CN102757503A - Preparation method and application of human prostate apoptosis response protein 4 and apoptin 2 ligand fusion protein - Google Patents

Abstract

The invention provides a human prostate apoptosis response 4(Par4) protein or a selective for apoptosis induction cancer cells domain (SAC) and apoptin 2 ligand (Apo2L) fused protein, namely Par4-Apo2L or SAC-Apo2L fused protein, a DNA (Deoxyribonucleic Acid) sequence for coding the protein, a vector containing the DNA sequence for coding, a host cell containing the vector, a method for preparing the protein by using the gene engineering, and a medicinal composition containing the protein. The Par4 or the SAC and the Apo2L have an effect of selectively inducing the apoptosis of cancer cells, so the fused protein can be used for preparing medicaments for treating diseases such as cancers.

Description

The method for making and the purposes of human prostate apoptotic response albumen 4 and apo 2 ligand fusion rotein

Technical field

The present invention relates to DNA recombinant technology and biotech drug field.More specifically; The present invention relates to human prostate apoptotic response albumen 4 (Par4) or its selectivity apoptosis cancer cells district (SAC), with the albumen that apo 2 ligand (Apo2L) merges, the dna sequence dna of this fusion rotein of encoding; The carrier that contains this dna sequence dna; The host cell that contains this carrier, with the method for this fusion rotein of genetically engineered preparation, and this fusion rotein is like the application in the disease treatments such as tumour.

Background technology

Par4 be 1994 by human differential hybrid methods such as Sells; The apoptosis induction gene that from the prostate cancer cell of apoptosis, is separated to first; So called after: prostate gland apoptotic response albumen 4 (Prostate apoptosis response 4 protein; Par4) [Cell Growth Differ, 1994; 5 (4): 457-466.].The Par4 gene is positioned on the human chromosomal 12q21.2, is made up of 7 exons and 6 introns, and code sequence is shown 1023 Nucleotide (SEQ ID NO.1), and the Par4 protein of coding is formed (SEQ ID NO.2) by 340aa.In the world Par4 is unified called after at present: PAWR (PRKC, apoptosis, WT1, regulator), the ID in each mcroorganism DB is: HGNC:8614; Entrez Gene:5074; Ensembl:ENSG00000177425; UniProtKB:Q96IZ0., the protein deletion mutantion finds in analyzing, 59 amino acid that the 145-203aa of people Par4 forms, and the core active region for its performance apoptosis effect has promptly constituted the apoptosis-induced minimum working energy gap of people Par4.This nucleus is that Par4 gets into nucleus, FasL/Fas is positioned apoptosis-induced necessity and sufficient conditions such as cytolemma, the short apoptosis pathway of activation Fas, inhibition NF-kB activity.This zone not only can be positioned the nucleus of tumour cell, also can be positioned normally to reach the nucleus of immortality cell, but the apoptosis of inducing tumor cell optionally only.This explanation: the Par4 cell death inducing is realized by this nucleus.Because this nucleus is not apoptosis-induced in normal cell, but can induce various apoptosis of tumor cells, therefore be called: selectivity apoptosis cancer cells district (selective for apoptosis induction cancer cells domain, SAC).Par4 and SAC also can be in the extracellulars, effect [Cell, 2009 of performance selective induction cancer cell-apoptosis; 138 (2): 377-388.]

Nineteen ninety-five; External report is expressed the sequence label library from the people, and (expressed sequence tag screens a kind of gene of the anti-tumor protein matter of encoding in EST), and the protein of this genes encoding is called TRAIL (tumor necrosis factor-related apoptosis-inducing ligand; TRAIL); Claim again apo 2 ligand (apoptotin-2 ligand, Apo2L), it is through starting cell inherent apoptosis program; Induced tumor and transformant apoptosis effectively, and normal cell is not had influence.In the world Apo2L is unified called after at present: tumour necrosis factor (part) superfamily member 10 [tumor necrosis factor (ligand) superfamily, member 10], the ID in each mcroorganism DB is: HGNC:11925; Entrez Gene:8743; Ensembl:ENSG00000121858; UniProtKB:P50591.The code sequence of people Apo2L gene is shown 846 Nucleotide (SEQ ID NO.3), and the Apo2L protein of coding is formed (SEQ ID NO.4) by 281 amino acid.Proved already that Apo2L was typical II type transmembrane protein, its N-holds the extracellular region of the 95th or 114 beginning can form free soluble necrohormone 2 ligand albumen, has the effect of special startup tumor death equally.

Since Par4 and Apo2L to the induced tumor apoptotic effect in different links, with suitable amino acid connecting arm [Protein Eng, 2003; 15 (11): 871-879] make both amalgamation and expressions, the recombinant fusion protein that is produced thus both can suppress tumour through Par4 selective induction apoptosis of tumor cells, can also be through the special startup tumour cell of Apo2L inherent apoptosis program and antitumor.Therefore, the resulting fused protein of the present invention can strengthen antineoplastic effect down both collaborative, also can reduce both and distinguish administration and give patient's institute's trouble caused and misery, for treatment of diseases such as antitumor provides new medicine or preparation.Therefore, this area presses for that exploitation is new to have Par4 and a bioactive medicine of Apo2L.In addition, this area also presses for the production technique that cost of development is cheap and/or step is easy.

Summary of the invention

One object of the present invention just provides a kind of new have Par4 and the bioactive medicine of Apo2L, and it is the fusion rotein of Par4 or its active fragments SAC and Apo2L.

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 provide a kind of with low cost with or the easy said Par4 of production of step or the method for SAC and Apo2L fusion rotein.

In first aspect of the present invention, a kind of fusion rotein is provided, it comprises:

(a) human prostate apoptotic response albumen 4 (Par4) element, this element has the aminoacid sequence of people Par4 or its active fragments SAC;

(b) people's apo 2 ligand (Apo2L) element, this element has the aminoacid sequence of people Apo2L or its active fragments; And

(c) 0-20 between people Par4 element and Apo2L element amino acid whose catenation sequence.

Better, described Par4 element has the aminoacid sequence of 1-340 position among the SEQ ID NO.2 or 145-203 position;

Described Apo2L element has the aminoacid sequence of 95-281 position among the SEQ ID NO.4 or 114-281 position;

And described catenation sequence is for containing 4-14 amino acid whose joint peptide;

Better, described fusion rotein has the aminoacid sequence shown in SEQ ID NO.6 or 9.

In second aspect of the present invention, a kind of isolated DNA molecule is provided, 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 SEQ ID NO.6 or 9.Better, described dna molecular comprises SEQ ID NO.5 or 7, the nucleotide sequence shown in 8.

In third aspect present invention, carrier that contains above-mentioned dna molecular and the host cell that contains above-mentioned carrier are provided.

In fourth aspect 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 said fusion rotein of expression, cultivate above-mentioned host cell, thereby give expression to described fusion rotein; With the described fusion rotein of separation and purification.

Aspect the of the present invention the 5th, 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.

Aspect the of the present invention the 6th, the purposes of fusion rotein of the present invention is provided, it is used to prepare the medicine of treating tumour.

Description of drawings

Fig. 1 has shown the different connecting methods of Par4, amino acid connecting arm and the Apo2L of different lengths.Wherein:

expression: Apo2L aminoacid sequence;

expression: Par4 aminoacid sequence; ... expression: amino acid connecting arm sequence; expression: disappearance partial amino-acid.

Fig. 2 has shown the electrophoretic analysis (SDS-PAGE) of the recombinant expressed and purge process of a kind of distinctive Par4-Apo2L fusion rotein.A wherein: the bacteria breaking supernatant of abduction delivering Par4-Apo2L after 3 hours; B: the bacteria breaking supernatant of abduction delivering Par4Apo2L not; C: the full bacterium of abduction delivering Par4-Apo2L after 2 hours; D: the full bacterium of abduction delivering Par4-Apo2L after 1 hour; E: the reorganization Par4-Apo2L fused protein elutant of metal affinity purification; F: ammonium sulfate precipitation is expressed the active ingredient of supernatant; G: the active peak of the reorganization Par4-Apo2L fused protein of metal affinity purification; The Par4-Apo2L that H:S200 is further purified; I: molecular weight protein marker (kD)

Fig. 3 has shown the electrophoretic analysis (SDS-PAGE) of the recombinant expressed and purge process of a kind of distinctive SAC-Apo2L fusion rotein.A wherein: molecular weight protein marker (kD); B: the SAC-Apo2L that is further purified; C: the active peak of metal affinity purification reorganization SAC-Apo2L fused protein; D: the elutant during the metal affinity purification; E: the broken supernatant of the SAC-Apo2L engineering bacteria that thermal induction is expressed; F: the bacteria breaking supernatant before the abduction delivering SAC-Apo2L not

Embodiment

The inventor merges Par4 encoding sequence or its active fragments SAC encoding sequence through extensive and deep research with the Apo2L encoding sequence, produce by the Par4-Apo2L of suitable amino acid connecting arm connection or the fusion rotein of SAC-Apo2L.Said fused protein has both biological functions, both can pass through optionally inducing apoptosis of tumour cell of Par4 or SAC, can pass through the special startup apoptosis of tumor cells of Apo2L program killing tumor cells again.Therefore; Resulting Par4-Apo2L of the present invention or SAC-Apo2L fused protein can be in both antineoplastic effects of collaborative enhancing down; Also can reduce both and distinguish administration and give patient's institute's trouble caused and misery, for treatment such as antitumor provides new compound.Accomplished the present invention on this basis.

Definition

As used herein; Term " prostate gland apoptotic response albumen 4 and apo 2 ligand fusion rotein " and " Par4-Apo2L fusion rotein " interchangeable use; All refer to merge the albumen that forms by the aminoacid sequence of human prostate apoptotic response albumen 4 elements and the aminoacid sequence of people's apo 2 ligand, wherein between can have or not have the connection peptides sequence.In addition, said fusion rotein can have or not have initial methionine(Met) or signal peptide.

As used herein; " prostate gland apoptotic response albumen 4 elements " and " Par4 element " interchangeable use in the term fusion rotein; The a part of aminoacid sequence of finger in said fusion rotein; This sequence has substantially the same aminoacid sequence with total length human prostate apoptotic response albumen 4 or its active fragments SAC natural or variation, and has the biological activity substantially the same with the natural prostate gland apoptotic response of people albumen 4.Preferred Par4 element is a human prostate apoptotic response albumen 4, and better is the human prostate apoptotic response albumen 4 and its selectivity apoptosis cancer cells district of total length, like the aminoacid sequence of 1-340 position and 145-203 position among the SEQ ID NO.2.

As used herein; " apo 2 ligand element " or " Apo2L element " interchangeable use in the term fusion rotein; The a part of aminoacid sequence of finger in said fusion rotein; This sequence has substantially the same aminoacid sequence with apo 2 ligand or its solubility active fragments natural or variation, and has the biological activity substantially the same with natural apo 2 ligand.Preferred Apo2L element is people's apo 2 ligand, and better is people's apo 2 ligand solubility active fragments, like the aminoacid sequence of 95-281 position or 114-281 position among the SEQ ID NO.4.

The sequence of prostate gland apoptotic response albumen 4 and apo 2 ligand can be derived from the people, also can be derived from inhuman animal, is on all four the same like the aminoacid sequence with SAC mouse, rat source the people source just.Yet, people's native sequences preferably.

As used herein; Term " connection peptides " or " amino acid connecting arm " interchangeable use refer to small peptide or amino acid between the aminoacid sequence of the aminoacid sequence of the aminoacid sequence of prostate gland apoptotic response albumen 4 elements and apo 2 ligand element and SAC element, that play ligation.The length of connection peptides is generally 0-20 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; Protein Eng, 2000; 13 (5): 309-312; Protein Eng, 2003; 15 (11): documents such as 871-879] design connection peptides.Usually, connection peptides does not influence or the aminoacid sequence of not serious aminoacid sequence that influences prostate gland apoptotic response albumen 4 elements and apo 2 ligand element forms correct folding and space conformation.

Preferred connection peptides example comprises (but being not limited to): 1. become separate structural domain in order to help protein folding; Is suitable with sequences such as GGGGSGGGGS as connecting arm, like 61-70 position among 341-350 position and the SEQ ID NO.9 among the SEQ ID NO.6; 2. cut into two independently protein moleculars to Par4-Apo2L in order to help proteolytic enzyme; The restriction enzyme site PLGLWA of the restriction enzyme site IEGR of the available active X factor, the metalloprotease of tomour specific high expression level connects arm, and is similar, and chymotrypsin 1; Papain; Plasmin, thrombin, the restriction enzyme site of enzymes such as trypsin also can design as the amino acid connecting arm; 3. in order to help purifying; Can be 6His as purification tag or as connecting arm, to use the metal affinity chromatography purified fusion protein, similar; Maltose binding protein (MBP), gsh-S transferring enzyme (GST) etc. also can design as the label of purifying or as connecting arm; The combination of 4. 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 like the NVVVHQAHHHHHHEFTYK connecting arm.

The dna sequence dna of code book invention fusion rotein, whole synthetic, also available pcr amplification or synthetic method obtain, even purchase (can come the Cat.No.SC110969 to Origene like the Par-4 encoding sequence from market; The Apo2L encoding sequence can come the Cat.No.RC207596 to Origene), 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 the suitable host cell again over to.At last, the host cell after the culture transformation obtains new fusion rotein of the present invention through 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 in eukaryotic cells such as eukaryotic cell such as CHO, COS series, express; The carrier that can in yeast saccharomyces cerevisiae or pichia yeast, express 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 as precursor expression and participate in the secretion of polypeptide, the DNA of coded signal peptide (secretion leader sequence) 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.

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 through 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 comprises (but being not limited to): conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, supersound process, ultra centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HPLC) is technological with other various LCs and the combination of these methods.

In another aspect of this invention, a kind of pharmaceutical composition also is provided.Pharmaceutical composition of the present invention comprises the of the present invention novel Par-Apo2L or the SAC-Apo2L albumen of significant quantity, and at least a pharmaceutically acceptable carrier, thinner or vehicle.When these compsns of preparation, usually with activeconstituents and mixed with excipients, or with the vehicle dilution, or wrap in the carrier that exists with capsule or anther sac form.Do the time spent when vehicle plays thinner, it can be solid, semisolid or the fluent material medium as vehicle, carrier or activeconstituents.Therefore, compsn 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, Vinylpyrrolidone polymer, Mierocrystalline cellulose, water, etc.Preparation also can comprise: wetting agent, emulsifying agent, sanitas (like methyl hydroxybenzoate and propyl ester), sweeting agent etc.

Compsn 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 through conventional route, comprising (but being not limited to): intramuscular, intraperitoneal, intravenously, subcutaneous, intracutaneous, oral 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 being no more than about 10 mg/kg body weight, this preferable dosage is that about 10 micrograms/kg body weight is to about 1 mg/kg body weight.Certainly, concrete dosage is factor such as considered route of administration, patient health situation also, 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, like IFN, TNF, IL-2 etc.; Various tumor chemotherapeutic drugs; Influence biological nucleic acid synthetic medicine like 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 the medicine of protein synthesis; The medicine that microtubules such as taxol suppress reaches various kinds of drug such as some hormone.

In sum, major advantage of the present invention is following:

(1) Par4-Apo2L, SAC-Apo2L fusion rotein had both had the function of the selective induction apoptosis of tumor cells of Par4 or SAC, had the function that the Apo2L specificity starts apoptosis of tumor cells again, were a kind of newtype drugs of treating 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 property.Par4 or SAC can transport Apo2L to tumor by local and play a role in the selective induction apoptosis of tumor cells; Same, the Apo2L specific action transports Par4 or SAC to effect that tumor tissues is brought into play its selective induction apoptosis of tumor cells when tumour cell.

(4) strengthen proteic stability.Behind both amalgamation and expressions, vitro half-lives is obviously elongated, and the stability in the aqueous solution is become one or two month (all under 4 ℃) after the fusion each about week by both; Believe the prolongation 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 are used to explain the present invention, rather than be used to limit scope of the present 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: the condition described in the laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989), or the condition of advising according to manufacturer.

Embodiment 1

Set up the cDNA library, screening obtains Par4 and Apo2L gene

1. prepare the total RNA of human peripheral blood single nucleus cell

By routine with lymphocyte separation medium separation of human peripheral blood lymphocyte; With RPMI-1640 substratum (GIBCO company); Add 10% newborn calf serum (Hangzhou SIJIQING biotech firm) and penicillium mould and Streptomycin sulphate be cultured to adherent, mononuclearcell, then with the intracellular toxin stimulation 2h of the intestinal bacteria R595 of 10 μ g/L; 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, screening Apo2L gene

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 EcoR I 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 made the multiple die on the nitrocellulose filter of 20 * 20cm.With the theoretical sequences Design random primer of Apo2L, 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% Vinylpyrrolidone polymer 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 Not I, 1% agarose gel electrophoresis, and the Southern trace is confirmed and probe hybridization.When selecting with above-mentioned probe hybridization, the maximum clone (about 1.7kp) of radioactive intensity carries out the DNA purifying.These clones' insertion portion is by the Not I site of subclone to pSPORT1 (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, according to the translation initiation site requirement of Kozak definition, 88-90 position Nucleotide (ATG; The coding methionine(Met)) be translation initiation site; So it comprises the 5 ' non-translational region of 87bp, 846bp ORFs and 3 ' non-translational region (contain polyadenylation signal or claim the polyA tail), encoding sequence wherein such as SEQ ID NO.3.Obtain coded 281 aminoacid sequences such as SEQ IDNO.4 thus.

3. screen the Par4 gene

With step 1 and 2 similar methods, from the human prostate cell cdna library, screen encoding sequence such as the SEQ ID NO.1 that obtains Par4, coded 340 aminoacid sequences such as the SEQ ID NO.2 of deduction.

Embodiment 2

Reverse-transcription polymerase chain reaction method (being RT-PCR) obtains the encoding sequence of Par4

1.RT obtain cDNA first chain of Par4

Human prostate cell total rna to buy from Clontech company (U.S.) is a template, is primer with P1, and Par4 cDNA first chain is synthesized in reversed transcriptive enzyme catalysis.Primer:

P1：5’-CCA?CAT?TGA?GTC?TTG?AAT?CC-3’(SEQ?ID?NO.11)

2. polymerase chain reaction (PCR) amplification obtains the Par4 encoding sequence

With above-mentioned cDNA first chain is template, and P1, P2 are primer, the synthetic and amplification Par4 encoding sequence of Tag archaeal dna polymerase catalysis.Through sequential analysis, the Par4 encoding sequence that is shown in gained dna sequence dna and each big DB is consistent, has promptly obtained the Par4 coding nucleotide sequence.Primer:

P2：5’-TAC?AAG?CTC?CTC?CAA?GC-3’(SEQ?ID?NO.12)

Embodiment 3

RT-PCR obtains the encoding sequence of Apo2L

1. prepare the total RNA of human peripheral blood single nucleus cell

(1) obtains mononuclearcell by ordinary method isolated lymphocytes from human peripheral, and then with adherent method;

(2) activate mononuclearcell with bacterial endotoxin and express more gene and the abundance that improves RNA, with RNA extraction agent box (Qiagen company) extracted total RNA with irritation cell.

2. rt (RT) obtains cDNA first chain of Apo2L

With above-mentioned total RNA is template, is primer with P3, and Apo2L cDNA first chain is synthesized in reversed transcriptive enzyme catalysis.Primer:

P3：5’-TCC?AGG?TCA?GTT?AGC?CAA?C-3’(SEQ?ID?NO.13)

3. polymerase chain reaction (PCR) amplification obtains the Apo2L encoding sequence

With above-mentioned cDNA first chain is template, and P3, P4 are primer, the synthetic and amplification Apo2L encoding sequence of Tag archaeal dna polymerase catalysis.Through sequential analysis, the gained dna sequence dna is consistent with the Apo2L encoding sequence that each big DB is shown, has promptly obtained the Apo2L coding nucleotide sequence.Primer:

P4：5’-CTG?ACT?TAC?AGC?AGT?CAG-3’(SEQ?ID?NO.14)

Embodiment 4

Synthetic combines with PCR and obtains the SAC encoding sequence

1. design and synthetic SAC coding correlated series

According to e. coli codon preferences and codon merger property, each codon of design coding SAC divides 4 sections to entrust biotech company's (worker is given birth in Shanghai) chemosynthesis.The characteristics of these 4 sections sequences are: P5 is the part of SAC template strand, and P6 is the part of coding strand, and the 3 ' terminal sequence of P5 and P6 is complementary, i.e. following stroke of collinear sequence among P5, the P6; 5 ' of P7 adds restriction enzyme EcoR I restriction enzyme site; Be 6 nucleotide sequences that add frame among the P7; The distance of having optimized between SD sequence and the foreign gene is 7 Nucleotide (italicized item), introduces the synthetic initiator codon ATG of albumen, and 3 ' terminal sequence is identical with the 5 ' sequence of P5; Be the sequence of following stroke wavy line among the P7, the centre is a SAC part encoding sequence; 5 ' of P8 adds another restriction enzyme BamH I restriction enzyme site, promptly adds 6 nucleotide sequences of frame among the P8, and 3 ' terminal sequence is identical with the 5 ' sequence of P6, i.e. the sequence of following stroke wavy line among the P8, and the centre is a SAC part encoding sequence.

P5：5’- GGC?CAG?ATT?GAA?AAA?CGT?AAA?CTG?CGT?GAG?AAA?CGT?CGCAGC?ACC?GGC?GTG?GTC?AAC?ATC?CCG?GCC?GCA? GAA?TGC?TTA?GAT?GAA-3’(SEQ?ID?NO.15)

P6：5’- AAT?AGT?GTT?CTG?TTG?GGT?AAT?TGC?ATC?TTC?ACG?TTT?ACGTTC?TTT?CTG?GCC?TGC?TTC?ATC?ATC?TTC?GTA? TTC?ATC?TAA?GCA?TTC-3’(SEQ?ID?NO.16)

P7：5’-AC ACA?ATG?CGT?AAA?GGC?AAA? GGC?CAG?ATT?GAA AAA?C-3’(SEQ?ID?NO.17)

P8：5’-AC

TTA?AGC?CTC? ATT?CTG?AAT?AGT?GTT?CTG?TTG?G-3’(SEQ?ID?NO.18)

2. polymerase chain reaction (PCR) amplification SAC encoding sequence

P5, P6 be mutually as primer, pcr amplification SAC part encoding sequence.Be template then with the amplified production, P7, P8 are primer, pcr amplification once more, and product is connected with T vector (TaKaRa company), dna sequence analysis, consistent with design, i.e. SEQ ID NO.19.Obtained thus forming by the intestinal bacteria preference codon, and 5 ' and 3 ' have different restriction endonuclease sites the SAC encoding sequence, the pcr template when can be used for making up pBV-SAC among the embodiment 5 and making up the SAC-Apo2L fusion protein expression plasmid.

Embodiment 5

Construction expression Expression of Fusion Protein carrier and engineering bacteria

Through reconstructed vector pBV220, make up the recombinant plasmid that efficiently expresses SAC, the prokaryotic expression carrier of constructed expression SAC, called after pBV-SAC.The recombinant expressed of corresponding fusion proteins of the present invention implemented with same method.The practical implementation step is following:

1. design and synthesize the oligonucleotide two strands

Synthetic oligonucleotide is double-stranded, and sequence (does not provide the sequence of complementary strand) as follows:

P9：5’-AA A?GAT?CTC?TCA?CCT?ACC?AAA?CAA?TGC?CCC?CCT?GCA?AAA?AATAAA?TTC?ATA?TAA?AAA?ACA?TAC?AGA?TAA?CCA?TCT?GCG?GTG?ATA?AATTAT?CTC?TGG?CGG?TGT?TGA?CAT?AAA?TAC?CAC?TGG?CGG?TGA?TAC?TGA?GCACAT?CAG?CAG?GAC?GCA?CTG?ACC?ACC?ATG?AAG?GTG?ACG?CTC?TTA?AAAATT?AAG?CCC?TGA?AGA?AGG?GCA?GCA?TTC?AAA?GCA?GAA?GGC?TTT?GGGGTG?TGT?GAT?ACG?AAA?CGA?AGC?ATT?GGT?TAA?AAA?TTA?AGG?AG G?AAT TCA?CA-3’(SEQ?ID?NO.20)

Wherein, italicized item 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 EcoR I restriction enzyme site GAATTC;

P10：5’-AA G?GAT?CCG?TCG?ACC?TGC?AGC?CAA?GCT?TGG?CTG?TTT?TGGCGG?ATG?AGA? GAA?GAT?TTT?CAG-3’(SEQ?ID?NO.21)

Wherein, 5 ' end has been introduced BamH I restriction enzyme site GGATCC, follows by Sal I, Pst I and HindIII restriction enzyme site, and 3 ' introduces Xmn I restriction enzyme site GAANNNNTTC.

2. construction of expression vector pBV-SAC

With the SAC encoding sequence that pcr amplification among the embodiment 4 obtains, cut (toolenzyme is all available from U.S. NEB company) with EcoR I and BamH I enzyme.Cut P9 with Bgl II and EcoR I enzyme, BamH I and Xmn I enzyme are cut P10.With Bgl II and Xmn I double digestion pBV220 plasmid.

Above-mentioned each endonuclease bamhi sepharose reclaims the fragment of corresponding size, connects each fragment with the T4 dna ligase, and resulting recombinant plasmid is the expression vector in selectivity apoptosis cancer cells district, called after pBV-SAC.

PBV-SAC compares pBV220 and has following characteristic: the SAC encoding sequence receives upper reaches lambda particles phage P _LP _RTandem promoter control, the ribosome binding sequence of optimization (SD sequence) inserts EcoR I restriction enzyme site and translation initiation codon ATG, optimized simultaneously and the SD sequence between distance be 7 Nucleotide etc.This plasmid and pBV220 are same, in promoter sequence, contain the cIts857 binding 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.Characteristic nucleotide sequences such as the SD sequence of upstream regulatory sequence, optimization, SAC encoding sequence and translation termination sequence are shown in SEQ ID NO.10.

The SAC encoding sequence can be a template with the natural Par4 encoding sequence that is obtained in embodiment 1 and 2; With table a kind of cited primer SEQ ID NO.23,25; PCR can obtain natural SAC encoding sequence, uses above-mentioned same method then, also can make up pBV-SAC.Though the recombinant plasmid of gained coding SAC sequence is different, the product sequence of coding is identical, i.e. 145-203 position among the SEQ ID NO.2.

3. each fragment is introduced restriction enzyme site construction of fusion protein expression vector

With above-mentioned same method, be primer (listed primer only part enumerated listed 8 kinds of fused proteins among amplification Fig. 1) with the listed sequence of table 1, with the foregoing description or commercially availablely (can come Cat.No.SC110969 to Origene like the Par-4 encoding sequence; The Apo2L encoding sequence can come the Cat.No.RC207596 to Origene) the corresponding encoded sequence of gained is template; Difference pcr amplification corresponding encoded dna fragmentation; The connecting arm encoding sequence (flexible amino acid connecting arm) that each PCR product of respective limits property endonuclease digestion and SEQ ID NO.38,39 form; The T4 dna ligase connects into the pBV220 carrier of the above-mentioned reconstruction of cutting with restriction enzyme EcoR I and Pst I enzyme, the transformed competence colibacillus cell, and screening contains the clone of recombinant plasmid; Determined dna sequence confirms, can obtain expressing the expression engineering bacteria of each fused protein.

Same, the metalloprotease restriction enzyme site encoding sequence of the codes for tumor high expression level that also can form with SEQ ID NO.40,41 makes up each Expression of Fusion Protein engineering bacteria as the amino acid connecting arm.

4. overlapping PCR construction of fusion protein expression vector

Similar, can be respectively with SEQ ID NO.42,43 and 44,45, as overlapping PCR primer; SAC that obtains with embodiment 4 simultaneously and embodiment 1,2 or commercially available Apo2L encoding sequence are template, add other corresponding primers, 1 pcr amplification; The metalloprotease restriction enzyme site that can obtain by flexible amino acid or tumour high-expression is the encoding sequence of the SAC-Apo2L fusion rotein of amino acid connecting arm, cuts with EcoR I and Pst I enzyme again, and the T4 dna ligase connects on the carrier that same enzyme cuts; The transformed competence colibacillus cell; Screening contains the clone of recombinant plasmid, and determined dna sequence confirms, can obtain the expression engineering bacteria of expressed fusion protein matter.

Similar, other expressing fusion protein engineering bacterias also can make up equally.Same, connect with other amino acid connecting arms between the fusion rotein, make up too.

Obviously, compare, between the fusion rotein through overlapping PCR structure, lacked 4 amino acid of 2 restriction enzyme digestion sites codings with above-mentioned introducing restriction enzyme digestion sites.But amino acid connecting arm sequence is identical.

The tabulation of table 1 primer nucleotides sequence

First group: the Par4 in the coded fused protein that increases holds at fusion rotein N; (p representes primer to A-D as shown in Figure 1, and P representes that with embodiment 1,2 or commercially available Par4 be template, and A representes that with embodiment 1,2 or commercially available Apo2L be template; S representes that with embodiment 4 synthetic SAC be template; Sandwich digit is represented coded amino acid whose initial or final position, and f representes forward primer, and r representes reverse primer)

Title	Sequence	Sequence number	Restriction enzyme site
				?pP1f	5’-at gaa ttc aca atg gcg acc ggt ggc tac c-3’	SEQ?ID?NO.22	EcoR?I
?pP145f	5’-gc gaa ttc aca atg agg aaa ggc aag ggg c-3’	SEQ?ID?NO.23	EcoR?I
				?pS1f	5’-gc gaa ttc aca atg cgt aaa ggc aag ggc c-3’	SEQ?ID?NO.24	EcoR?I
PP203r and pS59r are general	5’-gc?gga?tcc?agc?ttc?att?ctg?aat?ag-3’	SEQ?ID?NO.25	BamH?I
				?pP340r	5’-aa?gga?tcc?cct?ggt?cag?ctg?acc?c-3’	SEQ?ID?NO.26	BamH?I
?pA95f	5’-tc?gtc?gac?acc?tct?gag?gaa?acc?att?tc-3’	SEQ?ID?NO.27	Sal?I
				?pA114f	5’-aa?gtc?gac?gtg?aga?gaa?aga?ggt?cct?c-3’	SEQ?ID?NO.28	Sal?I
?pA281r	5’-ccg?ctg?cag?tta?gcc?aac?taa?aaa?ggc-3’	SEQ?ID?NO.29	Pst?I

Second group: the Apo2L in the coded fused protein that increases is at the N of fusion rotein end, a-d as shown in Figure 1 (be first group of primer of difference, shown in add 1 after the primer title)

The 3rd group: introduce the amino acid connecting arm

^*Corresponding f, r handle through simple sex change, renaturation, and it is double-stranded to form the oligonucleotide that has the restriction enzyme site cohesive end, are used for each fragment and introduce restriction enzyme site construction of fusion protein expression vector

A series of recombinant plasmids of expressing the Par4-Apo2L fused protein of different lengths and mode of connection have respectively been obtained thus.Special, the 8 kinds of Par4-Apo2L 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 Par4-Apo2L fused protein is shown in SEQ ID NO.5, and the aminoacid sequence of being inferred is shown in SEQ ID NO.6.

Another kind of preferred nucleotide sequence of expressing the SAC-Apo2L fused protein can be shown in SEQ IDNO.7, also can be shown in the codon optimized SEQ ID NO.8 of SAC, and the aminoacid sequence that they are inferred is consistent, shown in SEQ ID NO.9.

So far, all Nucleotide or aminoacid sequence involved in the present invention are all mentioned, see Nucleotide or aminoacid sequence table.The sequence table numbering is explained as follows:

SEQ ID NO.1:Par4 coding nucleotide sequence

SEQ ID NO.2:Par4 aminoacid sequence

SEQ ID NO.3:Apo2L coding nucleotide sequence

SEQ ID NO.4:Apo2L aminoacid sequence

SEQ ID NO.5:Par4-Apo2L coding nucleotide sequence

SEQ ID NO.6:Par4-Apo2L aminoacid sequence

SEQ ID NO.7: natural SAC-Apo2L coding nucleotide sequence

SEQ ID NO.8: synthetic SAC-Apo2L coding nucleotide sequence

SEQ ID NO.9:SAC-Apo2L aminoacid sequence

SEQ ID NO.10:pBV-SAC characteristic nucleotide sequence

SEQ ID NO.11-45: the nucleotide sequence of primer and joint

Embodiment 5

Transformed into escherichia coli is set up engineering bacteria

By a series of recombinant expression plasmid transformed into escherichia coli BL21s such as pBV-SAC [genotype: hsdS gal (λ cIts857ind1 Sam7 nin5 lacUV5-T7)] (can available from Shanghai give birth to worker bio-engineering corporation) of routine with acquisition among the embodiment 4; 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 Par4-Apo2L fused protein

Various substratum are following: 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 ₂O: 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 7.2.Add penbritin final concentration to 100 μ g/ml behind the various substratum high-temperature sterilizations.

After the first order seed overnight cultures, the ratio with 1: 20～100 is forwarded to secondary seed, and fermentor tank is gone into the secondary seed of overnight cultures by 1～5% inoculation of working volume.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%.Can obtain the wet thallus of about 30-100 grams per liter fermented liquid.

Ultrasonication fermentation thalline, centrifuging and taking supernatant, the filter membrane of 0.22 μ m excessively; With NTA Supperflow (Qiagen company) or the TALON Metal Affinity Rsins capable affinity chromatographys of metal affinity chromatography post such as (Clontech companies); 5～10mmol/L imidazoles, pH7.0 wash-out foreign protein is again with 80～100mmol/L imidazoles; PH7.0, the wash-out recombinant protein; Elutriant is crossed the CM Mierocrystalline cellulose, collects active ingredient; Be further purified after SephacrylS-200, can obtain highly purified recombination fusion protein.

The result: carrying out protokaryon recombinant expressed with 2 kinds of fusion roteins shown in Fig. 1-B, the D (being Par4-Apo2L and SAC-Apo2L) is example; Constructed engineering bacteria is expressed through thermal induction; After the ultrasonication, the capable SDS-PAGE of the centrifugal isolating supernatant of institute, visible expressed fusion protein accounts for more than 10% of supernatant total protein; It is pure to reach electrophoresis after being further purified, and the result sees Fig. 2,3.Can obtain similar results after all the other independent Par4, SAC, Apo2L or both are merged abduction delivering, obtain the recombinant protein of corresponding molecular weight size.

Embodiment 7

The BA of fused protein is confirmed

In the present embodiment, measure the active and Par4 activity of Apo2L that fusion rotein had.

The activity of Apo2L: external can human pancreas's cancerous ductal epithelial cell 1990 strain cells, the cell lung cancer NCI-H460 of the National People's Congress, murine melanoma B16 etc. be target cell; Confirm the BA of Apo2L; In the body with the human colon carcinoma HCT-8 transplanted tumor that suppresses nude mice etc. external responsive to Apo2L, to become the mice-transplanted tumor of knurl in vivo be model, the biological function of checking Apo2L.

The activity of Par4: observation in vitro Par4 can the cell lung cancer NCI-H460 of the National People's Congress, human prostata cancer PC-3, murine melanoma B16 etc. be target cell; Confirm the BA of Par4; In the body with the human colon carcinoma HCT-8 transplanted tumor that suppresses nude mice etc. external responsive to Par4, to become the mice-transplanted tumor of knurl in vivo be model, the biological function of checking Par4.

Concrete test operation is following:

1, Par4, Apo2L activation analysis: kill and wound various tumour cells

Various target cells derive from cell research institute of the Chinese Academy of Sciences or Shanghai Changhai Hospital.Mainly contain 1990,8898 strains of people's pancreas cancerous ductal epithelial cell; 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, SK-N-SH human neuroblastoma cells strain, people's laryngocarcinoma Hep-2 strain, human nasopharyngeal carcinoma CNE-2 strain, HEC CEV304 strain, human fibroblasts's strain, human colon carcinoma AT-29, HOC 3AO, mouse become fiber L929 strain, human glioma U251, people's breast cancer, people's liver cancer HepG-2, SMMU7721, human blood to learn tumour (U937, Jukart, HL60 etc.), melanin tumour b16-MB, Ehrlich ascitic tumor, Lewis sarcoma etc.

With the H460 cell is example, with culturing cell with 2 * 10 ⁸Cell/L kind is gone into 96 porocyte culture plates, 37 ℃ of 5%CO ₂Hatched in the incubator 4-6 hour, and abandoned 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/LNaCl, 200mL/L ethanol) dyeing 15 minutes, zero(ppm) water flush away Viola crystallina was 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 couplet appearance and read the A value in wavelength 595nm, be the A background with the blank well that does not add cell.Suspension cell is confirmed 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 at Par4, or Apo2L, or is the typical apoptotic form under the fusion rotein effect, as under 10 μ g/L apo 2 ligand effects, promptly observes cell generation typical change in 2 hours.

2. 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 is drawn mouse ascites down, and using PBS to regulate cell concn 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 is with computes:

Result: the result such as the table 2 of human colon cancer cell HCT-8 transplanted tumor.

Table 2HCT-8 table with test results

Visible by table 2, recombinant expressed fusion rotein has good inhibitory effect to the colorectal carcinoma transplanted tumor.

Same, to other transplanted tumors, like the cell lung cancer NCI-H460 of the National People's Congress, human pancreas cancer SW1990; 8898, 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.

All documents in that the present invention mentions are all quoted as a reference in this application, are just quoted such as a reference separately as each piece document.Should be understood that in addition after having read above-mentioned teachings of the present invention, those skilled in the art can do various changes or modification to the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Claims (10)

1. fused protein is characterized in that it is made up of following elements:

(a) human prostate apoptotic response albumen 4 (Par4) element, this element has people Par4 or its active fragments---and the aminoacid sequence in selectivity apoptosis cancer cells district (SAC) promptly has the aminoacid sequence of 1-340 position among the SEQ ID NO.2 or 145-203 position;

(b) people's apo 2 ligand (Apo2L) element, this element has the aminoacid sequence of people's apo 2 ligand or its active fragments, promptly has the aminoacid sequence of 95-281 among the SEQ ID NO.4 or 114-281 position; And

(c) 0-20 between people Par4 element and Apo2L element amino acid whose catenation sequence.

2. fusion rotein as claimed in claim 1 is characterized in that, described catenation sequence contains 4-14 amino acid.

3. fusion rotein as claimed in claim 1 is characterized in that, described fusion rotein has SEQ IDNO.6, the aminoacid sequence shown in 9.

4. the dna molecular of a synthetic is characterized in that, the described fused protein of its coding claim 1.

5. dna molecular as claimed in claim 4 is characterized in that, it has SEQ ID NO.5,7,8, the nucleotide sequence shown in 10.

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, it is characterized in that step is following: the cDNA library is set up in (1), and screening obtains Par4 and Apo2L gene; (2) RT-PCR obtains the encoding sequence of Par4 and the encoding sequence of Apo2L, or chemosynthesis has the encoding sequence of the SAC of e. coli codon preferences; (3) construction of fusion protein expression vector; (4) transformed into escherichia coli is set up engineering bacteria; (5) preparation fused protein; (6) BA of fused protein is confirmed.

9. a pharmaceutical composition is characterized in that, by pharmaceutically acceptable carrier or vehicle or thinner, and the described fusion rotein of the claim 1 of significant quantity is formed.

10. the purposes of the described fusion rotein of claim 1 is characterized in that, is used to prepare the medicine of treating tumour.

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