CN1257187C - Calreticulin-tumor necrosis factor correlated apoptosis inducing ligand fusion protein and its prepn and use - Google Patents

Calreticulin-tumor necrosis factor correlated apoptosis inducing ligand fusion protein and its prepn and use Download PDF

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CN1257187C
CN1257187C CN 200310108083 CN200310108083A CN1257187C CN 1257187 C CN1257187 C CN 1257187C CN 200310108083 CN200310108083 CN 200310108083 CN 200310108083 A CN200310108083 A CN 200310108083A CN 1257187 C CN1257187 C CN 1257187C
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fusion rotein
calreticulin
trail
sequence
dna
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CN1609124A (en
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王梁华
周劲松
张国钧
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Shanghai Song Bomeng Biological Technology Co ltd
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SHANGHAI QIA'ER BIOTECHNOLOGY CO Ltd
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Abstract

The present invention provides fusion protein (Calreticulin-TRAIL fusion protein) for a human calreticulin-tumor necrosis factor relevant apoptosis inducing ligand. A DNA sequence for encoding the fusion protein contains the carrier of the DNA sequence, the host cells of the carrier, a method for preparing the fusion protein by using genetic engineering and a medical composition containing the fusion protein. The Calreticulin-TRAIL fusion protein of the present invention has the function of inhibiting the new blood vessel endothelial cell growth of tumor of Calreticulin and the function of inducing tumor apoptosis of TRAIL. The Calreticulin-TRAIL fusion protein can be used for treating diseases, such as tumor, etc.

Description

The fusion rotein of calprotectin-tumor necrosin relative death inducing ligand and method for making thereof and purposes
Technical field
The present invention relates to DNA recombinant technology and pharmaceutical field.More specifically, the present invention relates to the fusion rotein of people's calprotectin (Calreticulin) and apoptosis induction ligand related to human tumor necrosis factor (hereinafter to be referred as TRAIL), the encode dna sequence dna of this fusion rotein, the carrier that contains this dna sequence dna, the host cell that contains this carrier, prepare the method for this fusion rotein with genetically engineered, and this fusion rotein is as the application in the disease treatments such as tumour.
Background technology
Calprotectin (Calreticulin) is found existing more than ten years so far, and its major function is a storage calcium.The code sequence of people's calprotectin gene is shown 1251 Nucleotide (SEQ ID NO:1), and nucleotide sequence is seen GenBank No.:NM 004343, and the calprotectin of coding is formed (SEQ ID NO:2) by 417 amino acid.In recent years find that its N-end has antineoplastic action, has proved that the N-of calprotectin holds 1-180 amino acids [J Exp Med, 1998; 188 (12): 2349-2356] or 120-180 amino acids [Blood, 1999; 94 (7): 2461-2468] antitumor action is brought into play in the growth of the new vessel endotheliocyte that the protein of Zu Chenging can be by suppressing tumour.At present this gene can from biotech company ( Http:// www.invivogen.com) obtain.
Nineteen ninety-five, external report from the people express the sequence label library ( eXpressed sEquence tAg screens a kind of gene of the anti-tumor protein matter of encoding in EST), the protein of this genes encoding be called tumor necrosin relative death inducing ligand ( tUmor necrosis factor- rElated aPoptosis- iNducing lIgand, TRAIL), claim again Tumor wilting extract ( ApopTotin- 2 lIgand is called for short Apo-2L), be tumour necrosis factor ( tUmor nEcrosis fActor, TNF) one of family member, it is by starting cell inherent apoptosis program, induced tumor and transformant apoptosis effectively, and normal cell is not had influence.The code sequence of people's trail dna is shown 843 Nucleotide (SEQ ID NO:3), and nucleotide sequence is seen GenBank No.:NM_003810, and encoded protein matter TRAIL forms (SEQ ID NO:4) by 281 amino acid.Proved already that TRAIL was typical II type transmembrane protein, its N-holds the extracellular region of the 95th or 114 beginning can form free solubility tumor apoptosis fibroin, has the tumor death effect equally.At present this gene can from biotech company ( Http:// www.invivogen.com) obtain.
Therefore, this area presses for that exploitation is new to have Calreticulin and a bioactive medicine of TRAIL.In addition, this area also presses for cheap and or the easy production technique of step of cost of development.
Summary of the invention
One object of the present invention just provides a kind of new have Calreticulin and the bioactive medicine of TRAIL, and it is the fusion rotein of Calreticulin and TRAIL.
Another object of the present invention provide encoding said fusion protein DNA, contain the carrier of this dna sequence dna, contain the host cell of this carrier.
Another object of the present invention provides the method for the described Calreticulin-TRAIL fusion rotein of a kind of production with low cost and/or that step is easy.
In a first aspect of the present invention, a kind of fusion rotein is provided, it comprises:
(a) calprotectin element, this element has the aminoacid sequence of people's calprotectin or its active fragments;
(b) tumor necrosin relative death inducing ligand element, this element has the aminoacid sequence of apoptosis induction ligand related to human tumor necrosis factor or its active fragments; And
(c) 1-20 between calprotectin element and tumor necrosin relative death inducing ligand element amino acid whose connection peptides sequence.
More preferably, described calprotectin element has the aminoacid sequence of 1-417 position, 1-180 position or 119-180 position among the SEQ ID NO:2;
Described tumor necrosin relative death inducing ligand element has the aminoacid sequence of 1-281 position, 95-281 position, 114-281 position among the SEQ ID NO:4;
And described joint peptide sequence contains 4-10 amino acid.
More preferably, described fusion rotein comprises the aminoacid sequence shown in the SEQ ID NO:6.
In a second aspect of the present invention, provide a kind of isolated DNA molecule, the fusion rotein that the invention of its code book is above-mentioned.Preferably, described dna molecule encode comprises the fusion rotein of the aminoacid sequence shown in the SEQ ID NO:6.More preferably, described dna molecular comprises the nucleotide sequence shown in the SEQ ID NO:5.
In a third aspect of the present invention, carrier that contains above-mentioned dna molecular and the host cell that contains above-mentioned carrier are provided.
In a fourth aspect of the present invention, a kind of method that produces fusion rotein of the present invention is provided, it comprises step:
Under the condition that is fit to the described fusion rotein of expression, cultivate above-mentioned host cell, thereby give expression to described fusion rotein; With separate described fusion rotein.
In a fifth aspect of the present invention, a kind of pharmaceutical composition is provided, it comprises pharmaceutically acceptable carrier or vehicle or thinner, and the fusion rotein of the present invention of significant quantity.
In a sixth aspect of the present invention, the purposes of fusion rotein of the present invention is provided, it is used to prepare the medicine for the treatment of tumour.
Description of drawings
Fig. 1 has shown the different connecting methods of Calreticulin, amino acid connecting arm and the TRAIL of different lengths.
Fig. 2 has shown the electrophoretic analysis (SDS-PAGE) behind the recombinant expressed and purifying of a kind of distinctive Calreticulin-TRAIL fusion rotein.Wherein, each swimming lane is as follows: A: molecular weight protein marker; B: the recombinant C alreticulin-TRAIL fused protein of purifying; C: the Calreticulin-TRAIL that protokaryon is recombinant expressed.
Embodiment
The inventor merges Calreticulin gene and trail dna through extensive and deep research, produces the fusion rotein of the Calreticulin-TRAIL that is connected by suitable amino acid connecting arm.Described fusion rotein has both biological functions, both can be antitumor by Calreticulin N-end inhibition endothelial cells in tumor neogenetic blood vessels growth, can also suppress tumour by the TRAIL inducing apoptosis of tumour cell.Therefore, the resulting Calreticulin-TRAIL fused protein of the present invention can strengthen antineoplastic effect down both collaborative, also can reduce both respectively administration give trouble and the misery that the patient brought, for treatment such as antitumor provides new compound.Finished the present invention on this basis.
Definition
As used herein, term " fusion rotein of calprotectin and tumor necrosin relative death inducing ligand ", " Calreticulin-TRAIL fusion rotein " etc. are used interchangeably, all refer to merge the albumen that forms by the aminoacid sequence of calprotectin element and the aminoacid sequence of tumor necrosin relative death inducing ligand element, wherein between can have or not have the connection peptides sequence.In addition, described fusion rotein can have or not have initial methionine(Met) or signal peptide.
As used herein, " calprotectin element " refers to a part of aminoacid sequence in described fusion rotein in the term fusion rotein, this sequence has substantially the same aminoacid sequence with total length calprotectin or its active fragments natural or variation, and has the biological activity substantially the same with the natural calcium plectin.Preferred calprotectin element is people's calprotectin, more preferably is people's calprotectin or its active fragments of total length, as the aminoacid sequence of 1-417 position, 1-180 position or 119-180 position among the SEQ IDNO:2.
As used herein, " tumor necrosin relative death inducing ligand element " or " TRAIL element " is used interchangeably in the term fusion rotein, the a part of aminoacid sequence of finger in described fusion rotein, this sequence has substantially the same aminoacid sequence with total length tumor necrosin relative death inducing ligand or its active fragments natural or variation, and has the biological activity substantially the same with natural tumor necrosin relative death inducing ligand.Preferred TRAIL element is an apoptosis induction ligand related to human tumor necrosis factor, more preferably be tumor necrosin relative death inducing ligand or its active fragments of total length, as the aminoacid sequence of 1-281 position, 95-281 position, 114-281 position among the SEQ ID NO:4.
The sequence of calprotectin and tumor necrosin relative death inducing ligand can be derived from the people, also can be derived from inhuman animal.Yet, people's native sequences preferably.
As used herein, term " connection peptides " or " amino acid connecting arm " are used interchangeably, and refer to small peptide between the aminoacid sequence of the aminoacid sequence of calprotectin element and TRAIL element, that play ligation.The length of connection peptides is generally 1-20 amino acid, preferably is 3-10 amino acid, is 4-6 amino acid best.The technician can be according to this area ordinary method (as referring to PNAS 1998; 95:5929-5934; ProteinEng, 2000; 13 (5): 309-312; Protein Eng, 2003; 15 (11): design connection peptides documents such as 871-879).Usually, connection peptides does not influence or the aminoacid sequence that has a strong impact on the aminoacid sequence of calprotectin element and TRAIL element forms correct folding and space conformation.
Preferred connection peptides example comprises (but being not limited to): becoming separate structural domain in order to help protein folding, is suitable (170-181 position among the SEQ ID NO:6) with sequences such as SGGGGSGGGG as connecting arm; In order to help proteolytic enzyme Calreticulin-TRAIL is cut two independently protein moleculars, the restriction enzyme site of the available active X factor (IEGR) connects arm.Similar, the restriction enzyme site of Chymotrypsin 1, papoid, Tryptase, Taka-proteinase, trypsinase etc. also can design as the amino acid connecting arm; In order to help purifying, can be 6His as connecting arm, to use metal affinity chromatography purifying Calreticulin-TRAIL fusion rotein; The combination of above-mentioned three kinds of schemes also can be designed to new amino acid connecting arm, has merged protease cutting site (NIa proteolytic enzyme) and metal affinity chromatography site 6His exactly as NVVVHQAHHHHHHEFTYK (SEQ ID NO:34) connecting arm.
The dna sequence dna of code book invention fusion rotein, all synthetic.Also available pcr amplification or synthetic method obtain calprotectin and or the DNA sequences encoding of tumor necrosin relative death inducing ligand, then it is stitched together, form the dna sequence dna of code book invention fusion rotein.
Having obtained it to be connected into suitable expression vector after code book invents the dna sequence dna of new fusion rotein, change proper host cell again over to.At last, cultivate the host cell after transforming, obtain new fusion rotein of the present invention by separation and purification.
As used herein, term " carrier " comprises plasmid, clay, expression vector, cloning vector, virus vector etc.Representational state comprises (but being not limited to): the carrier that can express in eukaryotic cells such as eukaryotic cell such as CHO, COS series, the carrier that can express in yeast saccharomyces cerevisiae or pichia yeast can be at the carrier and the prokaryotic expression carrier of expressed in insect cells such as silkworm.
In the present invention, can select various carrier known in the art such as commercially available carrier for use.Such as, select commercially available carrier for use, the nucleotide sequence of then code book being invented new fusion rotein operationally is connected in expression regulation sequence, can form protein expression vector.
As used herein, " operationally being connected in " refers to a kind of like this situation, and promptly some part of linear DNA sequence can influence the activity of same other parts of linear DNA sequence.For example, if signal peptide DNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion leader sequence) DNA operationally is connected in polypeptid DNA so; If transcribing of promotor control sequence, it is operationally to be connected in encoding sequence so; When if ribosome bind site is placed in the position that can make its translation, it is operationally to be connected in encoding sequence so.Generally, " operationally being connected in " means adjoining, then means in reading frame adjacent for the secretion leader sequence.
In the present invention, term " host cell " comprises prokaryotic cell prokaryocyte and eukaryotic cell.The example of prokaryotic host cell commonly used comprises intestinal bacteria, Bacillus subtilus etc.Eukaryotic host cell commonly used comprises yeast cell, insect cell and mammalian cell.Preferably, this host cell is an eukaryotic cell, more preferably is bombyx mori cell.
After obtaining transformed host cells, can under the condition that is fit to expression fusion rotein of the present invention, cultivate this cell, thereby give expression to fusion rotein.Can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, superly handle, the combination of super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.
Calreticulin-TRAIL fusion rotein of the present invention had both had the function of the inhibition endothelial cells in tumor neogenetic blood vessels growth of Calreticulin, had the effect of the inducing apoptosis of tumour cell of TRAIL again.
In another aspect of this invention, also provide a kind of pharmaceutical composition.Pharmaceutical composition of the present invention comprises the novel C alreticulin-TRAIL fusion rotein of the present invention of significant quantity, and at least a pharmaceutically acceptable carrier, thinner or vehicle.In preparation during these compositions, usually with activeconstituents and mixed with excipients, or with the vehicle dilution, wrap in can capsule or the carrier that exists of anther sac form in.Do the time spent when vehicle plays thinner, it can be solid, semisolid or the fluent material medium as vehicle, carrier or activeconstituents.Therefore, composition can be tablet, pill, pulvis, solution, syrup, sterilizing injecting solution etc.The example of suitable vehicle comprises: lactose, glucose, sucrose, sorbyl alcohol, N.F,USP MANNITOL, starch, Microcrystalline Cellulose, polyvinylpyrrolidone, Mierocrystalline cellulose, water, etc.Preparation also can comprise: wetting agent, emulsifying agent, sanitas (as methyl hydroxybenzoate and propyl ester), sweeting agent etc.
Composition can be made into unit or polynary formulation.Each formulation comprises the active substance that calculates predetermined amount in order to produce desired treatment effect, and the proper drug vehicle.
The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intraperitoneal, intravenously, subcutaneous, intracutaneous or topical.
When making pharmaceutical composition, be that fusion rotein of the present invention or its antibody with safe and effective amount is applied to the people, wherein this safe and effective amount is usually at least about 1 microgram/kg body weight, and in most of the cases be no more than about 8 mg/kg body weight, preferably this dosage is about 1 microgram/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.
In addition, fusion rotein of the present invention also can with the other treatment drug combination, comprising (but being not limited to): various cytokines, as IFN, TNF, IL-2 etc.; Various tumor chemotherapeutic drugs, influence biological nucleic acid synthetic medicine as 5-Fu, methotrexate etc., alkylating agent such as mustargen, endoxan class, Zorubicin, dactinomycin etc. disturb transcription to stop RNA synthetic medicine, and vincristine(VCR), camptothecin influence various kinds of drug such as the medicine of protein synthesis and some hormone.
In sum, major advantage of the present invention is as follows:
(1) Calreticulin-TRAIL fusion rotein had both had the function of the inhibition endothelial cells in tumor neogenetic blood vessels growth of Calreticulin, had the effect of the inducing apoptosis of tumour cell of TRAIL again, was a kind of newtype drug for the treatment of tumour.
(2) convenient drug administration.Because both useful effect dosage is in most of the cases approaching, so give simultaneously with fusion rotein, has made things convenient for the proportioning of single medicine, the experimenter has also reduced misery simultaneously.
(3) has target.Calreticulin can transport TRAIL to tumor by local in the tumor neovasculature while of inhibition and play a role; Same, when TRAIL acted on tumour cell, the new vessel that Calreticulin is transported to tumor tissues was brought into play its restraining effect.
(4) strengthen proteic stability.Behind both amalgamation and expressions, vitro half-lives is obviously elongated, and the stability in the aqueous solution is become month (all under 4 ℃) after the fusion each about week by both; Believe the increase that in vivo transformation period also can be suitable.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1
Set up the cDNA library, screening obtains TRAIL and Calreticulin gene
1. prepare the total RNA of human peripheral blood single nucleus cell
Use lymphocyte separation medium separation of human peripheral blood lymphocyte routinely, with RPMI-1640 substratum (GIBCO company), adding 10% newborn calf serum (Hangzhou folium ilicis chinensis biotech firm) and penicillin and Streptomycin sulphate is cultured to adherent, get mononuclearcell, intracellular toxin with the intestinal bacteria R595 of 10 μ g/L stimulates 2h then, to activate mononuclearcell, collect 10 7Cell with total RNA extraction agent box (Qiagen company) extracted total RNA, gets the total RNA of human peripheral blood single nucleus cell.
2. set up the cDNA library, the screening trail dna
Total RNA of above-mentioned gained, get total mRNA with Oligo-dT post (Qiagen company) purifying, carry out the synthetic of cDNA first chain and second chain with cDNA synthetic agent box (Clontch company) by specification, be connected in the λ gt10 carrier after adding the EcoRI joint, and be packaged into the lambda particles phage library with lambda particles phage package kit (Clontech company), build up λ gt10 cDNA library, the titre in library is 6 * 10 6
This λ gt10 cDNA library is with 1 * 10 5Bed board on bacterium colony/LB flat board is manufactured the multiple die on the nitrocellulose filter of 20 * 20cm.With the theoretical sequences Design random primer of TRAIL, preparation gene probe, screening by hybridization library.The repetition die that contains bacterium colony is containing 10% T 500,100 μ g/ml tRNA and 6 * 10 565 ℃ of hybridization are spent the night in the plate screening damping fluid of cpm/ml probe (50mmol/L Tris-HCl pH 7.5,1mol/L NaCl, 0.1% trisodium phosphate, 0.2% polyvinylpyrrolidone and 0.2%Ficoll).65 ℃ of plate screenings, and 2 * SSC (0.3mol/L NaCl, the 30mmol/L Trisodium Citrate, pH7.0), the 0.1%SDS damping fluid washes twice.Closely contact with film at-40 ℃ then, screened 40 hours.
Double male sample is chosen corresponding bacterium colony from main flat board, forwards the damping fluid (100mmol/L NaCl, the 10mmol/L MgSO that have added gelatin to 4, 50mmol/L Tris-HCl, pH 7.5) the LB flat board on, obtain 12 positive plaques.The λ DNA of 12 purifying digests with Notl, 1% agarose gel electrophoresis, and the Southern trace is confirmed and probe hybridization.When selecting with above-mentioned probe hybridization, the clone of radioactive intensity maximum (about 1.7kp) carries out the DNA purifying.These clones' insertion portion is by the NotI site of subclone to pSPORTl (GIBCO company).Measure the dna sequence dna of insertion portion in the plasmid.From the result of sequential analysis, obtain, insertion sequence in one of them plasmid is 1769bp, 5 ' the non-translational region that comprises 87bp, 843bp open reading frame and 3 ' non-translational region (contain polyadenylation signal or claim the polyA tail), this fragment is a trail dna, encoding sequence wherein such as SEQ ID NO:3.According to the translation initiation site requirement of Kozak definition, 88-90 position Nucleotide (ATG, coding methionine(Met)) is translation initiation site, 281 aminoacid sequences that the maximum open reading frame that obtains thus is coded such as SEQ ID NO:4.
3. screen the Calreticulin gene
With step 1 and 2 similar methods, screening obtains the encoding sequence such as the SEQ ID NO:1 of Calreticulin gene from people myocardial cell cDNA library, coded 417 aminoacid sequences such as the SEQ IDNO:2 of deduction.
Embodiment 2
Reverse transcription polymerase chain reaction method (being RT-PCR) obtains the encoding sequence of Calreticulin
1.RT obtain Calreticulin cDNA first chain
With the total RNA of people's liver cell from Clontech company (U.S.) purchase is template, is primer with P1, and Calreticulin cDNA first chain is synthesized in reversed transcriptive enzyme catalysis.
2. polymerase chain reaction (PCR) amplification obtains the Calreticulin encoding sequence
With above-mentioned cDNA first chain is template, and P1, P2 are primer, the synthetic and amplification Calreticulin encoding sequence of Tag archaeal dna polymerase catalysis.Through sequential analysis, the shown Calreticulin encoding sequence of the sequence (NM_004343) of gained dna sequence dna and GenBank registration is consistent, has promptly obtained the Calreticulin coding nucleotide sequence.
Primer:
P1：TTT TAA AGG GCC CGC GCG TTG(SEQ ID NO：26)
P2：ATT TGG CGC GGC AAG CTC AGC(SEQ ID NO：27)
Embodiment 3
RT-PCR obtains the encoding sequence of TRAIL
1. prepare the total RNA of human peripheral blood single nucleus cell
(1) isolated lymphocytes from human peripheral according to a conventional method, and then obtain mononuclearcell with adherent method;
(2) activate the abundance that mononuclearcell is expressed more gene with irritation cell and improved RNA with bacterial endotoxin, with RNA extraction agent box (Qiagen company) extracted total RNA.
2. reverse transcription (RT) obtains TRAIL cDNA first chain
With above-mentioned total RNA is template, is primer with P3, and TRAIL cDNA first chain is synthesized in reversed transcriptive enzyme catalysis.
3. polymerase chain reaction (PCR) amplification obtains the TRAIL encoding sequence
With above-mentioned cDNA first chain is template, and P3, P4 are primer, the synthetic and amplification TRAIL encoding sequence of Tag archaeal dna polymerase catalysis.Through sequential analysis, the shown TRAIL encoding sequence of the sequence (NM_003810) of gained dna sequence dna and GenBank registration is consistent, has promptly obtained the TRAIL coding nucleotide sequence.
P3：GAC TTA CAG CAG TCA GAC TCT GAC(SEQ ID NO：28)
P4：TAT TGC TTT TTC TTT CCA GGT CAG(SEQ ID NO：29)
Embodiment 4
Construction expression Calreticulin-TRAIL fused protein expression vector
With pBV220 carrier (can available from Inst. of Viruses, China Preventive Medicine Science Academy and Shanghai Sangon Biological Engineering Technology And Service Co., Ltd), the recombinant plasmid of construction expression TRAIL 114-281 amino acids, the prokaryotic expression carrier of constructed expression TRAIL 114-281 amino acids, called after pBV-TRAIL.Accordingly, the recombinant expressed of fused protein carries out with same method.Concrete implementation step is as follows:
1. design and synthesize primer and oligonucleotide two strands
Primer P5, the P6 of design amplification coding TRAIL 114-281 amino acids, its sequence is:
P5：AC G AAT TCA CA A TGG TGA GAG AAA GAG GTC(SEQ ID NO：30)；
P6：AC G GAT CCT TAG CCA ACT AAA AAG(SEQ ID NO：31)
The distance that P5 has optimized between SD sequence and the foreign gene is 8 Nucleotide (italicized item), introduces synthetic initiator codon ATG of albumen and EcoRI restriction enzyme site GAATTC; P6 introduces BamHI restriction enzyme site GGATCC.
Synthetic oligonucleotide two strands, sequence following (not providing the sequence of complementary strand):
P7：AA A GAT CTC TCA CCT ACC AAA CAA TGC CCC CCT GCA AAA AAT AAATTC ATA TAA AAA ACA TAC AGA TAA CCA TCT GCG GTG ATA AAT TAT CTCTGG CGG TGT TGA CAT AAA TAC CAC TGG CGG TGA TAC TGA GCA CAT CAG CAGGAC GCA CTG ACC ACC ATG AAG GTG ACG CTC TTA AAA ATT AAG CCC TGAAGA AGG GCA GCA TTC AAA GCA GAA GGC TTT GGG GTG TGT GAT ACG AAA CGAAGC ATT GGT TAA AAA TTA AGG AG G AAT TCA CA(SEQ ID NO：32)
Wherein, the italicized item of P7 sequence is λ P LP RTandem promoter subsequence, dash area are cI arrestin binding site, and black matrix is the SD sequence for optimizing partly, and 5 ' end is introduced BglII restriction enzyme site AGATCT, and 3 ' end is introduced EcoRI restriction enzyme site GGATTC;
P8：AA G GAT CCG TCG ACC TGC AGC CAA GCT TGG CTG TTT TGG CGG ATGAGA GAA GAT TTT CAG(SEQ ID NO：33)
Wherein, 5 of P8 sequence ' end has been introduced BamHI restriction enzyme site GGATCC, 3 ' introducing XmnI restriction enzyme site GAANNNNTTC.
2. construction of expression vector pBV-TRAIL, specific as follows:
With P5, P6 is primer, is template with the trail dna of embodiment 3, pcr amplification trail dna encoding sequence, and product is cut (toolenzyme is all available from U.S. NEB company) with EcoRI and BamHI enzyme.Cut P3 with BglII and EcoRI enzyme, BamHI and XmnI enzyme are cut P4.With BglII and XmnI double digestion pBV220 plasmid.
Above-mentioned each endonuclease bamhi sepharose reclaims the fragment of corresponding size, connects each fragment with the T4DNA ligase enzyme, and resulting recombinant plasmid is the expression vector of Tumor wilting extract, called after pBV-TRAIL.
PBV-TRAIL compares pBV220 and has following feature: the TRAIL encoding gene is subjected to upstream lambda particles phage P LP RTandem promoter control, the ribosome binding sequence of optimization (SD sequence) inserts EcoRI restriction enzyme site and translation initiation codon ATG, optimized simultaneously and the SD sequence between distance be 8 Nucleotide; Insert BamHI restriction enzyme site etc. in Tumor wilting extract encoding gene downstream.This plasmid and pBV220 are same, contain the cIts857 binding sequence in promoter sequence; Have ampicillin resistance gene; Transcription termination sequence rrnB; Contain coding temperature sensitive protein factor cIts857 gene on the plasmid, this coded product inactivation in the time of 42 ℃, thus losing inhibition to promotor, the downstream foreign gene of promotor control is expressed.Feature nucleotide sequences such as the SD sequence of upstream regulatory sequence, optimization, TRAIL encoding sequence and translation termination sequence are shown in SEQ ID NO:7.
With above-mentioned same method, with the listed sequence of table 1 is primer (listed primer only part has been enumerated listed 18 kinds of fused proteins among amplification Fig. 1), corresponding gene with above-mentioned gained is a template, amplification corresponding encoded dna fragmentation, digestion with restriction enzyme, T4DNA ligase enzyme connect on the carrier that enzyme is cut equally, the transformed competence colibacillus cell, screening contains the clone of recombinant plasmid, and order-checking is identified and confirmed.
The tabulation of table 1 primer nucleotides sequence
First group: the Calreticulin in the coded fused protein that increases holds at N, (p represents primer to A-I as shown in Figure 1, and C represents Calreticulin, and sandwich digit is represented coded amino acid whose initial or final position, f represents forward primer, and r represents reverse primer)
pC1f 5′-gcg aat tca cat atg ctg cta tcc gtg cc-3′ SEQ ID NO：8
pC119f 5′-gcg aat tca cat atg aca gac atg cac gga gac-3′ SEQ ID NO：9
pC180r aag gat ccg tct ggc cgc aca atc ag-3′ SEQ ID NO：10
PC417r aag gat ccc agc tcg tcc ttg gcc tg-3′ SEQ ID NO：11
pT1f 5′-aac tgc aga tgg cta tga tgg agg tc-3′ SEQ ID NO：12
pT95f 5′-aac tgc aga cct ctg agg aaa cca tt-3′ SEQ ID NO：13
pT114f 5′-aac tgc agg tga gag aaa gag gtc ctc-3′ SEQ ID NO：14
pT281r 5′-tca agc tta gcc aac taa aaa ggc-3′ SEQ ID NO：15
Second group: the TRAIL in the coded fused protein that increases holds at N, a-i as shown in Figure 1
pT1f1 5′-gcg aat tca cat atg gct atg atg gag gtc-3′ SEQ ID NO：16
pT95f1 5′-gcg aat tca cat atg acc tct gag gaa acc att-3′ SEQ ID NO：17
pT114f1 5′-gcg aat tca cat atg gtg aga gaa aga gg-3′ SEQ ID NO：18
pT281r 5′-aag gat cca act aaa aag gcc ccg-3′ SEQ ID NO：19
pC1f1 5′-aac tgc aga tgc tgc tat ccg tgc cg-3′ SEQ ID NO：20
pC119f1 5′-aac tgc aga cag aca tgc acg ga-3′ SEQ ID NO：21
pC180r1 5′-tca agc tta gtt gtc tgg ccg cac aat c-3′ SEQ ID NO：22
pC417r1 5′-tca agc tta cag ctc gtc ctt ggc c-3′ SEQ ID NO：23
The 3rd group: the encoding sequence of amino acid connecting arm can form double-stranded DNA through simple sex change and renaturation
pL1 5′-ga tcc ggc gga ggc ggg agc ggc ggg ggc gga agc ctg ca-3′ SEQ ID NO：24
pL2 5′-ggc ttc cgc ccc cgc cgc tcc cgc ctc cgc cg-3′ SEQ ID NO：25
A series of recombinant plasmids of expressing different lengths Calreticulin-TRAIL fused protein have respectively been obtained thus.Especially, the 18 kinds of Calreticulin-TRAIL recombinant fusion proteins as shown in Figure 1 that prepare with cited primer can directly show at external killing tumor cell, suppress the biological action of the tumor growth of transplanting in vivo.
A kind of nucleotide sequence of preferred expression Calreticulin-TRAIL fused protein is shown in SEQ IDNO:5, and the aminoacid sequence of being inferred is shown in SEQ ID NO:6.
Embodiment 5
Transformed into escherichia coli is set up engineering bacteria
Routinely with a series of recombinant expression plasmid transformed into escherichia coli BL21[genotype such as pBV-TRAIL that obtain among the embodiment 4: hsdS gal (λ cIts857indl Sam7 nin5 lacUV5-T7)] (can give birth to worker bio-engineering corporation) available from Shanghai, isolated plasmid dna from ammonia benzyl resistance bacterium colony, enzyme is cut evaluation, order-checking confirms that the positive colony of gained is the engineering bacteria of expressing the respective egg white matter.
Embodiment 6
Preparation Calreticulin-TRAIL fused protein
Various substratum are as follows: the LB substratum is as the test tube seed culture, and 2 * YT substratum is cultivated as secondary seed, and semisynthetic medium is used for fermentation, and fed-batch adds to be cultivated.
LB culture medium prescription (g/L): peptone: yeast powder: NaCl=10: 5: 5;
2 * YT culture medium prescription (g/L): peptone: yeast powder: NaCl=16: 10: 5;
Prescription (g/L) in the semi-synthetic fermention medium: peptone: yeast powder: KH 2PO 4: K 2HPO 4: Na 2HPO 4.12H 2O: (NH 4) 2SO 4: NH 4Cl=5: 5: 2: 4: 7: 1.2: 0.2;
Various trace element solutions, concentration are (g/L): MnSO 4.5H 2O: CaCl 2.6H 2O: Na 2MoO 4.2H 2O: ZnCl 2: CuSO 4.5H 2ZO: H 3BO 4: FeSO 4.7H 2O: CaCl 2.2H 2O: MgSO 4.7H 2O=0.001: 0.004: 0.002: 0.002: 0.001: 0.0005: 0.02: 0.02: 0.3;
Feeding medium during fermentation prescription (g/L): glucose: yeast powder: peptone: MgSO 4.7H 2O=200: 70: 70: 5.7.
The pH value of substratum all is adjusted to 7.0.Add penbritin final concentration to 100 μ g/ml behind the various substratum high-temperature sterilizations.
After the first order seed overnight incubation, be forwarded to secondary seed with 1: 50 ratio, fermentor tank is pressed the secondary seed that 5% of working volume inserts overnight incubation.Cultivate and divide two stages to carry out, cultivated 5-7 hour for 32 ℃; Being warming up to 42 ℃ cultivated 4-5 hour.Constant flow pump adds feed supplement, and dissolved oxygen is controlled between the 30-50%.Obtain the wet thallus of about 20-30 grams per liter fermented liquid.
Ultrasonication fermentation thalline, the centrifuging and taking supernatant is crossed the filter membrane of 0.22 μ m; With NTA Supperflow (Qiagen company) or TALON Metal Affinity Rsins (Clontech company) row metal affinity chromatography, 10mmol/L imidazoles, pH7.0 wash-out foreign protein, again with the 100mmol/L imidazoles, pH7.0, wash-out recombinant protein; Elutriant is crossed the CM Mierocrystalline cellulose, collects active ingredient; Be further purified after Sephacryl S-200, can obtain highly purified recombination fusion protein.
The result: with a kind of fusion rotein shown in Fig. 1-i (is TRAIL 114-281-Calreticulin 119-180) protokaryon is recombinant expressed is example, constructed engineering bacteria is expressed through thermal induction, the capable SDS-PAGE of supernatant after the ultrasonication, and visible expressed fusion protein accounts for more than 30% of supernatant total protein, and it is pure to reach electrophoresis after being further purified, and the results are shown in Figure 2.The calculating molecular weight is 27.6kD, and the theoretical iso-electric point of deduction is 8.4.All the other independent TRAIL, Calreticulin or both can obtain similar results after merging abduction delivering, obtain the recombinant protein of corresponding molecular weight size.
Embodiment 7
The biologic activity of fused protein is determined
In the present embodiment, the institute of mensuration fusion rotein has the active and Calreticulin activity of TRAIL.
The activity of TRAIL: external can human pancreas's cancerous ductal epithelial cell 1990 strain cells, the cell lung cancer NCI-460 of the National People's Congress, murine melanoma B16 etc. are target cell, determine the biologic activity of TRAIL, in the body with the human colon carcinoma HCT-8 transplanted tumor that suppresses nude mice etc. external to the TRAIL sensitivity, to become the mice-transplanted tumor of knurl in vivo be model, the biological function of checking TRAIL.
The activity of Calreticulin: in the restraining effect of observation in vitro Calreticulin to the newborn endotheliocyte (human umbilical vein, bovine aortic, induced lung source capillary vessel etc.) of cultivation in the presence of alkaline epithelial cell growth factor (bFGF), observe Calreticulin in vivo the human colon carcinoma HCT-8 transplanted tumor of chicken embryo bird cyst membrane new vessel growth, the growth of tame rabbit cornea new vessel, nude mice etc. is model, the biological function of checking Calreticulin.
Concrete test operation is as follows:
1, TRAIL activation analysis: kill and wound various tumour cells
Various target cells derive from cell research institute of the Chinese Academy of Sciences or Chinese Shanghai Changhai hospital.Mainly contain people's pancreas cancerous ductal epithelial cell 1990,8898 strains, the cell lung cancer NCI-460 of the National People's Congress, human colon carcinoma HCT-8, people's cancer of the stomach M85 strain, the SK-N-SH human neuroblastoma cells strain, people's laryngocarcinoma Hep-2 strain, human nasopharyngeal carcinoma CNE-2 strain, human endothelial cell CEV304 strain, human fibroblasts's strain, human colon carcinoma AT-29, human ovarian cancer 3AO, mouse becomes fiber L929 strain, human glioma U251, people's breast cancer, people's liver cancer HepG-2, SMMU7721, human blood is learned tumour (U937, Jukart, HL60 etc.), melanin tumour b16-MB, the Ehrlich ascitic tumor, Lewis sarcoma etc.
With 1990 cells is example, with culturing cell with 2 * 10 8Cell/L kind is gone into 96 porocyte culture plates, 37 ℃ of 5%CO 2Hatch 4-6h in the incubator, abandon supernatant; Add cell plate after with perfect medium sample being done gradient dilution; Observe to add behind the 1.5 μ g/ml dactinomycins influence simultaneously to various factor killer cell; Activity unit definition: making cell 50% death of cultivating in the plate hole is a unit, tires promptly to reach the dilution inverse of 50% sample when killing and wounding.Do quantitative analysis with the Viola crystallina method: attached cell abandons supernatant, and with Viola crystallina stationary liquid (5g/L Viola crystallina, 80mL/L formaldehyde, 1g/L NaCl, 200mL/L ethanol) dyeing 15min, distilled water flush away Viola crystallina is dried, and the person of being colored is a viable cell.Every hole adds the acetate of 200 μ L 330mL/L again, and the shaking table rotation makes the Viola crystallina dissolving, and put enzyme connection instrument and read the A value in wavelength 595nm, be the A background with the blank well that does not add cell.Suspension cell is determined viable count with mtt assay.Its activity is tired and is done straight-line regression with the mean of the logarithm of diluted sample degree and A595nm, obtains constant A, B, is 50% to kill and wound terminal point (Y) with (A contrast-A background)/2.Calculate the activity unit of sample, i.e. X value by Y=A+BlgX.Simultaneously, this cell is typical apoptosis form under the TRAIL effect, under the effect of 10mg/L Tumor wilting extract, promptly observes cell generation typical change in 2 hours.
2, the cultivation of new born bovine aortic endothelial cell
The about 10cm of clip new born bovine aorta section is long totally two under the aseptic condition; Indoor at aseptic laminar flow, with the aorta trimmed, arterial bifurcation is sentenced the cotton rope ligation; 0.1mol/L PBS with pH7.2 washes aorta lumen, thoroughly removes remaining hemocyte; Pour into about 10ml Digestive system (0.2%II Collagen Type VI enzyme is a solvent with the DMEM substratum) behind cotton rope ligation aorta one end, room temperature leaves standstill digestion 20min; Take out Digestive system,, collect washing fluid and mix, put into aseptic 50ml centrifuge tube with Digestive system with 0.1mol/L PBS flushing aorta inside pipe wall; The centrifugal 10min of 500 * g abandons supernatant; The precipitation (containing cell) 0.1mol/L PBS suspends, the centrifugal 10min of 500 * g repeats once; After the DMEM nutrient solution suspension that contains 20%FBS, 200 μ g/ml ECGF, add the plastics Tissue Culture Flask, put 37 ℃, 5%CO 2Cell culture incubator is cultivated; Microscopy has a large amount of cell attachments existence back (needing 3 days approximately) to change liquid, changes liquid once in later per 2 days, treats that the cell growth is individual layer and converges the cultivation of going down to posterity of back routine.
3, the cultivation of induced lung source capillary endothelial cell
The art pre-treatment: get SD rat heavy about 150g, abdominal injection 0.5% heparin sodium aqua 3ml with the stifling anesthetized animal of ether, is dipped in 75% alcohol disinfecting 3min in the sealing cylinder; Lung is got in operation: open chest, cut off left ventricle, right ventricle injection PBS liquid 10ml, flushing lung blood vessel to the left ventricle effluent liquid colourless till, cut lobe of the lung edge, put in the PBS liquid and wash, the lung tissue of cutting is cut into fritter about 2 * 2mm, in the DMEM nutrient solution that contains 20% calf serum, soak 5min; The fritter lung tissue is placed aseptic, exsiccant plastics Tissue Culture Flask, in 37 ℃, 5%CO 2Cultivate 1.5h in the incubator, make the lung tissue piece adherent; Just putting cultivation: the careful DMEM 10ml that contains 20%FBS, 200 μ g/ml ECGF that adds, tissue block is dashed from the bottle wall, put 37 ℃, 5%CO 2Incubator is cultivated; Carefully remove tissue block and change liquid, changed liquid once in later 2 days, cell grows to the cultivation of going down to posterity after individual layer converges.
4. fused protein suppresses various cell proliferation tests
Various cells (mouse source pulmonary capillary endothelial cell, new born bovine aortic endothelial cell, B16 melanoma cell) growth converges back (in the culturing bottle), and with 0.125% trysinization, the DMEM that contains 20%FBS suspends and adjusting concentration to 5 * 10 4/ ml is inoculated into 96 porocyte culture plates with 100 μ l/ holes, puts 37 ℃, 5%CO 2Incubator is cultivated.(need 4～5h) to give sample approximately: the protein initial concentration is 2 μ g/ holes to the adherent back of inverted phase contrast microscope observation of cell, makes 2 times of gradient dilutions.Put 37 ℃, 5%CO 2Cultivate in the incubator.Add MTT solution 15 μ l/ holes behind the 48h, 37 ℃, 5%CO 2Cultivate 4h; Add 100 μ l/ hole mtt assay reaction terminating liquids, put 1h for 37 ℃.With the abundant mixing in each hole and remove the rearmounted enzyme connection of bubble instrument and detect the 595/630nm absorption value.
5. fused protein inhibition of endothelial cell proliferation test
Prepare new born bovine aortic endothelial cell, mouse lung source capillary endothelial cell suspension with preceding method, regulate concentration to 6 * 10 4/ ml is inoculated in the 96 porocyte culture plates with 50 μ l/ holes, puts 37 ℃ of 5%CO 2Incubator is cultivated.Treat that (about 4～5h) give sample: establish six concentration gradients of every hole 0,2.5,5.0,10,20,40 μ l/ml, each concentration gradient is established 10 parallel holes behind the cell attachment.Add 15 μ l/ hole MTT solution, 37 ℃, 5%CO behind the 48h 2Incubator is cultivated 4h.Add 100 μ l/ hole stop buffers, 37 ℃ of temperature are bathed 1h.With the abundant mixing in each hole and remove the rearmounted enzyme connection of bubble instrument and detect 595nm/630nm double wave absorption value.Calculate fused protein 50% inhibition concentration.
6. the inhibition test of the new vessel that induces of fusion rotein confrontation rabbit cornea
Sample is coated in a kind of slowly-releasing thing (EVA, promptly ELvax40 is the multipolymer of a kind of ethene and ethene acetate), implants tame rabbit cornea, observe the situation that its inhibition induces angiogenic growth.Sample discharges sustainable release 100d with the speed of 0.1～1 μ g/d.
50mg EVA is soaked in 75% alcohol 10min, sterilization is placed in the 2ml dichloromethane solution, stirring makes its dissolving, add bacterial endotoxin 8mg and laboratory sample, fully stirring makes its mixing leave standstill 1h in EVA solution, forms the EVA glued membrane, be cut into small pieces, every heavy 1mg, uviolizing 30min, it is standby to sterilize.Rabbit is after intravenous anesthesia (3% vetanarcol, 40 μ g/kg body weight), and 1/3 place makes radial incision (long 2～3mm outside cornea, dark 0.3～0.5mm), with flaggy separator isolated cornea flaggy, make cornea form 3 * 3mm lacuna, insert a treated EVA.Every day observer's rabbit corneal growing state, postoperative 10d Taking Pictures recording analytical results.
7. the inhibition test of fusion rotein confrontation chick chorioallantoic membrane new vessel
It is the same that sample is embedded in EVA, but do not add intracellular toxin.Get instar chicken embryo on the 9th, aseptic dH 2O cleans, and after the 1/4000 formaldehyde wiping, dries, and puts 37 ℃ of incubations.Next day, the chicken embryo to be put under the ovoscopy lamp seek the embryo head, the chorion projection position between the 1cm before distance embryo head, two anterior vitelline veins is with wax crayon 1 * 1.5cm rectangle of drawing.Bore the aperture of about 1.0～2.0mm and penetrate the air chamber shell membrane at the chick embryo air sac end.In the eggshell position of windowing, behind 75% alcohol disinfecting, cut chorion (being sure not to injure the shell membrane of below) along the sideline mill of rectangle with the coil file cutter, clamp rectangle chorion edge with the ophthalmology tweezer, parallelly upwards mention, expose the shell membrane of below, the chorion dust inclines.Follow the shell membrane fiber direction with injection needles and scratch diameter 1mm aperture, can not injure the chorioallantoic membrane (CAM) of below.On the shell membrane window, drip stroke-physiological saline solution a little, dialled the shell membrane of little bore edges then gently with the ophthalmology tweezer, allow between liquid immersion shell membrane and the CAM, treat the CAM sinking after, wipe out rectangular frame endochorion film.The EVA embedded block is directly placed CAM go up avascular area.Seal the ovum window with the sterile transparent adhesive tape, seal the air chamber aperture, hatch for 37 ℃ with paraffin.Remove CAM plane above chorion and shell membrane with the ophthalmology tweezer after three days, note not injuring the CAM blood vessel.On the CAM plane, drip an amount of methyl alcohol, acetone balanced mix stationary liquid, at room temperature fixing 15min.After treating that vessel inner blood on the CAM solidifies, the CAM of test zone is cut, place to fill dH 2In the dish ware of O, after the expansion of elbow suction pipe, the dH in the ware that inclines 2O shakeouts CAM at the bottom of ware, upset is affixed on the filter paper Taking Pictures recording result, analytical results.
8. fused protein suppresses mice-transplanted tumor
The amplification of the inside and outside of various tumour cells: the tumour cell recovery back of liquid nitrogen cryopreservation is cultivated or Kunming mouse abdominal cavity (10 is gone in inoculation 7Cell/mouse).Put to death mouse after 10 days, aseptic condition absorbs mouse ascites down, and regulating cell concn with PBS is 10 8/ ml.It is subcutaneous to be inoculated in experiment mice with this cell suspension 0.2ml.Random packet, every group of 6 mouse, the mouse of subcutaneous vaccination give 0 (PBS, control group), 10,100,1000 μ g (sample)/kg (mouse body weight), administration every other day, 2 weeks respectively.After 1 week of drug withdrawal, put to death mouse, peel off tumour and weigh, calculate every group of heavy and tumour inhibiting rate of average knurl, analytical results.Tumour inhibiting rate calculates with following formula:
The result: the result of the cell lung cancer NCI-H460 of National People's Congress transplanted tumor is as follows.To other transplanted tumors, as human colon carcinoma HCT-8, human pancreas cancer SW1990,8898 strains, people's cancer of the stomach M85, people's laryngocarcinoma Hep-2, human nasopharyngeal carcinoma CNE-2, human colon carcinoma AT-29, human glioma U251, people's liver cancer HepG-2, melanin tumour b16-MB, the Ehrlich ascitic tumor can be obtained similar result on the models such as Lewis sarcoma.Table 2 has been listed prepared as stated above high purifying TRAIL, calreticulin or the various different fusion molecule of both amalgamation and expressions, respectively under 10,100,1000 μ g/kg administrations to the tumour inhibiting rate of the cell lung cancer NCI-H460 of National People's Congress mice-transplanted tumor.
Table 2NCI-H460 table with test results
Protein structure 10 μ g/kg tumour inhibiting rates (%) 100 μ g/kg tumour inhibiting rates (%) 1000 μ g/kg tumour inhibiting rates (%) Remarks
Fig. 1-A - - - The prokaryotic expression amount is extremely low
Fig. 1-B 28.6 33.8 40.5
Fig. 1-C 29.3 32.6 41.8
Fig. 1-D - - - The prokaryotic expression amount is extremely low
Fig. 1-E 25.2 31.6 39.9
Fig. 1-F 35.7 39.7 43.6
Fig. 1-G - - - The prokaryotic expression amount is extremely low
Fig. 1-H 33.4 40.8 51.6
Fig. 1-I 35.7 43.8 49.2
Fig. 1-a - - - The prokaryotic expression amount is extremely low
Fig. 1-b - - - The prokaryotic expression amount is extremely low
Fig. 1-c - - - The prokaryotic expression amount is extremely low
Fig. 1-d 28.3 34.9 39.6
Fig. 1-e 27.4 33.4 38.6
Fig. 1-f 34.2 38.7 44.5
Fig. 1-g 30.1 35.6 42.7
Fig. 1-h 32.6 40.6 49.7
Fig. 1-i 36.2 43.2 50.9
calreticulin 1-180aa 24.3 30.5 37.6
TRAIL 114-281aa 27.4 31.6 38.7
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Shanghai Qiaer biotechnology Co. Ltd
＜120〉fusion rotein of calprotectin-tumor necrosin relative death inducing ligand and method for making thereof and purposes
<130>035621
<160>34
<170>PatentIn version 3.1
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＜213〉homo sapiens (Homo sapiens)
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Ala Glu Pro Ala Val Tyr Phe Lys Glu Gln Phe Leu Asp Gly Asp Gly
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Lys Glu Glu Asp Glu Glu Glu Asp Val Pro Gly Gln Ala Lys Asp Glu
405 410 415
Leu
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＜213〉homo sapiens (Homo sapiens)
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ggctaa 846
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＜213〉homo sapiens (Homo sapiens)
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＜213〉artificial sequence
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＜223〉fusion rotein encoding sequence
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ccagacaact aa 732
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＜213〉artificial sequence
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＜223〉fusion rotein
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35 40 45
His Ser Phe Leu Ser Asn Leu His Leu Arg Asn Gly Glu Leu Val Ile
50 55 60
His Glu Lys Gly Phe Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe Arg Phe
65 70 75 80
Gln Glu Glu Ile Lys Glu Asn Thr Lys Asn Asp Lys Gln Met Val Gln
85 90 95
Tyr Ile Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro Ile Leu Leu Met Lys
100 105 110
Ser Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu Tyr Gly Leu Tyr
115 120 125
Ser Ile Tyr Gln Gly Gly Ile Phe Glu Leu Lys Glu Asn Asp Arg Ile
130 135 140
Phe Val Ser Val Thr Asn Glu His Leu Ile Asp Met Asp His Glu Ala
145 150 155 160
Ser Phe Phe Gly Ala Phe Leu Val Gly Ser Gly Gly Gly Gly Ser Gly
165 170 175
Gly Gly Gly Ser Leu Gln Thr Asp Met His Gly Asp Ser Glu Tyr Asn
180 185 190
Ile Met Phe Gly Pro Asp Ile Cys Gly Pro Gly Thr Lys Lys Val His
195 200 205
Val Ile Phe Asn Tyr Lys Gly Lys Asn Val Leu Ile Asn Lys Asp Ile
210 215 220
Arg Cys Lys Asp Asp Glu Phe Thr His Leu Tyr Thr Leu Ile Val Arg
225 230 235 240
Pro Asp Asn
<210>7
<211>1288
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(1288)
＜223〉the feature nucleotide sequence of pBV-TRAIL
<400>7
aaattcttca acgctaactt tgagaatttt tgtaagcaat gcggcgttat aagcatttaa 60
tgcattgatg ccattaaata aagcaccaac gcctgactgc cccatcccca tcttgtctgc 120
acgtgcgtcc tcaagctgct cttgtgttaa tggtttcttt tttgtgctca tacgttaaat 180
ctatcaccgc aagggataaa tatctaacac cgtgcgtgtt gactatttta cctctggcgg 240
tgataatggt tgcatgtact aaggaggttg tatggaacaa cgcataaccc tgaaagatta 300
tgcaatgcgc tttgggcaaa ccaagacagc taaagatctc tcacctacca aacaatgccc 360
ccctgcaaaa aataaattca tataaaaaac atacagataa ccatctgcgg tgataaatta 420
tctctggcgg tgttgacata aataccactg gcggtgatac tgagcacatc agcaggacgc 480
actgaccacc atgaaggtga cgctcttaaa aattaagccc tgaagaaggg cagcattcaa 540
agcagaaggc tttggggtgt gtgatacgaa acgaagcatt ggttaaaaat taaggaggaa 600
ttcacaatgg tgagagaaag aggtcctcag agagtagcag ctcacataac tgggaccaga 660
ggaagaagca acacattgtc ttctccaaac tccaagaatg aaaaggctct gggccgcaaa 720
ataaactcct gggaatcatc aaggagtggg cattcattcc tgagcaactt gcacttgagg 780
aatggtgaac tggtcatcca tgaaaaaggg ttttactaca tctattccca aacatacttt 840
cgatttcagg aggaaataaa agaaaacaca aagaacgaca aacaaatggt ccaatatatt 900
tacaaataca caagttatcc tgaccctata ttgttgatga aaagtgctag aaatagttgt 960
tggtctaaag atgcagaata tggactctat tccatctatc aagggggaat atttgagctt 1020
aaggaaaatg acagaatttt tgtttctgta acaaatgagc acttgataga catggaccat 1080
gaagccagtt tttttggggc ctttttagtt ggctaaggat ccgtcgacct gcagccaagc 1140
ttggctgttt tggcggatga gagaagattt tcagcctgat acagattaaa tcagaacgca 1200
gaagcggtct gataaaacag aatttgcctg gcggcagtag cgcgggtggt cccacctgac 1260
cccatgccga actcagaagt gaaacccc 1288
<210>8
<211>29
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>8
gcgaattcac atatgctgct atccgtgcc 29
<210>9
<211>33
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>9
gcgaattcac atatgacaga catgcacgga gac 33
<210>10
<211>26
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>10
aaggatccgt ctggccgcac aatcag 26
<210>11
<211>26
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>11
aaggatccca gctcgtcctt ggcctg 26
<210>12
<211>26
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>12
aactgcagat ggctatgatg gaggtc 26
<210>13
<211>26
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>13
aactgcagac ctctgaggaa accatt 26
<210>14
<211>27
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>14
aactgcaggt gagagaaaga ggtcctc 27
<210>15
<211>24
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>15
tcaagcttag ccaactaaaa aggc 24
<210>16
<211>30
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>16
gcgaattcac atatggctat gatggaggtc 30
<210>17
<211>33
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>17
gcgaattcac atatgacctc tgaggaaacc att 33
<210>18
<211>29
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>18
gcgaattcac atatggtgag agaaagagg 29
<210>19
<211>24
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>19
aaggatccaa ctaaaaaggc cccg 24
<210>20
<211>26
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>20
aactgcagat gctgctatcc gtgccg 26
<210>21
<211>23
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>21
aactgcagac agacatgcac gga 23
<210>22
<211>28
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>22
tcaagcttag ttgtctggcc gcacaatc 28
<210>23
<211>25
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>23
tcaagcttac agctcgtcct tggcc 25
<210>24
<211>40
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>24
gatccggcgg aggcgggagc ggcgggggcg gaagcctgca 40
<210>25
<211>32
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>25
ggcttccgcc cccgccgctc ccgcctccgc cg 32
<210>26
<211>21
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>26
ttttaaaggg cccgcgcgtt g 21
<210>27
<211>21
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>27
atttggcgcg gcaagctcag c 21
<210>28
<211>24
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>28
gacttacagc agtcagactc tgac 24
<210>29
<211>24
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>29
tattgctttt tctttccagg tcag 24
<210>30
<211>30
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>30
acgaattcac aatggtgaga gaaagaggtc 30
<210>31
<211>24
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>31
acggatcctt agccaactaa aaag 24
<210>32
<211>275
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉oligonucleotide
<400>32
aaagatctct cacctaccaa acaatgcccc cctgcaaaaa ataaattcat ataaaaaaca 60
tacagataac catctgcggt gataaattat ctctggcggt gttgacataa ataccactgg 120
cggtgatact gagcacatca gcaggacgca ctgaccacca tgaaggtgac gctcttaaaa 180
attaagccct gaagaagggc agcattcaaa gcagaaggct ttggggtgtg tgatacgaaa 240
cgaagcattg gttaaaaatt aaggaggaat tcaca 275
<210>33
<211>60
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>33
aaggatccgt cgacctgcag ccaagcttgg ctgttttggc ggatgagaga agattttcag 60
<210>34
<211>18
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉peptide linker
<400>34
Asn Val Val Val His Gln Ala His His His His His His Glu Phe Thr
1 5 10 15
Tyr Lys

Claims (10)

1. a fusion rotein is characterized in that, it is by forming with the lower section:
(a) calprotectin element, this element has the aminoacid sequence of people's calprotectin;
(b) tumor necrosin relative death inducing ligand element, this element has the aminoacid sequence of apoptosis induction ligand related to human tumor necrosis factor; And
(c) 1-20 between calprotectin element and tumor necrosin relative death inducing ligand element amino acid whose connection peptides sequence.
2. fusion rotein as claimed in claim 1 is characterized in that, described calprotectin element has the aminoacid sequence of 1-417 position, 1-180 position or 119-180 position among the SEQ ID NO:2;
Described tumor necrosin relative death inducing ligand element has the aminoacid sequence of 1-281 position, 95-281 position, 114-281 position among the SEQ ID NO:4;
And described joint peptide sequence contains 4-10 amino acid.
3. fusion rotein as claimed in claim 1 is characterized in that, described fusion rotein is the aminoacid sequence shown in the SEQID NO:6.
4. an isolated DNA molecule is characterized in that, the described fusion rotein of its coding claim 1.
5. dna molecular as claimed in claim 4 is characterized in that, it is the nucleotide sequence shown in the SEQ ID NO:5.
6. a carrier is characterized in that, it contains the described dna molecular of claim 4.
7. a host cell is characterized in that, it contains the described carrier of claim 6.
8. method that produces the described fusion rotein of claim 1 is characterized in that it comprises step:
Being fit to express under the condition of described fusion rotein, cultivate the described host cell of claim 7, thereby give expression to described fusion rotein; With separate described fusion rotein.
9. a pharmaceutical composition is characterized in that, comprises pharmaceutically acceptable carrier or vehicle or thinner, and the described fusion rotein of the claim 1 of significant quantity.
10. the purposes of the described fusion rotein of claim 1 is characterized in that, is used to prepare the medicine for the treatment of tumour.
CN 200310108083 2003-10-22 2003-10-22 Calreticulin-tumor necrosis factor correlated apoptosis inducing ligand fusion protein and its prepn and use Expired - Fee Related CN1257187C (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102775491A (en) * 2011-05-09 2012-11-14 中国人民解放军第二军医大学 Preparation method and application of single-chain human apoptosis-2 ligand (Apo2L) trimer protein

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
PL391627A1 (en) * 2010-06-25 2012-01-02 Adamed Spółka Z Ograniczoną Odpowiedzialnością Anticancer fusion protein
PL219845B1 (en) 2011-01-05 2015-07-31 Adamed Spółka Z Ograniczoną Odpowiedzialnością Anticancer fusion protein
PL394618A1 (en) 2011-04-19 2012-10-22 Adamed Spólka Z Ograniczona Odpowiedzialnoscia Anticancer fusion protein
PL397167A1 (en) * 2011-11-28 2013-06-10 Adamed Spólka Z Ograniczona Odpowiedzialnoscia Anti-tumor fusion protein
CN104177500B (en) * 2013-05-24 2018-05-25 江苏靶标生物医药研究所有限公司 A kind of tumour putrescence gene related apoptosis ligand fusion protein and its preparation method and purposes

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102775491A (en) * 2011-05-09 2012-11-14 中国人民解放军第二军医大学 Preparation method and application of single-chain human apoptosis-2 ligand (Apo2L) trimer protein

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