WO2016138618A1 - TRAIL穿膜肽样突变体MuR5、制备方法及应用 - Google Patents

TRAIL穿膜肽样突变体MuR5、制备方法及应用 Download PDF

Info

Publication number
WO2016138618A1
WO2016138618A1 PCT/CN2015/073504 CN2015073504W WO2016138618A1 WO 2016138618 A1 WO2016138618 A1 WO 2016138618A1 CN 2015073504 W CN2015073504 W CN 2015073504W WO 2016138618 A1 WO2016138618 A1 WO 2016138618A1
Authority
WO
WIPO (PCT)
Prior art keywords
trail
protein
mutant
transmembrane peptide
mur5
Prior art date
Application number
PCT/CN2015/073504
Other languages
English (en)
French (fr)
Inventor
陈守春
闫娟
徐琦
黄先洲
魏利佳
胡海洋
Original Assignee
成都华创生物技术有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 成都华创生物技术有限公司 filed Critical 成都华创生物技术有限公司
Priority to PCT/CN2015/073504 priority Critical patent/WO2016138618A1/zh
Priority to EP15883675.9A priority patent/EP3266796B1/en
Priority to CN201580000086.0A priority patent/CN106132986B/zh
Publication of WO2016138618A1 publication Critical patent/WO2016138618A1/zh
Priority to US15/591,139 priority patent/US10000552B2/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/525—Tumour necrosis factor [TNF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to the field of genetic engineering medicines, in particular to a TRAIL transmembrane peptide-like mutant MuR5, a preparation method and application thereof.
  • Tumor necrosis factor-related apoptosis-inducing ligand is a member of the Tumor necrosis factor (TNF) superfamily.
  • TNF Tumor necrosis factor
  • the gene sequences were respectively in 1995 by Wiley et al. It was independently cloned by Pitti et al. in 1996, which was named as apoptotic 2 ligand (Apo2Ligand, Apo2L). Later studies confirmed that Apo2L is essentially the same protein as TRAIL, so it is customary to call it Apo2L/TRAIL.
  • TRAIL The function of TRAIL is firstly used as a regulator of congenital or acquired immunity in organisms, and secondly as an anti-tumor effect in immunocytochemistry in an exogenous apoptotic pathway.
  • the greatest advantage of TRAIL is that it can selectively induce apoptosis of a variety of tumor cells with little toxicity to normal cells.
  • Apo2L/TRAIL is a human tumor cell line of various origins, including in vitro or in vivo, including nodules, lung cancer, breast cancer, prostate cancer, pancreatic cancer, kidney cancer, central nervous system tumors, Thyroid cancer, lymphoma, leukemia, and multiple myeloma all have the effect of inducing apoptosis.
  • TRAIL has been developed as an important potential anti-tumor drug.
  • the clinical trial of TRAIL has entered Phase II abroad and has completed Phase III in China.
  • a large number of in vitro and in vivo tests have confirmed that TRAIL has tumor-specific cytotoxicity, especially when it is combined with low-dose chemotherapeutic drugs, it shows significant synergy and synergy.
  • studies have found that TRAIL tolerance caused by loss of apoptotic machinery in the body is clearly associated with rapid growth and metastasis of tumor cells.
  • Tumor is a group of highly heterogeneous diseases. Traditionally, according to the classification methods of tissues and organs and pathological changes, it has not been suitable for the diagnosis and treatment of tumors. The current research direction is to clarify the gene expression and molecular typing of different tumor cells and give patients more Targeted treatment.
  • the deep understanding of anti-tumor drugs has led to the understanding that whether cytotoxic drugs, molecularly targeted drugs or monoclonal antibodies play a role in the activation of tumor cell apoptosis pathways, signals that induce tumor cell apoptosis Access pathways are the hub and central link in which these drugs work, and apoptosis avoidance is an important mechanism for tumor development and drug resistance.
  • TRAIL TRAIL-sensitive tumor cells
  • TRAIL-resistant tumor cell has defects in some links and factors in the apoptotic signaling pathway.
  • mutations these defects and mutations make these drug-resistant tumor cell apoptosis threshold abnormally elevated, easier to escape from apoptosis, and thus continue to grow and proliferate.
  • apoptotic signaling pathway to regulate the expression of DRs, enhance the aggregation and redistribution of DRs in the lipid raft microdomains on the cell membrane, enhance the endocytosis of TRAIL/DRs complex in the cell membrane, and promote the synthesis of DISC to TRAIL/DRs Recruitment, activation of the initial phase of Caspase (Caspase 8) activity, inhibition of apoptosis antagonistic factors FLIP, XIAP and IAPs activity, etc.) or mitochondrial apoptosis signaling pathway (such as enhanced mitochondrial membrane potential depolarization, promote mitochondria Increased permeability and release of Cyt c, Smac or ARTs, which promotes the cleavage of Bid to tBid, promotes the oligomerization of Bax and Bad, inhibits apoptosis and antagonizes Bcl-2, Bcl-xL, Bcl-w, Mcl-1, and surviv
  • other cell survival signaling pathways such as ERK/PI3K/AKt, MEK, Jak-STAT 3, MAPK, NF- ⁇ B, etc.
  • TRAIL and its receptor agonistic monoclonal antibody drug development process is temporarily frustrated, with the complete elucidation of the apoptotic signaling pathway pathway, the complete relationship between apoptosis/tolerance transmutation, the targeting based on apoptotic signaling pathway Anti-tumor drug development has not stopped.
  • TRAIL and cytotoxic drugs belong to two different types of drugs, there are differences and differences in drug dosages, routes of administration, and modes of action. It is less likely to develop a single, stable, and controllable new drug, and TRAIL and cytotoxicity. After the combination of drugs, its toxic side effects still exist, so its advantages are not obvious.
  • the object of the present invention is to provide a novel TRAIL transmembrane peptide which can greatly enhance the anti-tumor activity of TRAIL wild-type protein, in particular, can reverse the resistance of multi-drug resistant tumor cells to TRAIL wild-type protein.
  • Mutant mutant The prepared mutant protein can rapidly enter the cytoplasm by penetrating the cell membrane, and can promote the aggregation and internalization of the death receptor/mutein complex in the cell membrane lipid raft microdomain, and enhance the exogenous apoptosis signal. Transduction of pathways.
  • TRAIL transmembrane peptide-like mutants have superior therapeutic effects on many different types of tumors and are a promising new generation of highly effective tumor cell apoptosis drugs.
  • a TRAIL transmembrane peptide-like mutant wherein the mutant has an amino acid sequence of SEQ ID NO: 2.
  • the TRAIL transmembrane peptide-like mutant is characterized in that the 114th to the 118th amino acid coding sequence of the outer segment of the TRAIL wild-type protein is selectively transformed from VRRRG to RRRRR, that is, the 114th position is mutated by proline to Arginine, the 116th position is mutated to arginine by glutamate, and the 118th position is mutated to arginine by glycine, so that the N-terminus of the mutant protein becomes a continuous 5 arginine coding sequence, which becomes a transmembrane peptide. Protein-like structure.
  • the cDNA sequence encoding the mutant is SEQ ID NO: 1.
  • kits for amplifying the mutant comprising the following primers:
  • a method for preparing a TRAIL transmembrane peptide-like mutant comprises the following steps:
  • the step of constructing and identifying the expression vector in the step (2) comprises:
  • the prokaryotic expression vector described in the step (b) is pET 32a.
  • the induction temperature is 18 to 24 °C.
  • the step of purifying the TRAIL protein in the step (4) comprises:
  • the cation exchange resin SP Sepharose Fast Flow was purified as the first step to capture the target protein in the supernatant after the disruption;
  • the cation exchange resin Sephadex G-25medium is used as a second step for moderate purification to further increase protein purity and remove endotoxin;
  • the anion exchange resin Q Sepharose Fast Flow is used as a final step for fine purification to meet the needs of industrial scale-up and future clinical applications.
  • the mechanism of the invention is to induce apoptosis of tumor cells, and the TRAIL transmembrane peptide-like mutant can rapidly enter the tumor cells to induce apoptosis by transmembrane action.
  • TRAIL transmembrane peptide-like mutants can also effectively promote the aggregation, redistribution and/or internalization of TRAIL-DR4/DR5 complexes in the lipid raft microdomains of cell death receptors, and enhance exogenous apoptosis signals. Transduction of pathways.
  • the present invention provides a novel protein structure with minimal mutation sites, minimal effect on protein structure, and maximized function.
  • TRAIL transmembrane peptide-like mutants only pass through three non-contiguous site mutations. Since the site mutation occurs at the amino terminus of the protein, it has little effect on the biological activity and stability of the protein, but it has obtained a transmembrane peptide. The ability of the fusion protein to penetrate the membrane.
  • the expression vector can obtain higher than TRAIL wild-type protein expression level and soluble expression in the wide induction temperature range of 18 ⁇ 24 °C
  • the ratio of soluble protein is 80% to 100%.
  • TRAIL transmembrane peptide-like mutants have significantly improved anti-tumor activity in almost all tumor cell types tested, especially for TRAIL wild-type protein.
  • the tumor cell line of the drug can significantly reverse the tolerance of these cells to the wild type protein of TRAIL, and has a stronger therapeutic effect.
  • FIG. 1 Electrophoresis pattern of TRAIL-MuR5 fragment PCR product; electrophoresis conditions: 3% Agarose, voltage 100V, 20min; Lane 1: TRAIL-MuR5 fragment PCR product electrophoresis band; M: DL2000 (strand molecular weight from top to bottom : 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp), the amount of the sample was 5 ⁇ l, and the amount of the PCR product loaded was 5 ⁇ l.
  • Figure 2 TRAIL-MuR5 and pET32a plasmid NdeI, EcoRI digestion and electrophoresis; electrophoresis conditions: 1% Agarose, voltage 150V, 25min; Lane 1: TRAIL-MuR5 enzyme digestion after gel recovery band; Lane2: pET32a digestion Post-gel recovery electrophoresis band; M: GeneRuler1kb DNA Ladder (band molecular weight from top to bottom: 10000bp, 8000bp, 6000bp, 5000bp, 4000bp, 3500bp, 3000bp, 2500bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp ), the amount of sample was 5 ⁇ l; the amount of PCR product loaded was 3 ⁇ l.
  • Figure 3 pET32a/TRAIL-MuR5 plasmid XbaI and EcoRI digestion and identification of electrophoresis; electrophoresis conditions: 1% Agarose, voltage 150V, 30min; Lane 1 ⁇ 10: pET32a / TRAIL-MuR5 strain extracted by enzyme digestion and electrophoresis M: GeneRuler1kb DNA Ladder (the molecular weight of the band is 10000 bp, 8000 bp, 6000 bp, 5000 bp, 4000 bp, 3500 bp, 3000 bp, 2500 bp, 2000 bp, 1500 bp, 1000 bp, 750 bp, 500 bp, 250 bp) from the top to the bottom; The amount was 10 ⁇ l and the Marker load was 5 ⁇ l.
  • Figure 4 SDS-PAGE electrophoresis pattern of pET32a/TRAIL-MuR5 expression; electrophoresis conditions: 15% gel, 200V, 35min; Lane 1: pET32a/TRAIL-MuR5 pre-induction electrophoresis band, Lane 2: pET32a/TRAIL-MuR5 induction Post-electrophoresis band, Lane 3: pET32a/TRAIL-MuR5, supernatant after electrophoresis, Lane 4: pET32a/TRAIL-MuR5, post-cracking electrophoresis band; M: Unstained Protein Molecular Weight Marker From top to bottom: 116.0KDa, 66.2KDa, 45.0KDa, 35.0KDa, 25.0KDa, 18.4KDa, 14.4KDa), Marker sample loading is 5 ⁇ l, and other samples are loaded with 20 ⁇ l.
  • Figure 5 SDS-PAGE electropherogram of the cation exchange process; electrophoresis conditions: 15% gel, 200V, 50 min.
  • Lane 1 cation exchange stock solution
  • Lane 2 cation exchange permeate
  • Lane 3 cation exchange 600 mM NaCl eluate
  • Lane 4 cation exchange 1.2 M NaCl eluate
  • Lane 5 cation exchange NaOH eluate
  • M Unstained Protein Molecular Weight Marker From top to bottom: 116.0KDa, 66.2KDa, 45.0KDa, 35.0KDa, 25.0KDa, 18.4KDa, 14.4KDa).
  • the sample loading Marker was 5 ⁇ l, and the others were 20 ⁇ l.
  • Figure 6 SDS-PAGE electrophoresis pattern of anion exchange process; electrophoresis conditions: 15% gel, 200V, 50 min.
  • Lane 1 anion exchange stock solution
  • Lane 2 anion exchange permeate
  • Lane 3 2M NaCl eluate
  • Lane 4 0.5 M NaOH eluate
  • M Unstained Protein Molecular Weight Marker (stripe molecular weight from top to bottom They are: 116.0 KDa, 66.2 KDa, 45.0 KDa, 35.0 KDa, 25.0 KDa, 18.4 KDa, 14.4 KDa).
  • the sample loading Marker was 5 ⁇ l, and the others were 20 ⁇ l.
  • Figure 7 Western blot analysis results
  • Lane 1 TRAIL-MuR5 western blot results
  • Lane2 TRAILwestern blot results
  • M PageRuler Prestained Protein Ladder (stripe molecular weight from top to bottom: 170KDa, 130KDa, 100KDa, 70KDa , 55KDa, 40KDa, 35KDa, 25KDa, 15KDa, 10KDa).
  • Penetrating peptide is a kind of short peptide with positive membrane cations of 20-30 amino acids with cell membrane penetrating function. It is a new drug transport and transmission technology developed in recent decades, also known as protein transduction. Protein transduction domain (PTD).
  • PTD Protein transduction domain
  • HAV-1 transactivator TAT can translocate across the membrane into the cytoplasm and nucleus.
  • arginine-rich TAT polypeptides GRKKRRQRRRGY
  • GRKKRRQRRRGY arginine-rich TAT polypeptides
  • Drosophila homologous transcription factor ANTP herpes simplex virus type I (HSV-1) transcription factor VP22, Transpotan, polyarginine and other sequences have cell membrane penetration ability, and hundreds of species have been found. Peptide function of membrane function.
  • transmembrane peptides can be divided into different species according to different criteria. In terms of structural characteristics, early transmembrane peptides were simply divided into: (1) transmembrane peptides with a large number of cations without typical structures, such as TAT and penetratin; (2) amphiphilic ⁇ derived from protein signal sequences Spiral peptide. From the source, some people have divided the transmembrane peptide into natural and synthetic, and further can be divided into three categories: (1) transmembrane peptides derived from proteins, such as penetratin, TAT and pVEC. They usually have the smallest effective fragments of the transporter, the protein transduction moiety and the membrane ectopic sequence.
  • Model transmembrane peptides such as MAP and Arg (7), etc., which are artificially synthesized to form an amphipathic alpha helix or to mimic the structure of a known transmembrane peptide.
  • polyarginine and polylysine According to the structure of the transmembrane peptide, polyarginine and polylysine have higher transmembrane activity than TAT protein.
  • the key structure of an amino acid having a membrane-performing peptide is that its main molecular composition is rich in basic amino acids such as arginine, lysine, and histidine.
  • Basic amino acids are an important feature of this type of transmembrane protein. These amino acids have a strong positive charge and may interact with negatively charged cell membrane lipid molecules to mediate the transmembrane process, where arginine residues play an important role in protein cell internalization.
  • polyarginine transduction protein into cells: one is to directly form a small pore into the cell by arginine in the cell membrane and lipid bilayer; Various forms of mediated endocytosis, including macrocytosis, small recess protein-mediated, clathrin-mediated, phagocytosis, and endocytic exchange, and other mechanisms of transduction of proteins into cells.
  • TRAIL-induced aggregation and redistribution of death receptors on lipid membrane microdomains of tumor cells recruiting Fas-Associated death domain (FADD) with or without endocytosis of TRAIL-DR4/5 complex
  • Caspase-8 assembled into a death inducing signaling complex (DISC), a cascade cascade that initiates apoptotic effects by cleaving caspase-8.
  • FADD Fas-Associated death domain
  • DISC death inducing signaling complex
  • Most literatures suggest that internalization of the TRAIL-DR4/5 complex is required for continuous amplification of apoptotic signals.
  • a foreign protein is expressed in fusion with a transmembrane peptide, and the expressed fusion protein may alter the spatial conformation of the protein molecule to lose its biological activity.
  • the fusion protein increases the antigenicity of the original protein molecule and poses a safety risk.
  • the TRAIL protein By selectively mutating the TRAIL protein soluble fragment (114-281aa) to encode several amino acids at the N-terminus of the amino acid sequence, the TRAIL protein forms a peptide-like peptide-like amino acid sequence, which is a transmembrane peptide-like mutation in TRAIL. Different transmembrane peptide-like mutants.
  • the invention breaks through the design idea of the original transmembrane peptide fusion protein, and selectively selects the 114th proline and the 116th glutamic acid at the N-terminus of the 114th to 281th amino acid coding sequences of the outer segment of the TRAIL wild type protein.
  • the 118th glycine was mutated to arginine, respectively, and the 114th to 118th amino acids of the TRAIL protein form 5 consecutive arginine sequences.
  • the endogenous 5 consecutive arginine sequences minimize the change in the N-terminal amino acid sequence of the TRAIL extracellular domain (retaining the original 115th and 117th arginine sequences), which maintains the blank conformation of TRAIL protein to the greatest extent.
  • biological activity, and constructed to form a continuous five arginine sequences with transmembrane function we named the transmembrane peptide-like mutant of the present invention as TRAIL-MuR5.
  • the penetrating peptide-like mutant is a new design idea of a transmembrane peptide fusion protein.
  • the amino acid coding sequence of the outer segment of the TRAIL wild-type protein was modified from VRRRG to RRRR, that is, the 114th position was changed from proline to arginine, and the 116th position was changed from glutamic acid to refined. Amino acid, the 118th position is mutated from arginine to arginine, and the mutation site is 3, so that the N-terminus of the mutant protein becomes a coding sequence of 5 consecutive arginine, and becomes a protein containing a penetrating peptide-like structure.
  • the cDNA encoding the mutant is SEQ ID NO: 1, and the mutant amino acid is SEQ ID NO: 2.
  • the upstream primer MuR5-TR-NdeI is represented by SEQ ID NO:3;
  • the downstream primer TR-Eco-R is set forth in SEQ ID NO:4.
  • the TRAIL-MuR5 fragment was amplified by PCR and ligated with pET32a, and the single colony of the ligated product was picked and identified.
  • the TRAIL-MuR5 fragment was amplified by PCR using the pMD19/TRAIL plasmid as a template.
  • the TRAIL-MuR5 fragment of interest and the vector pET32a were digested with NdeI and EcoRI, respectively.
  • the TRAIL-MuR5 fragment was ligated to the vector pET32a excised with the Trx fusion tag sequence, transformed into Top10 competent cells, picked up for monoclonal, and identified by double digestion with XbaI and EcoRI.
  • the primer design is shown in Example 1.
  • the original sequence of the pMD19/TRAIL plasmid was derived from the NCBI Reference Sequence: NM_003810.3, and the vector pET32a was derived from Invitrogen.
  • the MuR5-TR-NdeI/TR-Eco-R primer pair amplified the TRAIL-MuR5 target fragment, and prepared a reaction system according to Table 1, and the reaction system was 50 ⁇ l.
  • the PCR-amplified TRAIL-MuR5 target fragment was recovered by Omega Glue Recovery Kit, eluted with 50 ⁇ l of ultrapure water, electrophoresed, photographed, and used.
  • the target fragment TRAIL-MuR5 is ligated with pET32a plasmid after double digestion
  • the vector and the target gene fragment were digested with NdeI and EcoRI.
  • the enzyme digestion system is shown in Table 3, and the reaction system was 100 ⁇ l.
  • the OMEGA gel recovery kit was used for gel recovery, and the vector and the target fragment were eluted with 30 ⁇ l of ultrapure water, respectively. Electrophoresis, photography.
  • the target fragment of the rubber recovery is connected to the carrier, and the connection system is shown in Table 4.
  • the bacteria were blown well, all coated on Amp-containing LB solid medium, and cultured overnight at 37 °C.
  • step 5 Add the supernatant obtained in step 5 to the two HiBind Miniprep adsorption columns that have been packed into the collection tube. Be careful not to aspirate the precipitate, centrifuge at 10,000 g for 1 min, pour off the waste liquid in the collection tube, and re-dispose the adsorption column. Go back to the collection tube.
  • pET32a/TRAIL-MuR5 plasmid The plasmid was digested with XbaI and EcoRI. The enzyme digestion reaction system is shown in Table 5.
  • the TRAIL-MuR5 fragment was amplified by MuR5-TR-NdeI/TR-Eco-R primer pair, and the molecular weight of the fragment was about 500 bp. As shown in Fig. 1, the target gene was obtained according to the above PCR reaction conditions.
  • TRAIL–MuR5 and pET32a were digested with NdeI and EcoRI to obtain target fragments of about 500 bp and 5.4 kb, respectively. As shown in Fig. 2, the gel recovery after enzymatic cleavage yielded the expected single band. .
  • TRAIL-MuR5 target fragment is linked to pET32a and transformed results respectively
  • the plate has colonies growing and the density is normal.
  • the plasmid was identified by restriction enzyme digestion.
  • the pET32a/TRAIL-MuR5 plasmid was digested with XbaI and EcoRI. After ligating the plasmid, the vector fragment of about 5.4Kb and the target fragment of about 550bp should be observed. As shown in Fig. 3, pET32a/TRAIL-MuR5 was positive for 6 samples, and the positive plasmid was sent to the Huada gene for sequencing, and the correct plasmid was used to preserve the strain.
  • the plasmid with the correct sequencing was transformed into competent E. coli BL21 (DE3) in Example 2, and a single strain was picked for expression test to examine the expression effect.
  • the cells were centrifuged in 50 ml of culture medium, resuspended in 8 ml of 50 mM Na 2 HPO 4 solution, and disrupted by ultrasonic wave.
  • the bacteriostatic conditions were: ⁇ 6 probe, 200W pulsed for 2s, paused for 2s, and cycled for 10min. .
  • the bacterial solution was centrifuged at 12000 rpm for 10 min, and the supernatant and precipitate were separated. The pellet was resuspended in 1 ml of H 2 O. The supernatant and the pellet were each 20 ⁇ l. 30 ⁇ l of H 2 O and 50 ⁇ l of 2 ⁇ loading buffer were added to prepare for electrophoresis. sample.
  • the prepared electrophoresis sample was placed in a boiling water bath for 10 minutes, and centrifuged at 10,000 rpm for 10 minutes using a 5430R centrifuge, A-45-30-11 rotor, and 10 ⁇ l of each supernatant.
  • TRAIL-MuR5 The experimental electropherogram is shown in Figure 4 (pET32a/TRAIL-MuR5).
  • TRAIL-MuR5 has strong expression, and most of the expressed products are supernatant after sterilizing, and the soluble expression ratio is high.
  • TRAIL-MuR5 protein purification process.
  • SPSepharose Fast Flow gel chromatography column Sephadex G-25medium gel chromatography column
  • anion exchange penetration three-step method.
  • the TRAIL-MuR5 protein was purified in bulk to obtain samples for in vitro and in vivo activity assays.
  • the bacteria liquid was ultrasonically disrupted, and the bacteria breaking condition was as follows: using ⁇ 10 probe, 500W pulse was broken for 2s, and then suspended for 2s, and the bacteria were broken for 15min.
  • Cation exchange buffer A 20 mM Na 2 CO 3 -NaHCO 3 , 0.5 M NaCl, 5% glycerol, 0.1% Tween 80, 1 mM DTT, adjusted to pH 10.50.
  • Cation exchange buffer B 20 mM Na 2 CO 3 -NaHCO 3 , 1.2 M NaCl, 5% glycerol, 0.1% Tween 80, 1 mM DTT, adjusted to pH 10.20.
  • Desalting and anion exchange buffer 0.15 M NaCl, 0.3 M glycine, 0.2 M arginine.
  • the cation exchange purification was carried out in accordance with the following purification steps. All breakthrough and elution components were collected during purification for electrophoresis analysis:
  • Sample preparation and loading Take the supernatant and centrifuge the supernatant.
  • the second step of anion exchange purification was carried out according to the following purification procedure. All breakthrough and elution components were collected during purification for electrophoresis analysis:
  • Sample preparation and loading The cation exchange purification and elution samples were taken, and the Sephadex G-25medium chromatography column replacement buffer was used as an anion exchange buffer, and then loaded.
  • NaCl wash The column was washed with 2CV 2M NaCl to remove the protein bound to the column.
  • Reequilibration Re-equilibrate the column with anion exchange buffer.
  • the electrophoresis results of each step of the purification process are shown in Fig. 5 and Fig. 6: the eluent of the first step SP was collected 22 ml, the concentration was 5.33 mg/ml, the purity of the target protein was 85.35%, and the second step was desalted eluate 30 ml.
  • the concentration of 3.24 mg / ml has the effect of removing residual heteroprotein and part of the pyrogen, while the third step of anion exchange penetrating liquid is 42.85 ml, the concentration is 2.16 mg / ml, mainly depyrogen.
  • the experimental procedure of this example was repeated several times, and the amount of protein sufficient for evaluation of in vitro living activity was obtained, which was 183.39 mg.
  • TRAIL-MuR5 is a mutation in the N-terminal of the wild-type TRAIL, the determinant of TRAIL is still retained and can specifically bind to the polyclonal antibody of TRAIL, so it can be identified by TRAIL polyclonal antibody.
  • Example 5 The purified TRAIL-MuR5 protein was thawed at -20 ° C and diluted to 1 mg/ml with ultrapure water at the concentration provided. 50 ⁇ l of the sample was added to 50 ⁇ l of 2 ⁇ loading buffer to prepare an electrophoresis sample. Each 10 ⁇ l of electrophoresis was taken, that is, the loading amount was 5 ug.
  • Reference product TRAIL-20131204 lyophilized product (prepared by laboratory and compared with NIBSC international standard, activity higher than 10 7 IU/mg) dissolved in 1ml PBS, sampled 50 ⁇ l and added 50 ⁇ l 2 ⁇ loading buffer to make electrophoresis sample. Each 10 ⁇ l of electrophoresis was taken, that is, the loading amount was 5 ug.
  • the sample was separated by 15% SDS-PAGE and transferred to a PVDF membrane. First blocked overnight at 4 ° C, and then incubated with primary antibody [rabbit anti-human TRAIL polyclonal antibody (1:500)] for 2 hours at room temperature, then incubated with secondary antibody [goat anti-rabbit IgG-HRP (1:5000)] for 2 hours at room temperature. Finally, enhanced chemiluminescence (ECL) detection was used. Specific steps are as follows:
  • the protein was separated by 1.15% SDS-PAGE electrophoresis; the gel was removed, the edge of the gel was cut out, and immersed in TBST buffer for 15 min.
  • PVDF membrane transfer (wet transfer): PVDF membrane must be slightly wetted with methanol for 15 seconds before use, then immersed in distilled water for 1-3 minutes, then equilibrated in the transfer buffer; in the transfer clamp, by The negative electrode to the positive electrode are sequentially laid: sponge pad, filter paper (4-8 pieces), target glue, PVDF film, filter paper (4-8 pieces), sponge pad, after the bubble is exhausted, the clamp is placed in the transfer film slot, the voltage is 40V. , time 45min.
  • Blocking membrane The membrane was blocked overnight in blocking solution (3% BSA) at 4 ° C, and the next day was taken and shaken at room temperature for 30 min to block the non-specific binding site.
  • the primary antibody was diluted to the working concentration [rabbit anti-human TRAIL polyclonal antibody (1:500)] with a blocking solution, shaken with the membrane, and incubated for 2 h at room temperature.
  • wash the membrane Wash the membrane three times with TBST for 10 min each time.
  • the film of 10 ⁇ 10 cm requires more than 50 ml of washing liquid.
  • HRP-labeled secondary antibody was diluted to the working concentration [goat anti-rabbit IgG-HRP (1:5000)] with blocking solution, shaken with the membrane, and incubated for 2 h at room temperature.
  • wash the membrane Wash the membrane three times with TBST for 10 min each time.
  • the film of 10 ⁇ 10 cm requires more than 50 ml of washing liquid.
  • IC50 values of TRAIL-MuR5 and wild-type TRAIL 2 protein samples against 12 tumor cell lines were determined by CCK-8 test kit to evaluate its in vitro biological activity.
  • the cell lines for detection were obtained from Shanghai Institute of Cell Science or Wuhan Virus Institute of Chinese Academy of Sciences.
  • Fetal bovine serum (Code: FS101-02, full gold)
  • Two protein samples were diluted in sterile PBS buffer to a final concentration of 5 mg/ml and sterilized by filtration.
  • the protein sample to be tested was diluted to 5 mg/ml in sterile PBS buffer, and then diluted 8 times, and added to the cells at 25 ⁇ l/well.
  • the final concentration of the compound was diluted from 1 mg/ml to 0,3 times for a total of 10 concentration points; and according to the preliminary experimental results, the final concentration of the protein sample was adjusted accordingly.
  • tumor cell growth inhibition rate % [(Ac-As) / (Ac-Ab)] ⁇ 100%
  • the IC50 curve was fitted and the IC50 value was calculated using the software Graphpad Prism 5 and using the calculation formula log(inhibitor) vs.normalized response-Variable slope.
  • This experiment tested two protein samples (TRAIL-MuR5 and wild-type TRAIL) against three pancreatic cancer cell lines (CFPAC-1, BxPC-3, PANC-1) and two lung cancer cell lines (NCI-H460, A 549). ), 3 knot (straight) intestinal cancer cell lines (SW620, HT-29, HCT 116), 3 breast cancer cell lines (MDA-MB-231, MCF-7, T47D), 1 acute T cell leukemia cell In vitro anti-cell proliferation activity of strain (Jurkat). The experimental results are shown in the table below.
  • the TRAIL transmembrane peptide-like mutant TRAIL-MuR5 is compared to TRAIL wild-type protein in almost all tumor cell types tested [including multiple nodules (straight) intestinal cancer cells, various lung cancer cells, and various pancreatic cells. , a variety of breast cancer cells], its anti-tumor activity is significantly improved, especially for TRAIL wild-type protein-resistant tumor cell lines, can significantly reverse the tolerance of these cells to TRAIL wild-type protein, has a stronger therapeutic effect.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Cell Biology (AREA)
  • Plant Pathology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

提供了一种TRAIL穿膜肽样突变体MuR5、制备方法及应用，所述TRAIL穿膜肽样突变体是通过选择性地将TRAIL野生型蛋白胞膜外段第114～118位氨基酸编码序列由VRERG改造为RRRRR，成为包含穿膜样肽结构的蛋白。所述突变体可用于治疗肿瘤。

Description

TRAIL穿膜肽样突变体MuR5、制备方法及应用 技术领域
本发明涉及基因工程药物领域，特别是指一种TRAIL穿膜肽样突变体MuR5、制备方法及应用。
背景技术
1、Apo2L/TRAIL用于肿瘤治疗的进展与意义
肿瘤坏死因子相关凋亡诱导配体(Tumor necrosis factor-related apoptosis-inducing ligand，TRAIL)为肿瘤坏死因子(Tumor necrosis factor，TNF)超家族的成员，其基因序列分别于1995年由Wiley等人和1996年由Pitti等人独立克隆获得，后者将其命名为凋亡素2配体(Apo2Ligand，Apo2L)。后来的研究证实，Apo2L与TRAIL实质上是同一种蛋白质，因此习惯上可将其称为Apo2L/TRAIL。TRAIL的功能首先是作为生物体先天性或获得性免疫的调节剂，其次在细胞外源性凋亡途径中作为免疫监视发挥抗肿瘤的作用。TRAIL的最大优点是可以选择性地诱导多种肿瘤细胞凋亡而对正常细胞几乎没有毒性。研究资料表明，无论在体外还是体内，Apo2L/TRAIL对于各种来源的人肿瘤细胞系，包括结(直)肠癌、肺癌、乳腺癌、前列腺癌、胰腺癌、肾癌、中枢神经系统肿瘤、甲状腺癌、淋巴瘤、白血病以及多发性骨髓瘤等都具有诱导凋亡的作用。
从发现至今的近20年时间里，TRAIL一直被作为一种重要的潜在抗肿瘤药物开发，TRAIL的临床实验在国外已进入Ⅱ期，在国内已完成Ⅲ期。大量体内外试验均证实，TRAIL具有肿瘤特异性细胞毒性，尤其当它与小剂量化疗药物联用时即表现出明显的协同和增效作用。相反，研究发现机体中凋亡机制的缺失导致的TRAIL耐受与肿瘤细胞的快速生长和转移明确相关。
肿瘤是一组高度异质性的疾病，传统上按照组织器官、病理改变的分型方法已经不适合肿瘤的诊疗，目前的研究方向在于阐明不同肿瘤细胞的基因表达、分子分型，给予患者更具针对性的治疗。对于抗肿瘤药物认识的深入使人们了解到，无论细胞毒性药物、分子靶向药物还是单克隆抗体，其发挥作用的过程中均涉及到肿瘤细胞凋亡途径的激活，诱导肿瘤细胞凋亡的信号通路途径是这些药物发挥作用的枢纽和中心环节，而凋亡逃避正是肿瘤发生发展以及耐药的重要机制。
2、Apo2L/TRAIL用于肿瘤治疗的缺陷和对策
最近进展显示，仅依赖Apo2L/TRAIL治疗多种不同类型的肿瘤仍是远远不够的。尽管重组人Apo2L/TRAIL或TRAIL受体DR4/DR5的激动性单克隆抗体在Ⅰ期临床治疗中取得令人鼓舞的结果，但在随后进行的Ⅱ期临床研究中却没有显示出明确的临床受益。大量研究表明，正常细胞以及大约一半(甚至高达60％)以上的传代肿瘤细胞株对TRAIL表现出耐药。根据Roberta di peitro和Giorgia zauli的综述，Apo2L/TRAIL对已经研究的92株原代或传代肿瘤细胞中的61株敏感，敏感率为66.3％，而对其余的31株耐药，耐药率为33.7％。TRAIL对正常细胞的耐受有着其生理意义，TRAIL在体内保持着精准的调控，只在生长发育过程中清除衰老退化和转化细胞中发挥作用，而不至于对正常细胞产生杀伤。几乎所有的 TRAIL敏感肿瘤细胞在其凋亡信号通路中的各个环节和因素均具有相似的完整和功能，而每一种TRAIL耐药肿瘤细胞均在凋亡信号通路中的一些环节和因素存在缺陷和变异，这些缺陷和变异使得这些耐药的肿瘤细胞凋亡阈值异常升高，较易逃避凋亡清除，从而持续生长增殖。
大量研究证实，单用Apo2L/TRAIL对于许多肿瘤细胞并不产生高效的抑制和杀伤作用。究其原因，肿瘤细胞凋亡信号通路是一个十分复杂庞大的系统，其中既包含许多促凋亡因素，又包含大量的凋亡抑制因子，这两方面的因素的相互作用决定了肿瘤细胞的最后归宿。凋亡信号通路的健全和功能是肿瘤细胞凋亡的必要条件，但并不是充分条件。多种不同类型的药物、分子或基因干预均可增强TRAIL对肿瘤细胞的敏感性，这些药物包括不同类型的化疗药物、天然产物、小分子激酶抑制剂等。他们分别通过强化细胞外凋亡信号通路(如上调DRs表达，增强DRs在细胞膜上脂筏微区域的聚集和重分布，增强TRAIL/DRs复合物在细胞膜的内吞，促使DISC向TRAIL/DRs复合物的募集，激活起始阶段Caspase(Caspase 8)的活性，抑制凋亡拮抗因子FLIP、XIAP和IAPs的活性等)或线粒体凋亡信号通路(如增强线粒体膜电势的去极化，促使线粒体通透性增加并释放Cyt c、Smac或ARTs，促使Bid裂解为tBid，促使Bax、Bad寡聚化，抑制凋亡拮抗Bcl-2、Bcl-xL、Bcl-w、Mcl-1、survivin因子等)或抑制其他细胞生存信号通路(如ERK/PI3K/AKt、MEK、Jak-STAT 3、MAPK、NF-κB等)或几条通路的联合而增强TRAIL诱导的肿瘤细胞凋亡活性。
尽管TRAIL及其受体激动性单克隆抗体药物开发过程暂时受挫，但是随着细胞凋亡信号通路途径的完全阐明，凋亡/耐受相互转换关系的完全揭示，基于凋亡信号通路的靶向抗肿瘤药物研发并未停步。目前研究较多的是将TRAIL与细胞毒类药物的联合应用，但大多数实验显示这种联合仅能对TRAIL相对敏感的肿瘤细胞产生明显的协同和增效作用，而不能完全逆转多种不同耐药机制产生的耐药现象。由于TRAIL与细胞毒类药物分属两类不同药物，存在药物品种剂量、给药途径、作用方式的不同和差异，开发成单一、稳定、可控的新药可能性较小，且TRAIL与细胞毒类药物联用后，其毒副作用依然存在，故其优势并不明显。
发明内容
针对现有技术的缺陷，本发明的目的是提供一种能够大幅度增强TRAIL野生型蛋白抗肿瘤活性，尤其是能够逆转多种耐药肿瘤细胞对TRAIL野生型蛋白耐药的新型TRAIL穿膜肽样突变体。制备的突变体蛋白既能通过穿透细胞膜直接进入细胞浆而快速起效，又能促进死亡受体/突变蛋白复合物在细胞膜脂筏微区域的聚集和内化，增强外源性凋亡信号途径的转导。TRAIL穿膜肽样突变体对于多种不同类型的肿瘤具有优越的治疗作用，是极具潜力的新一代高效诱导肿瘤细胞凋亡药物。
本发明的技术方案是这样实现的：
一种TRAIL穿膜肽样突变体，其中，所述突变体的氨基酸序列如SEQ ID NO：2。
进一步的，所述TRAIL穿膜肽样突变体是通过选择性地将TRAIL野生型蛋白胞膜外段第114～118位氨基酸编码序列由VRERG改造为RRRRR，即第114位由缬氨酸突变为精氨酸，第116位由谷氨酸突变为精氨酸，第118位由甘氨酸突变为精氨酸，使得突变体蛋白的N端成为连续5个精氨酸编码序列，成为包含穿膜肽样结构的蛋白。
进一步的，编码所述突变体的cDNA序列如SEQ ID NO：1。
进一步的，扩增所述突变体的试剂盒，所述试剂盒包括如下引物：
上游引物MuR5-TR-NdeI：
GGTCATATGCGTCGTCGTCGTCGTCCGCAGCGTGTGGCTGCTCAC
下游引物TR-Eco-R：
GTTGAATTCT TATTAACCAA CAAGGAAAGC ACCGAAGAAA G。
一种TRAIL穿膜肽样突变体的制备方法，包括如下步骤：
(1)、cDNA片段扩增与克隆；其中cDNA序列如SEQ ID NO：1；
(2)、表达载体的构建与鉴定；
(3)、重组TRAIL蛋白的融合表达；
(4)、TRAIL蛋白的纯化；
(5)、TRAIL蛋白鉴定。
作为优选的技术方案，步骤(2)中所述表达载体的构建与鉴定步骤包括：
(a)、将原核表达载体中的融合标签序列切除；
(b)、将优化后编码TRAIL穿膜肽样突变体蛋白的cDNA序列如SEQ ID NO：1克隆于原核表达载体上以获得高效可溶性非融合表达。
作为优选的技术方案，步骤(b)中所述原核表达载体为pET 32a。
作为优选的技术方案，步骤(3)中所述重组TRAIL蛋白的融合表达时，其诱导温度为18～24℃。
作为优选的技术方案，步骤(4)中所述TRAIL蛋白的纯化的步骤包括：
阳离子交换树脂SP Sepharose Fast Flow作为第一步纯化以捕获破菌后上清中的目的蛋白；
阳离子交换树脂Sephadex G-25medium作为第二步中度纯化以进一步提高蛋白质纯度和去除内毒素；以及
采用阴离子交换树脂Q Sepharose Fast Flow作为最终步骤精细纯化以使产品满足工业化放大和将来临床应用所需。
上述一种TRAIL穿膜肽样突变体在制备抗肿瘤药物中的应用。
本发明诱导肿瘤细胞凋亡的作用机理，TRAIL穿膜肽样突变体能通过穿膜作用快速进入肿瘤细胞内发挥诱导细胞凋亡的作用。此外，TRAIL穿膜肽样突变体还能有效促进死亡受体在细胞膜上脂筏微区域的聚集、重分布和/或TRAIL-DR4/DR5复合物的内化作用，增强外源性凋亡信号途径的转导。
本发明的有益技术效果是：
1、本发明提供了一种全新的蛋白质结构，采用最少的突变位点，对蛋白结构影响最小，得到的功能却最大化。TRAIL穿膜肽样突变体仅通过三个非连续位点的突变，由于位点的突变发生在蛋白质的氨基端，对蛋白的生物活性和稳定性影响较小，但却获得了超过穿膜肽融合蛋白的穿膜能力。
2、高的蛋白表达及可溶性表达比例，采用高效原核表达载体pET32a的改造形式，表达载体在18～24℃的较宽诱导温度范围内均可获得高于TRAIL野生型蛋白的表达水平和可溶性表达比例，可溶性蛋白比例达80％～100％。
3、不同于TRAIL野生型蛋白的纯化制备工艺，本发明工艺的有效性、回收率和产品质量明显提高，由于不采用特异性的亲和层析纯化方法，纯化成本相应降低，可放大潜力显著，能完全满足将来临床所需。
4、广泛的体外生物活性，TRAIL穿膜肽样突变体与TRAIL野生型蛋白相比，在经过检测的几乎所有的肿瘤细胞类型中，其抗肿瘤活性均明显提高，尤其对于TRAIL野生型蛋白耐药的肿瘤细胞株，能明显逆转这些细胞对TRAIL野生型蛋白的耐受，具有更强的治疗作用。
附图说明
为了更清楚地说明本发明实施方案或现有技术中的技术方案，下面将对实施方案或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施方案，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。
图1：TRAIL-MuR5片段PCR产物电泳图；电泳条件：3％Agarose，电压100V，20min；Lane 1：TRAIL-MuR5片段PCR产物电泳条带；M：DL2000(条带分子量从上到下依次为：2000bp、1000bp、750bp、500bp、250bp、100bp)，上样量5μl，PCR产物上样量5μl。
图2：TRAIL-MuR5与pET32a质粒NdeI、EcoRI酶切后电泳图；电泳条件：1％Agarose，电压150V，25min；Lane 1：TRAIL-MuR5酶切后胶回收电泳条带；Lane2：pET32a酶切后胶回收电泳条带；M：GeneRuler1kb DNA Ladder(条带分子量从上到下依次为：10000bp、8000bp、6000bp、5000bp、4000bp、3500bp、3000bp、2500bp、2000bp、1500bp、1000bp、750bp、500bp、250bp)，上样量5μl；PCR产物上样量均为3μl。
图3：pET32a/TRAIL-MuR5质粒XbaI和EcoRI酶切鉴定电泳图；电泳条件：1％Agarose，电压150V，30min；Lane 1～10：pET32a/TRAIL-MuR5菌种所提取质粒酶切后电泳图；M：GeneRuler1kb DNA Ladder(条带分子量从上到下依次为：10000bp、8000bp、6000bp、5000bp、4000bp、3500bp、3000bp、2500bp、2000bp、1500bp、1000bp、750bp、500bp、250bp)；鉴定产物上样量均为10μl，Marker上样量为5μl。
图4：pET32a/TRAIL-MuR5表达SDS-PAGE电泳图；电泳条件：15％凝胶，200V，35min；Lane 1：pET32a/TRAIL-MuR5诱导前电泳条带，Lane 2：pET32a/TRAIL-MuR5诱导后电泳条带，Lane 3：pET32a/TRAIL-MuR5破菌后上清电泳条带，Lane 4：pET32a/TRAIL-MuR5破菌后沉淀电泳条带；M：Unstained Protein Molecular Weight Marker(条带分子量从上到下依次为：116.0KDa、66.2KDa、45.0KDa、35.0KDa、25.0KDa、18.4KDa、14.4KDa)，Marker上样量为5μl，其它样品上样量均为20μl。
图5：阳离子交换过程SDS-PAGE电泳图；电泳条件：15％凝胶，200V，50min。Lane 1：阳离子交换原液，Lane 2：阳离子交换穿透液，Lane 3：阳离子交换600mM NaCl洗脱液，Lane 4：阳离子交换1.2M NaCl洗脱液，Lane 5：阳离子交换NaOH洗脱液；M：Unstained Protein Molecular Weight Marker(条带分子量 从上到下依次为：116.0KDa、66.2KDa、45.0KDa、35.0KDa、25.0KDa、18.4KDa、14.4KDa)。样品上样量Marker为5μl，其它均为20μl。
图6：阴离子交换过程SDS-PAGE电泳图；电泳条件：15％凝胶，200V，50min。Lane 1：阴离子交换原液，Lane 2：阴离子交换穿透液，Lane 3：2M NaCl洗脱液，Lane 4：0.5M NaOH洗脱液；M：UnstainedProtein Molecular Weight Marker(条带分子量从上到下依次为：116.0KDa、66.2KDa、45.0KDa、35.0KDa、25.0KDa、18.4KDa、14.4KDa)。样品上样量Marker为5μl，其它均为20μl。
图7：western blot鉴定结果图；Lane 1：TRAIL-MuR5western blot结果图；Lane2：TRAILwestern blot结果图；M：PageRuler Prestained Protein Ladder(条带分子量从上到下依次为：170KDa、130KDa、100KDa、70KDa、55KDa、40KDa、35KDa、25KDa、15KDa、10KDa)。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
Apo2L/TRAIL穿膜肽样突变体的设计思路如下：
凋亡蛋白作用最终起效的核心部位在细胞膜内，细胞膜是治疗性生物活性物质向细胞内转运的生物屏障。由于凋亡蛋白的亲水性，生物活性分子不能自由进入细胞膜内，导致其作用发挥和实际应用受限。穿膜肽是一类具有细胞膜穿透功能的大小多在20～30个氨基酸的带正电的阳离子的短肽，是近几十年发展起来的药物新型转运和传输技术，又称蛋白质转导域(Protein transduction domain，PTD)。
1988年，Green和Frankel首次证实了人免疫缺陷病毒(HIV-1)的反式激活蛋白TAT能跨膜转移到细胞质和细胞核内。其中一个富含精氨酸的TAT多肽(GRKKRRQRRRGY)具有穿膜转导蛋白功能，并能介导多种多源性物质，如基因、蛋白、多肽、化学合成的纳米颗粒等进入细胞膜甚至细胞核内。后又相继发现果蝇同源异性转录因子ANTP、单纯疱疹病毒Ⅰ型(HSV-1)转录因子VP22、Transpotan、多聚精氨酸等序列具有细胞膜穿透能力，目前已发现上百种具有穿膜功能的肽段。
根据不同的标准，可将穿膜肽分成不同的种类。从结构特点上讲，早期有人将穿膜肽简单分为：(1)不具有典型结构的带大量阳离子的穿膜肽，如TAT和penetratin等；(2)来源于蛋白信号序列的两亲α螺旋肽。从来源上分，有人将穿膜肽分为天然存在和人工合成两种，更进一步的可以分为三类：(1)来源于蛋白的穿膜肽，如penetratin、TAT和pVEC等。它们通常具有转运蛋白最小的有效片段，即蛋白转导部分和膜异位序列。(2)模式(Model)穿膜肽，如MAP和Arg(7)等，它们是为了形成确定两亲性α螺旋或模拟已知穿膜肽结构而人工合成的。根据穿膜肽结构合成的多聚精氨酸和多聚赖氨酸，其穿膜能力比TAT蛋白的转导活性更高。(3)人工设计、合成的穿膜肽，如PEP-1、MPG和Transportan等，它们通常是嵌合性多肽，含有1个疏水部分和1个亲水部分，如PEP-1(KETWW ETWWT EWSQP KKKRK V)包含一个富含疏水性色氨酸基序的片段(KETWW ETWWT EW)，一个间隔区(SQP)，一个富含亲水性 赖氨酸基序的区域(KKKRKV)。这种肽段具有更多的优点，PEP-1无需与目的大分子共价连接，直接与目的大分子混合后即可将天然构象蛋白高效导入细胞。
具有穿膜功能肽的氨基酸的结构关键是其主要分子组成为富含碱性氨基酸，如精氨酸、赖氨酸、组氨酸。碱性氨基酸是这一类穿膜蛋白质组成的重要特征。这些氨基酸带有强正电荷，可能与带负电荷的细胞膜脂类分子相互作用从而介导穿膜过程，其中精氨酸残基在蛋白细胞内化过程中起了重要作用。目前，关于多聚精氨酸转导蛋白入胞的作用机制主要存在两种观点：一是通过精氨酸在细胞膜和脂质双层中短暂形成小孔直接转导蛋白入胞；二是通过多种形式介导的细胞内吞作用，包括巨胞饮作用、小凹蛋白介导型、网格蛋白介导型、吞噬作用及内吞体交流等机制转导蛋白入胞。TRAIL诱导死亡受体在肿瘤细胞膜脂筏微区域上的聚集和重分布，通过或不通过TRAIL-DR4/5复合物的细胞内吞作用，募集Fas相关死亡结构域(Fas-Associated death domain，FADD)和Caspase-8，组装为死亡诱导信号复合物(Death inducing signaling complex，DISC)，通过裂解Caspase-8从而启动凋亡效应的瀑布级联过程。大部分文献认为TRAIL-DR4/5复合物的内化是凋亡信号持续放大所必需。传统上将外源蛋白与穿膜肽融合表达，表达的融合蛋白可能改变蛋白分子的空间构象从而使其丧失生物活性。此外融合蛋白增加了原有蛋白分子的抗原性从而带来安全性风险。
我们通过选择性突变TRAIL蛋白可溶性片段(114～281aa)编码氨基酸序列N端的数个氨基酸，使TRAIL蛋白形成类似穿膜肽样氨基酸序列，即对TRAIL进行穿膜肽样突变，目前已经获得10几种不同的穿膜肽样突变体。本发明突破原有穿膜肽融合蛋白设计思路，选择性地将TRAIL野生型蛋白胞膜外段第114～281位氨基酸编码序列N端的第114位的缬氨酸、第116位的谷氨酸、第118位的甘氨酸分别突变为精氨酸，使TRAIL蛋白的第114～118位氨基酸形成5个连续的精氨酸序列。内源的5个连续精氨酸序列使得对于TRAIL胞膜外段N端氨基酸序列改变最小(保留了原第115位、第117位精氨酸序列)，既最大程度维持了TRAIL蛋白的空白构象和生物活性，又构建形成了具有穿膜功能的连续5个精氨酸序列，我们将本发明的穿膜肽样突变体命名为TRAIL-MuR5。穿膜肽样突变体是一种穿膜肽融合蛋白全新设计思路。
实施例1
TRAIL穿膜肽样突变体的序列及引物设计
选择性地将TRAIL野生型蛋白胞膜外段第114～118位氨基酸编码序列由VRERG改造为RRRRR，即第114位由缬氨酸突变为精氨酸，第116位由谷氨酸突变为精氨酸，第118位由甘氨酸突变为精氨酸，突变位点3处，使得突变体蛋白的N端成为连续5个精氨酸的编码序列，成为包含穿膜肽样结构的蛋白。
编码突变体的cDNA如SEQ ID NO：1，突变体氨基酸如SEQ ID NO：2。
引物合成如下：
上游引物MuR5-TR-NdeI如SEQ ID NO：3所示；
下游引物TR-Eco-R如SEQ ID NO：4所示。
实施例2
PCR扩增TRAIL-MuR5片段，并与pET32a连接，连接产物单菌落挑取及鉴定
以pMD19/TRAIL质粒为模板PCR突变扩增TRAIL-MuR5片段。分别用NdeI和EcoRI双酶切TRAIL-MuR5目的片段和载体pET32a。将TRAIL-MuR5片段与切除了Trx融合标签序列的载体pET32a连接，转化入Top10感受态细胞，挑取单克隆，用XbaI和EcoRI双酶切鉴定。引物设计见实施例1，pMD19/TRAIL质粒的原始序列来源于NCBI Reference Sequence:NM_003810.3，载体pET32a来源于Invitrogen。
实验步骤
一、PCR扩增TRAIL-MuR5目的片段
1.以pMD19/TRAIL质粒为模板，MuR5-TR-NdeI/TR-Eco-R引物对扩增TRAIL-MuR5目的片段，根据表1配制反应体系，反应体系为50μl。
表1.TRAIL-MuR5PCR反应体系(50μl)
Figure PCTCN2015073504-appb-000001
2.涡旋震荡混匀，短暂离心，将溶液收集到管底。
3.PCR扩增反应条件见表2。
表2.TRAIL-MuR5PCR反应条件
Figure PCTCN2015073504-appb-000002
4.电泳，照相。
5.将PCR扩增的TRAIL-MuR5目的片段用Omega胶回收试剂盒做胶回收，用50μl超纯水洗脱，电泳，照相，备用。
二.目的片段TRAIL-MuR5与pET32a质粒双酶切后连接
1.用NdeI和EcoRI双酶切载体与目的基因片段，酶切体系见表3，反应体系100μl。
表3.TRAIL-MuR5与pET32a双酶切反应体系(100μl)
Figure PCTCN2015073504-appb-000003
2.将Ep管放入多用恒温箱中，30℃，2小时。
3.用OMEGA的胶回收试剂盒做胶回收，载体和目的片段分别用30μl超纯水洗脱。电泳，照相。
4.将胶回收目的片段和载体连接，连接体系见表4。
表4.TRAIL-MuR5与pET32a连接反应体系(10μl)
Figure PCTCN2015073504-appb-000004
5.于16℃金属浴孵育过夜。
6.将连接产物10μl加入100μl Top10感受态细胞，冰浴30min。
7.在水浴42℃热击90秒。
8.置冰上孵育2分钟。
9.加入500μl SOC培养基，37℃振荡培养45分钟。
10.转化的感受态细胞离心后，在超净工作台，弃去400μl，余约100μl培养基。
11.将细菌吹匀，全部涂布于含Amp的LB固体培养基上，37℃培养过夜。
三.单菌落挑取及酶切鉴定
(一)单菌落挑取
1.准备多支已灭菌试管，每管加入氨苄青霉素LB液体培养基100ml。
2.将培养基分装于各个试管中，每管分装约4ml。
3.在已长好菌落的平皿上，使用经充分灼烧的镊子夹取无菌枪头，挑取平板所长出的菌落，pET32a/TRAIL-MuR5平皿挑取10个。将枪头投入装有LB培养基的试管中。
4.将各试管捆扎好，放入摇床夹具上充分固定。37℃、220rpm振摇过夜。
(二)质粒提取
1.将菌液各取1ml分别加入离心管中。10000g离心1min，尽量吸取上清。
2.向留有菌体沉淀的离心管中加入250μl Solution I(预先加入RNAase A)，彻底悬浮细菌沉淀。
3.加入250μl Solution II，温和地混匀，使菌体充分裂解，此时菌液变得清亮粘稠，并在5min内完成此步骤。
4.向离心管中加入350μl Solution III，立即颠倒混匀，此时出现白色絮状沉淀，13000g离心10min，此时在离心管底部形成沉淀。
5.将步骤5中所得上清均分加入到2个已装入收集管的HiBind Miniprep吸附柱中，注意不要吸出沉淀，10000g离心1min，倒掉收集管中的废液，将吸附柱重新放回收集管中。
6.向收集管中加入500μl Buffer HB，10000g离心1min，倒掉收集管中的废液，将吸附柱重新放回收集管中。
7.向收集管中加入700μl Wash Buffer，10000g离心1min，倒掉收集管中的废液，将吸附柱重新放回收集管中。
8.重复步骤7。
9.将吸附柱重新放回收集管中，13000g离心2min干燥吸附柱，倒掉收集管中的废液。
10.将各吸附柱置于一个新的1.5ml Ep管中，向每个吸附膜的中间部位悬空滴加65μl Elution Buffer，室温放置数分钟，13000g以上离心1min，将质粒溶液收集到1.5ml Ep管中。
11.各得到60μl质粒DNA。-20℃保存质粒。
(三)酶切鉴定
1.pET32a/TRAIL-MuR5质粒用XbaI和EcoRI双酶切质粒。酶切反应体系见表5。
表5.pET32a/TRAIL-MuR5酶切反应体系(10μl)
Figure PCTCN2015073504-appb-000005
Figure PCTCN2015073504-appb-000006
2.将Ep管放入多用恒温箱中，37℃，孵育2小时。
3.酶切结束后电泳鉴定。
(四)选择酶切正确的成功连接的菌种，保存甘油菌种，送测序。
实验结果
一.PCR扩增目的片段结果
以MuR5-TR-NdeI/TR-Eco-R引物对突变扩增TRAIL-MuR5目的片段，片段分子量大小为500bp左右，如图1所示，根据上述PCR反应条件得到目的基因。
二.理论上TRAIL–MuR5与pET32a用NdeI和EcoRI双酶切后，得到大小分别约为500bp、5.4kb左右的目的片段，如图2所示，酶切后胶回收均得到预期的单一条带。
三.TRAIL-MuR5目的片段分别与pET32a连接及转化结果
1.平皿有菌落长出，密度正常。
2.挑取的单菌落，第二日部分试管长出细菌，密度正常。
3.用酶切方法鉴定质粒，pET32a/TRAIL-MuR5质粒用XbaI和EcoRI双酶切鉴定，连接成功质粒酶切后应出现5.4Kb左右载体片段及550bp左右目的片段。如图3所示，pET32a/TRAIL-MuR5有6个样本阳性，阳性质粒送华大基因测序，测序正确的质粒保存菌种。
实施例3
pET32a/TRAIL-MuR5表达试验
将在实施例2中获得测序正确的质粒转化感受态大肠杆菌BL21(DE3)，挑取1个单菌进行表达试验，考察表达效果。
实验步骤
一.质粒转化及菌种保存
1.配制LB培养基100ml，121℃灭菌20min。
2.取pET32a/TRAIL-MuR5质粒1μl加入100μl BL21(DE3)感受态细胞中，冰浴30min。
3.在水浴42℃热击90秒。
4.置冰上孵育3分钟。
5.取20μl转化的感受态细胞全部涂布于含Amp的LB固体培养基上，37℃培养过夜。
6.次日平板长出菌落后，在平板上挑取一个单菌加入50ml LB(Amp+)中，37℃培养过夜。
7.保存甘油菌20支，甘油终浓度15％，-20℃保存。
二.菌种表达
1.取过夜培养的pET32a/TRAIL-MuR5培养液各1000μl接入50ml LB(Amp+)培养基中。接种后37℃，250rpm振摇培养3h后降低培养温度至24℃。按1％的比例加入0.1M IPTG诱导培养，诱导前取样0.5ml离心弃去上清，加入50μlH2O重悬后加入50μl 2×loading buffer制成诱导后电泳样品。
2.诱导过夜后收菌，检测A600值，取样150μl离心弃去上清，加入50μlH2O重悬后加入50μl 2×loadingbuffer制成诱导后电泳样品，剩余菌液使用5430R型离心机，12000rpm离心5min。
3.取50ml培养液离心获得菌体，使用8ml 50mM Na2HPO4溶液重悬，超声波破菌。破菌条件为：Φ6探头，200W脉冲破菌2s后暂停2s，循环共10min。。
4.破菌液取1ml 12000rpm离心10min，分离上清和沉淀，沉淀使用1ml H2O重悬，上清和沉淀重悬液各取20μl，加入30μl H2O及50μl 2×loading buffer，制成电泳样品。
5.将制成的电泳样品置于沸水浴中处理10min，使用5430R型离心机，A-45-30-11型转头，12000rpm离心10min，各取上清10μl电泳。
实验结果
实验电泳图见图4(pET32a/TRAIL-MuR5)，TRAIL-MuR5具有较强表达，并且大部分表达产物在破菌后上清，可溶性表达比例高。
实施例4
TRAIL-MuR5蛋白的纯化制备
根据对于TRAIL-MuR5大量小试工艺的探索，我们建立了TRAIl-MuR5蛋白纯化工艺，我们使用SPSepharose Fast Flow凝胶层析柱、Sephadex G-25medium凝胶层析柱、阴离子交换穿透三步法批量纯化TRAIL-MuR5蛋白，以获取样品供体内外活性分析用。
实验步骤
一.菌体破碎及离心
1.取80g MuR5菌体，加入Na2CO3、甘油、Tween80、DTT及NaCl，另加入H2O使总体积达到400ml，使以上物质终浓度分别为Na2CO320mM、甘油5％、Tween800.1％、DTT1mM、NaCl500mM。
2.将菌液进行超声波破菌，破菌条件为：使用Φ10探头，500W脉冲破菌2s后暂停2s，共破菌15min。
3.使用5430R型离心机，F-35-6-30型转头，7850rpm离心40min，取上清，使用0.45μm滤膜过滤后作为上柱样品。
二.蛋白质纯化溶液及柱准备
1.配制如下溶液：
(1)阳离子交换缓冲液A：20mM Na2CO3-NaHCO3，0.5M NaCl，5％甘油，0.1％Tween80，1mM DTT，调节pH至10.50。
(2)阳离子交换缓冲液B：20mM Na2CO3-NaHCO3，1.2M NaCl，5％甘油，0.1％Tween80，1mM DTT，调节pH至10.20。
(3)0.5M NaOH。
(4)2M NaCl。
(5)脱盐及阴离子交换缓冲液：0.15MNaCl，0.3M甘氨酸，0.2M精氨酸。
2.使用SP Sepharose Fast Flow凝胶层析柱，使用5CV纯水冲洗柱上残存的乙醇，然后使用5CV相应的平衡缓冲液平衡。
3.使用Sephadex G-25medium凝胶层析柱，使用5CV纯水冲洗柱上残存的乙醇，然后使用5CV阴离子交换缓冲液平衡。
4.使用Q Sepharose Fast Flow凝胶层析柱，使用5CV纯水冲洗柱上残留的乙醇，然后使用5CV阴离子交换缓冲液平衡。
三.阳离子交换纯化
按照如下纯化步骤进行阳离子交换纯化。纯化期间收集所有穿透及洗脱成分以备电泳分析：
1.平衡：使用阳离子交换A缓冲液平衡SP Sepharose Fast Flow层析柱至UV稳定。
2.样品制备及上样：取破菌离心上清，上样。
3.清洗：使用2CV阳离子交换缓冲液A洗涤柱子以除去残留的未结合蛋白。
4.洗脱：先使用2CV 14.3％阳离子交换缓冲液B洗脱杂蛋白，再使用2CV 100％阳离子交换缓冲液B洗脱目的蛋白。
5.NaOH清洗：使用2CV 0.5M NaOH溶液清洗柱子。
6.再平衡(Reequilibration)：使用5CV阳离子交换缓冲液A再平衡柱子。
四.阴离子交换纯化
按照如下纯化步骤进行第二步阴离子交换纯化。纯化期间收集所有穿透及洗脱成分以备电泳分析：
1.平衡：使用阴离子交换缓冲液平衡Q Sepharose Fast Flow层析柱至UV稳定。
2.样品制备及上样：取阳离子交换纯化洗脱样品，经Sephadex G-25medium层析柱置换缓冲液为阴离子交换缓冲液后，上样。
3.平衡液清洗：使用2CV阴离子交换缓冲液清洗柱子以获得未结合上柱子的目的蛋白。
4.NaCl清洗：使用2CV 2M NaCl洗涤柱子以除去结合在柱上的蛋白。
5.NaOH清洗：使用2CV 0.5M NaOH溶液清洗柱子。
6.再平衡(Reequilibration)：使用阴离子交换缓冲液再平衡柱子。
实验结果
每一步纯化过程样品的电泳结果见图5、6：第一步SP的洗脱液收集22ml，浓度5.33mg/ml，经检测目的蛋白的纯度为85.35％，第二步脱盐洗脱液30ml，浓度3.24mg/ml，具有去除剩余杂蛋白和部分热原的作用，而第三步阴离子交换穿透液42.85ml，浓度2.16mg/ml，主要是去热原。多次重复本实施例的实验操作，获得了足够体外生活学活性评价的蛋白量，为183.39mg。
实施例6
TRAIL-MuR5蛋白的Western Blot检测
由于TRAIL-MuR5为野生型TRAIL N端3个位点突变所得，TRAIL的抗原决定簇仍然保留，能够与TRAIL的多克隆抗体发生特异性结合，因此可用TRAIL多克隆抗体进行检测鉴定。
实验步骤
一.样品配制
1.实施例5纯化的TRAIL-MuR5蛋白从-20℃解冻后，按其提供的浓度用超纯水稀释成1mg/ml。取样本50μl加入50μl 2×loading buffer，制成电泳样品。各取10μl电泳，即上样量为5ug。
2.对照品TRAIL-20131204冻干品(由实验室制备，并与NIBSC国际标准品比较，活性高于107IU/mg)用1mlPBS溶解，取样本50μl加入50μl 2×loading buffer，制成电泳样品。各取10μl电泳，即上样量为5ug。
二.检测过程
样品经15％SDS-PAGE电泳分离后，转膜至PVDF膜上。首先4℃封闭过夜，再与一抗[兔抗人TRAIL多克隆抗体(1:500)]室温孵育2小时，然后与二抗[羊抗兔IgG-HRP(1:5000)]室温孵育2小时，最后使用增强型化学发光(ECL)检测。具体步骤如下：
1.15％SDS-PAGE电泳分离蛋白；取出凝胶，切去凝胶边缘，浸于TBST缓冲液中，时间15min。
2.使用PVDF膜转膜(湿转)：PVDF膜使用前必须用甲醇略微浸湿15秒，然后在蒸馏水中浸泡1～3min，随后在转膜缓冲液中平衡；在转膜夹中，由负极到正极依次铺上：海绵垫、滤纸(4～8张)、目的胶、PVDF膜、滤纸(4～8张)、海绵垫，排气泡后加紧夹子放入转膜槽中，电压40V，时间45min。
3.封闭膜：将膜在封闭液(3％BSA)中4℃条件下封闭过夜，次日取出在室温振摇30min，以封闭非特异结合位点。
4.一抗孵育：将一抗用封闭液稀释至工作浓度[兔抗人TRAIL多克隆抗体(1:500)]，与膜一起振摇，室温孵育2h。
5.洗膜：用TBST洗膜三次，每次10min。10×10cm的膜需洗涤液50ml以上。
6.二抗孵育：将HRP标记的二抗用封闭液稀释至工作浓度[羊抗兔IgG-HRP(1:5000)]，与膜一起振摇，室温孵育2h。
7.洗膜：用TBST洗膜三次，每次10min。10×10cm的膜需洗涤液50ml以上。
8.显色：(1)将等体积的Solution A和Solution B混合，制备足够的检测混合液(0.125ml/cm2)。检测混合液配置后立即使用，室温1h内可保持稳定。(2)沥去洗过的印迹膜上多余的洗液，但不可使膜干 燥。在膜上有蛋白的一面加上检测混合液，沥去多余的检测混合液，放在柯达凝胶成像Image Station 4000R上，用X-ray曝光，首次选择曝光时间1min，根据图像结果调整曝光时间。电脑记录图像。
9.结果判断：阳性结果应呈现明显色带。阴性结果不显色。
实验结果
如图7所示，TRAIL-MuR5及TRAIL对照品呈阳性反应，阴性对照呈阴性反应。
实施例7
蛋白TRAIL-MuR5及TRAIL生物活性分析
应用CCK-8检测试剂盒检测TRAIL-MuR5及野生型TRAIL 2个蛋白样品对12个肿瘤细胞株的体外抗增殖活性IC50值，以评价其体外生物活性。
材料和方法
检测用细胞株均来自中国科学院上海细胞所或武汉病毒所。
Figure PCTCN2015073504-appb-000007
试剂和耗材
Cell Counting Kit-8(Cat#CK04-13，Dojindo)
96孔培养板(Nest Biotech Co)
胎牛血清(Code：FS101-02，全式金)
培养基(购自GIBCO)
台式酶标仪Infinite F50(TECAN)
2个蛋白样品：通过实施例5制备或实验室自备。
实验步骤
1.试剂配制
培养基的配制
Figure PCTCN2015073504-appb-000008
蛋白样品的制备
用无菌的PBS缓冲液稀释2个蛋白样品使终浓度为5mg/ml，并过滤除菌。
2.IC50实验
a)收集对数生长期细胞，计数，用完全培养基重新悬浮细胞，调整细胞浓度至合适浓度(依照细胞密度优化试验结果确定)，接种96孔板，每孔加100μl细胞悬液。细胞(SW620细胞除外，不需要5％CO2)在37℃，100％相对湿度，5％CO2培养箱中孵育24小时。
b)用无菌PBS缓冲液将待测蛋白样品稀释至5mg/ml后梯度稀释8次，按25μl/孔加入细胞。化合物终浓度从1mg/ml至0，3倍梯度稀释，共10个浓度点；并根据初步的实验结果，对蛋白样品的作用终浓度进行相应的调整。
c)细胞(SW620细胞除外，不需要5％CO2)置于37℃，100％相对湿度，5％CO2培养箱中孵育48小时。
d)吸弃培养基，加入含10％CCK-8的完全培养基置于37℃培养箱中孵育2～4小时。
e)轻轻震荡后在SpectraMax M5Microplate Reader上测定450nm波长处的吸光度，以650nm处吸光度作为参比，计算抑制率。
3.数据处理
按下式计算药物对肿瘤细胞生长的抑制率：肿瘤细胞生长抑制率％＝[(Ac-As)/(Ac-Ab)]×100％
As：样品的OA/RLU(细胞+CCK-8+待测化合物)
Ac：阴性对照的OA/RLU(细胞+CCK-8)
Ab：阳性对照的OA/RLU(培养基+CCK-8)
运用软件Graphpad Prism 5并采用计算公式log(inhibitor)vs.normalized response-Variable slope进行IC50曲线拟合并计算出IC50值。
实验结果
本实验测试了2个蛋白样品(TRAIL-MuR5和野生型TRAIL)对3个胰腺癌细胞株(CFPAC-1、BxPC-3、PANC-1)，2个肺癌细胞株(NCI-H460、A 549)，3个结(直)肠癌细胞株(SW620、HT-29、HCT 116),3个乳腺癌细胞株(MDA-MB-231、MCF-7、T47D)，1个急性T细胞白血病细胞株(Jurkat)的体外抗细胞增殖活性。实验结果如下表所示。
Figure PCTCN2015073504-appb-000009
Figure PCTCN2015073504-appb-000010
TRAIL穿膜肽样突变体TRAIL-MuR5与TRAIL野生型蛋白相比，在经过检测的几乎所有的肿瘤细胞类型中[包括多种结(直)肠癌细胞、多种肺癌细胞、多种胰腺细胞、多种乳腺癌细胞]，其抗肿瘤活性均明显提高，尤其对于TRAIL野生型蛋白耐药的肿瘤细胞株，能明显逆转这些细胞对TRAIL野生型蛋白的耐受，具有更强的治疗作用。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种TRAIL穿膜肽样突变体，其特征在于，所述突变体的氨基酸序列如SEQ ID NO：2。
  2. 根据权利要求1所述的一种TRAIL穿膜肽样突变体，其特征在于，所述TRAIL穿膜肽样突变体是通过选择性地将TRAIL野生型蛋白胞膜外段第114～118位氨基酸编码序列由VRERG改造为RRRRR，即第114位由缬氨酸突变为精氨酸，第116位由谷氨酸突变为精氨酸，第118位由甘氨酸突变为精氨酸，使得突变体蛋白的N端成为连续5个精氨酸编码序列，成为包含穿膜肽样结构的蛋白。
  3. 根据权利要求1所述的一种TRAIL穿膜肽样突变体，其特征在于，编码所述突变体的cDNA序列如SEQ ID NO：1。
  4. 根据权利要求3所述的一种TRAIL穿膜肽样突变体，其特征在于，扩增所述突变体的试剂盒，所述试剂盒包括如下引物：
    上游引物MuR5-TR-NdeI：
    GGTCATATGCGTCGTCGTCGTCGTCCGCAGCGTGTGGCTGCTCAC
    下游引物TR-Eco-R：
    GTTGAATTCT TATTAACCAA CAAGGAAAGC ACCGAAGAAA G。
  5. 一种TRAIL穿膜肽样突变体的制备方法，包括如下步骤：
    (1)、cDNA片段扩增与克隆；其中cDNA序列如SEQ ID NO：1；
    (2)、表达载体的构建与鉴定；
    (3)、重组TRAIL蛋白的融合表达；
    (4)、TRAIL蛋白的纯化；
    (5)、TRAIL蛋白鉴定。
  6. 根据权利要求5所述的一种TRAIL穿膜肽样突变体的制备方法，其特征在于，步骤(2)中所述表达载体的构建与鉴定步骤包括：
    (a)、将原核表达载体中的融合标签序列切除；
    (b)、将优化后编码TRAIL穿膜肽样突变体蛋白的cDNA序列如SEQ ID NO：1克隆于原核表达载体上以获得高效可溶性非融合表达。
  7. 根据权利要求5所述的一种TRAIL穿膜肽样突变体的制备方法，其特征在于，步骤(b)中所述原核表达载体为pET 32a。
  8. 根据权利要求5所述的一种TRAIL穿膜肽样突变体的制备方法，其特征在于，步骤(3)中所述重组TRAIL蛋白的融合表达时，其诱导温度为18～24℃。
  9. 根据权利要求5所述的一种TRAIL穿膜肽样突变体的制备方法，其特征在于，步骤(4)中所述TRAIL蛋白的纯化的步骤包括：
    阳离子交换树脂SP Sepharose Fast Flow作为第一步纯化以捕获破菌后上清中的目的蛋白；
    阳离子交换树脂Sephadex G-25medium作为第二步中度纯化以进一步提高蛋白质纯度和去除内毒素； 以及采用阴离子交换树脂Q Sepharose Fast Flow作为最终步骤精细纯化。
  10. 一种权利要求1-9任一权利要求所述的TRAIL穿膜肽样突变体在制备抗肿瘤药物中的应用。
PCT/CN2015/073504 2015-03-02 2015-03-02 TRAIL穿膜肽样突变体MuR5、制备方法及应用 WO2016138618A1 (zh)

Priority Applications (4)

Application Number Priority Date Filing Date Title
PCT/CN2015/073504 WO2016138618A1 (zh) 2015-03-02 2015-03-02 TRAIL穿膜肽样突变体MuR5、制备方法及应用
EP15883675.9A EP3266796B1 (en) 2015-03-02 2015-03-02 Trail membrane-penetrating peptide-like mutant mur5, preparation method therefor, and application thereof
CN201580000086.0A CN106132986B (zh) 2015-03-02 2015-03-02 TRAIL穿膜肽样突变体MuR5、制备方法及应用
US15/591,139 US10000552B2 (en) 2015-03-02 2017-05-10 TRAIL cell-penetrating peptide-like mutant MuR5 and preparation method thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2015/073504 WO2016138618A1 (zh) 2015-03-02 2015-03-02 TRAIL穿膜肽样突变体MuR5、制备方法及应用

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US15/591,139 Continuation-In-Part US10000552B2 (en) 2015-03-02 2017-05-10 TRAIL cell-penetrating peptide-like mutant MuR5 and preparation method thereof

Publications (1)

Publication Number Publication Date
WO2016138618A1 true WO2016138618A1 (zh) 2016-09-09

Family

ID=56849151

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2015/073504 WO2016138618A1 (zh) 2015-03-02 2015-03-02 TRAIL穿膜肽样突变体MuR5、制备方法及应用

Country Status (4)

Country Link
US (1) US10000552B2 (zh)
EP (1) EP3266796B1 (zh)
CN (1) CN106132986B (zh)
WO (1) WO2016138618A1 (zh)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109069584A (zh) * 2017-06-29 2018-12-21 成都华创生物技术有限公司 一种trail类蛋白持续抑制肿瘤细胞生长的给药方法
CN109840889A (zh) * 2019-01-24 2019-06-04 华东交通大学 基于仿生算法的高精度视觉测量方法、装置和系统
WO2020037714A1 (zh) * 2018-08-23 2020-02-27 成都华创生物技术有限公司 Trail突变体在制备治疗痤疮药物中的应用及一种制剂

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1958794A (zh) * 2005-11-03 2007-05-09 成都地奥制药集团有限公司 人肿瘤坏死因子相关凋亡诱导配体突变体编码cDNA及制备方法和应用
CN103555729A (zh) * 2013-10-14 2014-02-05 成都华创生物技术有限公司 一种改造的trail基因序列、表达方法及应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8461311B2 (en) * 2010-06-08 2013-06-11 Washington University TRAIL trimers, methods and uses therefor
PL391627A1 (pl) * 2010-06-25 2012-01-02 Adamed Spółka Z Ograniczoną Odpowiedzialnością Przeciwnowotworowe białko fuzyjne
PL394618A1 (pl) * 2011-04-19 2012-10-22 Adamed Spólka Z Ograniczona Odpowiedzialnoscia Przeciwnowotworowe bialko fuzyjne
WO2014141094A1 (en) * 2013-03-14 2014-09-18 Adamed Sp. Z O.O. Anticancer conjugate

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1958794A (zh) * 2005-11-03 2007-05-09 成都地奥制药集团有限公司 人肿瘤坏死因子相关凋亡诱导配体突变体编码cDNA及制备方法和应用
CN103555729A (zh) * 2013-10-14 2014-02-05 成都华创生物技术有限公司 一种改造的trail基因序列、表达方法及应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP3266796A4 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109069584A (zh) * 2017-06-29 2018-12-21 成都华创生物技术有限公司 一种trail类蛋白持续抑制肿瘤细胞生长的给药方法
WO2020037714A1 (zh) * 2018-08-23 2020-02-27 成都华创生物技术有限公司 Trail突变体在制备治疗痤疮药物中的应用及一种制剂
CN109840889A (zh) * 2019-01-24 2019-06-04 华东交通大学 基于仿生算法的高精度视觉测量方法、装置和系统
CN109840889B (zh) * 2019-01-24 2022-09-02 华东交通大学 基于仿生算法的高精度视觉测量方法、装置和系统

Also Published As

Publication number Publication date
EP3266796B1 (en) 2018-12-19
EP3266796A4 (en) 2018-02-21
EP3266796A1 (en) 2018-01-10
CN106132986A (zh) 2016-11-16
US10000552B2 (en) 2018-06-19
CN106132986B (zh) 2019-08-30
US20170247427A1 (en) 2017-08-31

Similar Documents

Publication Publication Date Title
WO2016138625A1 (zh) TRAIL穿膜肽样突变体MuR6、制备方法及应用
WO2015103894A1 (zh) 一种trail穿膜肽样突变体、制备方法及应用
WO2016138618A1 (zh) TRAIL穿膜肽样突变体MuR5、制备方法及应用
US20180170994A1 (en) Bi-targeted Mutain MuR5S4TR of TRAIL and Preparation Method and Application Thereof
CN110144327A (zh) 一种靶向性抗肿瘤t细胞及其制备方法和应用
CN108026181B (zh) 一种TRAIL双靶点突变蛋白MuR6S4TR、其制备方法及其应用
CN112279921A (zh) 用于胞内递送分子的复合物
CN116891533A (zh) 补体抑制杂合蛋白
CN106810611A (zh) 抗cMet和CD3特异性双靶向抗体及其制备方法和应用、含该双靶向抗体表达盒的微环DNA及应用
WO2021143695A1 (zh) 一种抗肿瘤融合蛋白及其制法和应用
WO2003093431A2 (en) Ablated slam-dependent entry
JP6018079B2 (ja) 非天然ｍｉｃタンパク質
WO2023135264A1 (en) Binding proteins specific for cd32a
KR20230100212A (ko) 비바이러스 기반 고효율 핵산 형질주입용 조성물 및 그 용도
CN112063587A (zh) 超级抗原或其变体在car免疫细胞制备中的应用
CN118221829A (zh) 一种il-10单体融合蛋白
WO2004009811A1 (fr) Systeme d'entrainement de genes comprenant une sequence de noyau d'histone
KR20140046995A (ko) 인간 ｎｌｂｐ 단백질 유래의 ｎｐ1 폴리펩티드를 포함하는 세포 투과 펩티드 및 이를 이용한 카고 전달 시스템

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15883675

Country of ref document: EP

Kind code of ref document: A1

REEP Request for entry into the european phase

Ref document number: 2015883675

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE