL (114-281)-E263G effect, Apoptosis Rate is 38.4%, compared with the apoptosis rate (13.08%) under 1nM wild type TRAIL (114-281) effect, there is conspicuousness Difference (P < 0.01 * *)；Under 5nM TRAIL (114-281)-E263G effect, apoptosis rate 50.51% is wild with 5nM Apoptosis rate (20.86%) under type TRAIL (114-281) effect is compared, and has significant difference (P < 0.01 * *).Therefore, Flow cytomery result proves that the antitumous effect of TRAIL (114-281)-E263G is substantially better than wild type TRAIL (114-281)。
The rush apoptosis rate of table 1 TRAIL (114-281)-E263G and TRAIL (114-281) to tumour cell COLO 205
Drug concentration TRAIL(114-281) TRAIL(114-281)-E263G
0.5nM 7.28% 22.18%*
1nM 13.08% 38.4%**
5nM 20.86% 50.51%**
Note: 0.5nM TRAIL (114-281)-E263G administration group vs.0.5nM TRAIL (114-281) administration group, * P < 0.05；1nM TRAIL (114-281)-E263G administration group vs.1nM TRAIL (114-281) administration group, P < 0.01 * *；5nM TRAIL (114-281)-E263G administration group vs.5nM TRAIL (114-281) administration group, P < 0.01 * *.
The affinity costant K of 9 wild type TRAIL (114-281) of embodiment and its mutant and DR5DMeasurement
Using ForteBio Octet QKe interaction of biomacromolecules analysis-e/or determining wild type TRAIL (114- And the affinity costant K of mutant and DR5 281)D。
1, the preparation of DR5 extracellular region (1-130 amino acids), reference Biochem Biophys Res Commun 2005, 330:1205-1212 method carries out.Firstly, being template PCR expansion with DR5cDNA (Sino Biological Inc.) Increase DR5 extracellular region (1-130 amino acids) encoding gene, utilizes 1 (5-GGGAATTCCATATGGCTCTGATCACCCAA of primer CAAG-3, SEQ ID NO:8) and primer 2 (5-CCGCTCGAGTGATTCTTTGTGGACACATTC-3, SEQ ID NO:9) draw Enter I restriction enzyme site of Nde I and Xho, and is cloned into expression plasmid pET-21b.Correct recombinant expression plasmid pET-21b/ is sequenced SDR5 Transformed E .coli BL21 (DE3) host strain, 37 DEG C carry out IPTG inducing expression in LB.Thalline were collected by centrifugation after expression, Thallus is resuspended with the PBS buffer solution of the imidazoles containing 10mM, ultrasonication thallus is centrifuged 10min in 4 DEG C with 12000rpm/min, receives Collect supernatant, after 0.45 μm of membrane filtration, with 16/60 molecular exclusion chromatography of Ni-NTA affinity column and Sephacryl S100 Column (GE Health) purifies DR5 extracellular region destination protein.
2, affinity costant KDMeasurement.SD Buffer is added in the 1st column of 96 orifice plates and the 3rd column, and (PBS, pH7.4 contain 0.02%Tween 20,0.1%BSA), every 200 μ l of hole.Biotinylated sDR5 is diluted to about 2 μM with SD Buffer, so The 2nd column, every 200 μ l of hole are added to afterwards.TRAIL or mutant are diluted to 7 gradients: 6000nM with SD Buffer, 3000nM, 1500nM, 750nM, 375nM, 187.5nM, 93.75nM.It is then added into the 4th column of 96 orifice plates, every hole 200 μl；96 orifice plates are put into Octet QKe biomolecular interaction analysis instrument.It is pressed in ForteBio Octet QKe system SD buffer balance 60s, marked by streptavidin sensor with sDR5 ining conjunction with 300s, SD buffer balance 120s, and dash forward Variant TRAIL (114-281)-E263G or wild type TRAIL (114-281) combine 300s and the program of dissociation 600s to be tied It closes and dissociation measures (result is as shown in Figure 9), and K is calculated using ForteBio Octet QKe Data Analysis SoftwareDIt is worth (table 2)。
The affinity costant K of 2 TRAIL of table (114-281) and TRAIL (114-281)-E263G mutant and DR5D
Ligand Kon(M-1S-1) Koff(S-1) KD(M)
TRAIL(114-281) (7.72±0.21)×104 (1.51±0.1)×10-3 1.96±0.2×10-8
TRAIL(114-281)-E263G (1.32±0.06)×105 (1.02±0.12)×10-3 7.73±0.5×10-9
Thus it proves, the affinity of mutant TRAIL (114-281)-E263G and death receptor DR5 are wild type TRAIL 2.54 times of the affinity of (114-281) and death receptor DR5.

Claims (7)

1. a kind of mutant of apoptosis induction ligand related to human tumor necrosis factor, it is characterised in that the human tumour necrosis factor Apoptosis induction ligand related is wild type, the TRAIL of the film mating type of recombination or the TRAIL active tablet containing extracellular soluble outskirt Section, the mutant include the amino acid sequence as shown in SEQ ID No:1 or 2.
2. mutant as described in claim 1, it is characterised in that the variant amino acid sequence such as SEQ ID No:1 institute Show.
3. mutant as described in claim 1, it is characterised in that the variant amino acid sequence such as SEQ ID No:2 institute Show.
4. mutant as described in any one of claims 1-3 is in the anti-tumor drug for preparing TRAIL mediate tumor cell apoptosis In purposes.
5. purposes as claimed in claim 4, it is characterised in that the drug contains as described in any one of claims 1-3 Mutant is as active pharmaceutical ingredient and pharmaceutically acceptable carrier.
6. the purposes as described in claim 5, it is characterised in that the drug also contains other anti-tumor drugs and/or resists swollen Tumor medicine sensitizer.
7. the preparation method of mutant as described in any one of claims 1-3, it is characterized in that passing through artificial synthesized or gene gram The encoding gene of any one of the grand method preparation claim 1-3 mutant, and table is carried out in corresponding host cell It reaches.
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1380339A (en) * 2001-04-10 2002-11-20 成都地奥制药集团有限公司 Mutational human gene nucleotide sequence coded polypeptide
CN1417229A (en) * 2001-11-09 2003-05-14 上海中信国健药业有限公司 Human tumor necrosin relative death inducing ligand mutein and its prepn process and medicine composition
CN1958794A (en) * 2005-11-03 2007-05-09 成都地奥制药集团有限公司 Method for preparing mutant code cDNA of apoptosis induction ligand related to human tumor necrosis factor, and application
CN102119032A (en) * 2007-11-23 2011-07-06 格罗宁根大学 Improved cytokine design
CN102388062A (en) * 2007-12-17 2012-03-21 私人基金基因组调控中心(Crg) Trail variants for treating cancer
CN102775497A (en) * 2012-07-13 2012-11-14 浙江大学 TNF (Tumor Necrosis Factor)-related apoptosis-inducing ligand fusion protein and preparation method thereof

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1380339A (en) * 2001-04-10 2002-11-20 成都地奥制药集团有限公司 Mutational human gene nucleotide sequence coded polypeptide
CN1417229A (en) * 2001-11-09 2003-05-14 上海中信国健药业有限公司 Human tumor necrosin relative death inducing ligand mutein and its prepn process and medicine composition
CN1958794A (en) * 2005-11-03 2007-05-09 成都地奥制药集团有限公司 Method for preparing mutant code cDNA of apoptosis induction ligand related to human tumor necrosis factor, and application
CN102119032A (en) * 2007-11-23 2011-07-06 格罗宁根大学 Improved cytokine design
CN102388062A (en) * 2007-12-17 2012-03-21 私人基金基因组调控中心(Crg) Trail variants for treating cancer
CN102775497A (en) * 2012-07-13 2012-11-14 浙江大学 TNF (Tumor Necrosis Factor)-related apoptosis-inducing ligand fusion protein and preparation method thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Designed tumor necrosis factor-related apoptosis-inducing ligand variants initiating apoptosis exclusively via the DR5 receptor;Van der sloot等;《PNAS》;20060606;第103卷(第23期);全文
Receptor-selective mutants of apotosis-inducing ligand 2/tumor necrosis factor-related apoptosis-inducing ligand reveal a greater contribution of death receptor(DR)5 than DR4 to apoptosis signaling;Kelley 等;《The journal of biological chemistry》;20050121;第280卷(第3期);第2207页右侧第1段、第2211页倒数第1段、第2212左栏最后1段、图1、表III

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