CN104177500B - A kind of tumour putrescence gene related apoptosis ligand fusion protein and its preparation method and purposes - Google Patents

Abstract

The invention belongs to biological technical fields, specifically disclose a kind of tnf family cytokines apoptosis protein fusion protein and its preparation method and purposes.The fusion protein is made of annexin, connection peptide, tnf family cytokines apoptosis protein, and the encoding gene of the fusion protein is built by cloning.The tnf family cytokines apoptosis protein fusion protein has the effect of the inducing cell apoptosis significantly increased, enables to induce the apoptosis of tumor cells insensitive to Apoptosis, can reduce and obtain the required protein medicine-feeding dosage of therapeutic effect.

Description

A kind of tumour putrescence gene related apoptosis ligand fusion protein and its preparation method and purposes

Technical field

The invention belongs to biological technical fields, are related to a kind of tumour putrescence gene related apoptosis ligand fusion protein and its preparation method And purposes.

Background technology

Cancer is to cause dead second largest factor in the world.The method for the treatment of cancer is mainly changed in addition to operation at present Treatment and radiotherapy, but both approaches lack specificity, have apparent cytotoxicity.Preferable antitumor drug is can selectivity Tumour cell is killed without injuring normal cell in ground.Many chemotherapeutics are to kill tumour cell by apoptosis-induced, are swollen Oncocyte also can inhibit the mechanism of apoptosis by generating to cause the failure of chemotherapy.Therefore, it is antitumor to become development for apoptosis pathway The target spot of the most attraction of drug.

Since Aggarwal et al. is separated for 1984 and identifies first tumor necrosis factor（TNF）So far, this albumen Family has 19 members.Tnf family cytokines member by with its receptor family -- Tumor Necrosis Factor Receptors family phase Interaction plays important physiological action.Tumor necrosis factor and its receptor family system are in autoimmune disease, bacterium and disease It plays a crucial role in the generations and development of a variety of diseases such as malicious infection, tumour, diabetes, osteoporosis.Therefore, most of tumour Necrosin and its receptor family member carry out extensive exploitation by the drug target as a variety of diseases.U.S.'s food and medicine management Office（FDA）5 kinds of TNF blocking agent drug list marketings of approved, be mainly used for the inflammation such as arthritis, psoriasis, Crohn disease and Treating autoimmune diseases, wherein the sales volume there are three types of drug has ranked among U.S.'s " cookle "（Annual sales amount crosses 1,000,000,000 Dollar）Ranks become one of most successful biotechnology class drug.In November, 2010 FDA ratify tnf family cytokines into The humanized antibody of one of member-RANKL（Xgeva）For preventing the relevant osteoclasia illness of metastases；In the recent period, FDA is criticized again The neutralizing antibody of accurate another tnf family cytokines member BAFF（Benlysta）For systemic loupus erythematosus（SLE） Treatment.In addition, also kinds of tumors necrosin and its receptor family member's related drugs is in different clinical test ranks Section.Tumor necrosis factor and its receptor family become the star family in drug development, it is believed that have bigger in the near future Development.In tumor necrosis factor and its receptor family member, most of tnf family cytokines member can be expressed as II Type transmembrane protein, the extracellular region three parts of intracellular region, transmembrane region, c-terminus including aminoterminal.Tnf family cytokines into Member extracellular plot structure it is similar, typical β egg rolls structure is all formed by a plurality of antiparallel β-pleated sheet lamella, with Several regions that receptor combines, amino acid sequence is than more conservative.Most of transmembrane TNF family members extracellular region can be specific Enzyme hydrolysis generate soluble protein；It is soluble similar to receptor binding capacity to film combination type TNF family members, but can be with Different signal pathways is activated, mediates different physiology and pathological effect.

Up to the present, the member of about 19 kinds tumor necrosis factor superfamilies is cloned out, has part to make Such as TNF α is used for drug, TNF β are most of also in clinical stage, such as FasL, TRAIL, TWEAK, RANKL.Wherein, greatly Partial tumors necrosin family member（For example, FasL, TNF, TRAIL etc.）All it is that its biology is played by inducing cell apoptosis Learn and its treat function.Below only by taking tumour putrescence gene related apoptosis ligand as an example, illustrate tnf family cytokines member Effect, application and its problems faced of inducing cell apoptosis.

Tumour putrescence gene related apoptosis ligand（TRAIL）It is tumor necrosis factor（TNF）The another member of superfamily, from Since being found from nineteen ninety-five, because it can induce a series of apoptosis of tumor cells without influencing normal cell and as having The antineoplastic biological medicament of important application prospect（Walczak.H etc., 1999, Nat.Med5:157-163）.TRAIL is molecular weight For the II type transmembrane protein of 30KD, the soluble protein that molecular weight is 20KD can be processed by cysteine proteinase （S.M.Mariani etc., 1998, Eur.J.Immonol28:973-982）.The N-terminal region of TRAIL be it is not conservative, but C-terminal with Other members of the family have the conservative of height.TRAIL is albumen of the family uniquely containing cysteine Cys230, should Cys230 enables three TRAIL molecules to interact.With the zinc ion that the Cys is combined to maintain Trimeric structures, it is soluble with And normal biological function plays an important role（Mongkolsapaya J etc., Nat Struct Biol6:1048- 1053）.So-called TRAIL refers to soluble tumor necrosin relative death inducing ligand in the present invention, typically refer to but Tumor necrosin relative death inducing ligand 114-281 amino acid sequences are not limited to, which includes by tumor necrosis factor The apoptosis induction ligand related derivative of son（Mutein truncated, lengthen, mutation）.The member of TNF superfamilies The expression of FasL and TNF α be strictly control and only transient expression in some cells activated；But TRAIL is a variety of Normal structure includes lymphocyte, spleen, thymus gland, prostate, constitutive expression in the tissues such as ovary and intestines（In brain and liver It does not express）（Wiley, S.R. etc., 1995, Immunity3:673-682）.In vivo, when being administered systemically, FasL and TNF α remove Can induce can also cause inflammatory reaction and hepatotoxicity wind agitation outside apoptosis of tumor cells, but TRAIL will not（Ashkenazi, A. etc., 1998, Science281：1305-1308）.Therefore, TRAIL is a kind of promising antitumor drug.

Apoptosis is mainly caused by two accesses, i.e. extracellular signal access and intracellular signaling pathways, but this two accesses are final Activate identical apoptosis effect cysteine proteinase（Caspase）With apoptosis effect molecule.Intracellular apoptotic signal access be by Caused by serious DNA damage, anoxic and other pressure（Such as chemotherapy and radiation）.Intracellular signaling pathways be largely by The apoptosis of Bcl-2 protein families interacts what is mediated and regulate and control with anti-apoptotic member（Wei, M.C. etc., 2001, Science292:727–730）.Extracellular apoptotic signal access is caused by TNF superfamilies ligand binding to corresponding receptor 's.TRAIL is mainly by extracellular access come apoptosis-induced（Scaffidi C. etc., 1998, EMBO J.17：1675– 1687）.Other members are different from TNF superfamilies, and TRAIL can be combined with a series of receptors, the affinity combined with not isoacceptor Difference, caused result are also different.TRAIL-R1（DR4）And TRAIL-R2（DR5）It is apoptosis receptor, TRAIL can be induced Apoptotic signal passes into the cell（LeBlanc, H.N. etc., 2004, Cell Death Differ10：66–75）.TRAIL-R3 （DcR1）And TRAIL-R4（DcR2）False receptor, can antagonism TRAIL apoptotic signal.TRAIL-R3 there is no intracellular region and The death domain of TRAIL-R4 cut, therefore they cannot all conduct apoptotic signal.In addition, TRAIL can also be with OPG With reference to, but affinity is relatively low.

As other TNF superfamilies ligand members, tumour putrescence gene related apoptosis ligand it is apoptosis-induced first Step is the TRAIL and TRAIL-R1 of tripolymer and the combination of TRAIL-R2.The interaction of TRAIL and its receptor promote receptor into Cluster simultaneously recruits adaptor protein FADD, FADD and further recruits Casapase8 precursors by death effector region, and then is formed dead Signal induces compound（DISC）（Kischkel FC etc., 2000, Immunity12:611–20）.Casapase8 precursors are recruited It to after DISC, is activated by conformation change and self splicing, the Caspase8 after activation is further sheared and activating effect Caspase3,6,7 is so as to performing Apoptosis.According to whether cell can be divided into two by intracellular signaling pathways to perform apoptosis Type i.e. I type and II type.In I type cell, DISC activates Caspase8 and passes through direct activation Caspase3 completely, and 6,7 It is and apoptosis-induced（Gonzalvez F. etc., 2010, Oncogene29:4752–4765）.In II type cell, DISC activation Caspase8 is not enough to apoptosis-induced thus needs intracellular signaling pathways to amplify apoptotic signal.Intracellular signaling pathways be The Bid of Caspase8 mediations cuts into what is activated after activity form tBid.TBid rapid displacements are interior to mitochondria and activate Bax And Bak, so as to cause the change of mitochondrial membrane potential.So as to which antiapoptotic factors such as cromoci and Smac/DIABLO are discharged into In cytoplasm.Cromoci is combined with adaptor protein Apaf-1 and is recruited Caspase-9 into apoptosis compound.Activation Caspase-9 further activates Caspase-3, so as to cause complete apoptosis.

It is a kind of although many reports confirm that tumour putrescence gene related apoptosis ligand can induce kinds of tumor cells apoptosis Promising antitumor drug, but many primary tumours have resistant function to the apoptosis that TRAIL is induced（Dyer,M.J. Deng 2007, J.Clin.Oncol.25:4505-4506）.This resistance mechanisms be happened at TRAIL induction apoptosis pathway in from Receptor is to multiple levels of effector molecule.It is tied in acceptor levels, DR4 or DR5 important areas such as death receptor region and with TRAIL The apoptosis of TRAIL inductions can be inhibited by closing the mutation in region（MCDONALD ER3RD etc., 2001, J Biol Chem276:14939– 14945）.DcR1 and DR4 and DR5 competitive bindings TRAIL and inhibit the formation of DISC, and DcR2 is enrolled into together with DR5 In DISC and inhibit originate caspase activation（Merino D. etc., 2006, Mol Cell Biol26:7046–7055）. TRAIL DISC are horizontal, and cFLIP and Caspase8 competitive bindings FADD is so as to inhibiting the activation of caspase（Kataoka T. etc., 2005, Crit.Rev.Immunol.25:31–58）.The high expression of XIAP can directly inhibit Caspase3,7,9（Deveraux, Q.L etc., 1997, Nature388：300–304）.The anti-apoptotic proteins of Bcl-2 families such as Bcl-2, Bcl-X L and Mcl-1 can Inhibit the change of mitochondrial membrane potential and inhibit apoptosis（Tait S.W. etc., 2010, Nat.Rev.Mol.Cell Biol11: 621-632）.In addition, the activation of other survival signaling accesses including AKT and NF- κ B accesses also result in it is apoptosis-induced to TRAIL Inhibition（LoPiccolo J etc., 2008, Drug Resist Updat11:32–50）.Tumour cell is to the repellence of TRAIL The use of TRAIL is limited, therefore understands tumour cell to contribute to based on TRAIL's the intracellular molecular mechanism that TRAIL is resisted The development of anti-tumor method.

At present, it is side of the more common increase tumour cell to TRAIL sensibility that chemotherapy and radiation is used in combination with TRAIL Method, but radiation and chemotherapy also has a certain impact to normal cell.In addition, the unstable easy denaturation of TRAIL, and half-life period Short, these are applied to all have an impact.Therefore, how to be increased using the method for biology thin as the disease of representative using tumour cell Born of the same parents or pathological tissue tumour cell are to the sensibility of TRAILThis has great importance for the application of TRAIL.For with thin For born of the same parents' apoptosis activity is the tnf family cytokines ligand member of main biological function, inducing cell apoptosis is them Principal biological function and pharmacy value, over the course for the treatment of treated target cell for the resistance of Apoptosis for tumour The application of necrosin family apoptosis protein has great importance.

Annexin（Annexins）It is the calcium dependent cardiolipin binding protein superfamily of a major class, guarantor is respectively provided in structure Keep C- ends central domain and its N- terminal domains.Being defined as the standard of annexin is, can there are items in calcium ion The electronegative phosphatide of cell surface is reversibly incorporated under part；And the repetitive sequence with about 70 amino acid must be included, This repetitive sequence is folded referred to as annexin.Annexin gene superfamilies have unique, conservative four homologous heavy Complex sequences, the structure have calcium and phosphatide binding ability.From sequence alignment and evolution, annexin 1,2,3,9,10,3 ' 6 Very high homology and similar, annexin 4,5（V）, 8,5 ' 6 very high homologies and similar.But annexin I（Annexin 1）'s Space structure domain annexin V（Annexin5）It is almost overlapped, shows annexin in structure and function（With cell table The electronegative phosphatide in face combines）On high similarity.Annexin V（Annexin V）It is annexin（Annexin）Family One of member, can be with phosphatidylserine under the conditions of existing for calcium ion（PS）With reference to.Its expression is wide, particularly exists Bone, heart, smooth muscle, endothelial cell, cartilage cell and neuron.Annexin V takes part in the extracellular process of a variety of intracellulars, Including blood coagulation, signal transduction, anti-inflammatory response, film transport and ion channel activity（Rahman, M.M. etc., 1997, Anat.Embryol195:31-39）.In addition, nearest research also found, annexin V can close tumour cell autocrine Microcapsule bubble containing EGFR is so as to inhibiting tumor vascular new life（Khalid Al-Nedawi etc., 2009, Proc Natl Acad Sci106:3794–3799）.

The content of the invention

It is an object of the present invention to provide it is based on a kind of apoptosis protein by tnf family cytokines, overcome The albumen that target cell or treatment patient resist for the Apoptosis that tnf family cytokines member induces, it is film connection egg In vain（Annexin）Or with phosphatide combine activity derivatives of Annexin and tnf family cytokines apoptosis protein or The fusion protein that tnf family cytokines apoptosis protein derivative with activity inducing apoptosis is formed.

It is a further object to provide one kind with tumour putrescence gene related apoptosis ligand（TRAIL）Based on , overcome with target cell（For example, some tumour cells）Or patient（For example, tumor patient）To TRAIL resist albumen, it It is annexin V（Annexin V）Or have the annexin V derivative of phosphatide combination activity is related to tumor necrosis factor to wither Die ligand（TRAIL）Or the fusion that the tumour putrescence gene related apoptosis ligand derivative with activity inducing apoptosis is formed Albumen.

DNA it is a further object of the present invention to provide encoding said fusion protein, the carrier containing the DNA sequence dna and containing this The host cell of carrier.

It is a further object of the present invention to provide a kind of of low cost, methods for producing to significant effect the fusion protein.

It is thin that another object of the present invention is to provide induction of the fusion protein in preparation including antitumor drug Application in born of the same parents' apoptosis drug.

In the first aspect of the present invention, a kind of tumour putrescence gene related apoptosis ligand fusion protein, feature are provided It is its amino acid sequence with annexin or the derivatives of Annexin sequence with phosphatide combination activity, neoplasm necrosis The amino acid sequence of factor family apoptosis protein or the tnf family cytokines cell with activity inducing apoptosis Apoptotic proteins derivative sequence and positioned at annexin amino acid sequence and tnf family cytokines apoptosis protein ammonia Connection peptide sequence between base acid sequence is to combine activity by gene engineering method structure by annexin or with phosphatide Derivatives of Annexin, connection peptide, tnf family cytokines apoptosis protein or swollen with activity inducing apoptosis The tumour putrescence gene related apoptosis ligand variant of three part compositions of tumor necrosis factor family apoptosis protein derivative melts Hop protein matter, i.e., using conventional gene engineering method by it is artificial synthesized or clone construction of fusion protein encoding gene, can It recombinantly expresses dissolubility and easily isolates and purifies.

The annexin refers to combine the annexin family member of activity with phosphatide or combine with phosphatide to live The derivatives of Annexin of property, the tnf family cytokines apoptosis protein refer to have in tnf family cytokines There are the member protein matter of activity inducing apoptosis or the tnf family cytokines cell with activity inducing apoptosis to wither Die protein derivatives.The connection peptide sequence, which is one section, has flexible structure, the small peptide without branched-chain amino acid, wherein, Small peptide length is but is not limited to that 1-30 amino acid residue is mainly free of branch by glycine, alanine, serine etc. Amino acid forms.The derivative refers to that there is phosphatide to combine activity, and annexin truncated, lengthen, mutation is dashed forward Variant has activity inducing apoptosis, tnf family cytokines Apoptosis egg truncated, lengthen, mutation White mutant.

It is above-mentioned by annexin or thin with the active derivatives of Annexin of phosphatide combination and tnf family cytokines Born of the same parents' apoptotic proteins or tnf family cytokines apoptosis protein derivative with activity inducing apoptosis are formed Tumour putrescence gene related apoptosis ligand fusion protein can be by annexin or with the active annexin of phosphatide combination Derivative is placed in tnf family cytokines apoptosis protein or the tumor necrosis factor Zijia with activity inducing apoptosis The N-terminal of race's apoptosis protein derivative, one section of centre addition contain but are not limited to the company of 1-30 amino acid residue composition Connect peptide；It is thin tnf family cytokines can also to be placed in by annexin or with the active derivatives of Annexin of phosphatide combination Born of the same parents' apoptotic proteins or with activity inducing apoptosis tnf family cytokines apoptosis protein derivative C-terminal, in Between one section of addition contain but be not limited to the connection peptide that 1-30 amino acid residue forms.

The preferred fusion protein includes such as SEQ ID NO：6 or SEQ ID NO：Amino acid sequence shown in 1.

In the second aspect of the present invention, a kind of tumour putrescence gene related apoptosis ligand fusion protein is additionally provided, it is special Sign is the amino acid of its amino acid sequence with annexin V or the annexin V derivative with phosphatide combination activity Sequence, the amino acid sequence of tumour putrescence gene related apoptosis ligand or the tumor necrosis factor with activity inducing apoptosis The amino acid sequence of related apoptosis ligand derivative and positioned at annexin V amino acid sequence or with phosphatide combine live Property annexin V derivative amino acid sequence and tumour putrescence gene related apoptosis ligand amino acid sequence or with induction Connection peptide sequence between the amino acid sequence of the tumour putrescence gene related apoptosis ligand derivative of anti-apoptotic activity, is logical Cross annexin V derivative, connection peptide, tumour that gene engineering method structure combines activity by annexin V or with phosphatide Putrescence gene related apoptosis ligand or the tumour putrescence gene related apoptosis ligand derivative three with activity inducing apoptosis The tumour putrescence gene related apoptosis ligand variant fused protein of a part composition, i.e., passed through using conventional gene engineering method Artificial synthesized or clone's construction of fusion protein encoding gene soluble recombinantly expressed and easily isolated and purified.

The connection peptide sequence, which is one section, has flexible structure, the small peptide without branched-chain amino acid, wherein, small peptide is long It spends and is but is not limited to, 1-30 amino acid residue, mainly by the amino acid without branch such as glycine, alanine, serine Composition.

The preferred fusion protein includes such as SEQ ID NO：Amino acid sequence shown in 1-5.

The above-mentioned tumor necrosin relative death being made of annexin V and tumour putrescence gene related apoptosis ligand Ligand fusion protein can be placed in neoplasm necrosis by annexin V or with the active annexin V derivative of phosphatide combination Factor related apoptosis ligand or with activity inducing apoptosis tumour putrescence gene related apoptosis ligand derivative N-terminal, One section of centre addition contains but is not limited to the connection peptide of 1-30 amino acid residue composition；It can also be by annexin V or tool There is the annexin V derivative that phosphatide combines activity to be placed in tumour putrescence gene related apoptosis ligand or with inducing cell apoptosis The C-terminal of the tumour putrescence gene related apoptosis ligand derivative of activity, one section of centre addition contain but are not limited to 1-30 ammonia The connection peptide of base acid residue composition.

In the third aspect of the present invention, a kind of separated DNA molecular is provided, it encodes the above-mentioned fusion egg of the present invention In vain, preferably, the DNA molecular coding includes SEQ ID NO：The fusion protein of amino acid sequence shown in 1-6.More , the DNA molecular includes SEQ ID NO：Nucleotide sequence shown in 7-12.

In the fourth aspect of the present invention, the carrier containing above-mentioned DNA molecular is provided；Additionally provide the place containing above-mentioned carrier Chief cell.The host cell can select common engineering bacteria in genetic engineering, preferably Escherichia coli BL21.

In the fifth aspect of the present invention, a kind of method for generating fusion protein of the present invention is provided, it includes the following steps：

Under conditions of the expression fusion protein is suitble to, the host cell described in claim 7 is cultivated, so as to give expression to The fusion protein；The fusion protein with separation.

Specifically, the present invention provides one kind in Escherichia coli solubility expression, can easily be divided on a large scale Method from purifying, high-purity tumour putrescence gene related apoptosis ligand fusion protein.Tumor necrosis factor is related at present Apoptosis ligand is in the inclusion body products that expression in escherichia coli is mostly inactive, and the tumor necrosis factor phase in the present invention It is more increasingly complex than wild type tumor putrescence gene related apoptosis ligand to close molecular structure and the molecular weight of apoptosis ligand fusion protein, Therefore in expression in escherichia coli, inclusion body is formed will be increasingly complex by even more serious, purifying.Present invention employs low temperature Lower culture and the expression of induced expression tumour putrescence gene related apoptosis ligand fusion protein so that expression product is effectively kept away The formation of inclusion body is exempted from；The present invention make use of life of the tumour putrescence gene related apoptosis ligand with chelating metal ion simultaneously Object function is combined using ion-exchange chromatography and metal affinity chromatography so that tumour putrescence gene related apoptosis ligand merges egg White effective purifying, the protein for obtaining high-purity, and the purified product of having obtained has a high proportion of dimer form, Therefore with extraordinary bioactivity.Wherein low temperature refers to 10 DEG C -35 DEG C.Certainly, the cell of higher organism is utilized（It is yeast, true Bacterium, insect cell, mammalian cell etc.）Soluble fused protein product can be preferably generated than Escherichia coli.

After annexin V is merged with tumour putrescence gene related apoptosis ligand, tumor necrosis factor phase can be significantly improved The ratio of apoptosis ligand fusion protein polymer is closed, since the activity of tumour putrescence gene related apoptosis ligand depends on polymer （Especially tripolymer）Formation, therefore tumour putrescence gene related apoptosis ligand fusion protein inducing cell apoptosis is active To significantly improve.

The cDNA of open encoding tumor necrosis factor related apoptosis ligand fusion protein simultaneously, it can pass through neoplasm necrosis It the cDNA of factor related apoptosis ligand and annexin V cDNA sequence and is connected the DNA sequence dna of peptide and prepares.Above-mentioned composition The cDNA of tumour putrescence gene related apoptosis ligand fusion protein can be used for gene therapy.

The tumour putrescence gene related apoptosis ligand fusion protein of the present invention can be by using polyethylene glycol, aliphatic acid Change, recombinate and modified plus the methods of antibody Fc fragment or albumin, so as to extend tumour putrescence gene related apoptosis ligand fusion The half-life period of albumen reaches better pharmacokinetics effect.

In the sixth aspect of the present invention, fusion protein as described above answering in anti-tumor medicine is prepared is provided With.The application including the fusion protein be individually used for preparing anti-tumor medicine or with existing anti-tumor medicine group Into composition or with existing chemotherapy, radiotherapy, Chinese medicine treatment, biological therapy the methods of in oncotherapy conjunctive use.

In the seventh aspect of the present invention, a kind of pharmaceutical composition is provided, including pharmaceutically acceptable carrier or figuration Agent or diluent and a effective amount of fusion protein described in claim 1.

In the eighth aspect of the present invention, due to tumor necrosis factor and its receptor family evolve, protein structure and its The very high homology of the mode of action, biological function etc. and similar, therefore, technical solution disclosed by the invention is not only limited to Spread out in tumour putrescence gene related apoptosis ligand or the tumour putrescence gene related apoptosis ligand with activity inducing apoptosis Biology is also applied for the other members of tnf family cytokines or the tnf family cytokines with activity inducing apoptosis Apoptosis protein derivative.

In the ninth aspect of the present invention, due to annexin family evolve, protein structure and its mode of action, biology Learn the very high homology of function etc. and similar, therefore, technical solution disclosed by the invention be not limited solely to annexin V or The annexin V derivative of activity is combined with phosphatide, be also applied for the other members of annexin family or is combined with phosphatide The derivatives of Annexin of activity.

Compared with existing tumour putrescence gene related apoptosis ligand and its variant, the present invention has following beneficial effect Fruit：

（1）Preferably form the ability of aggressiveness：With wild type tumor putrescence gene related apoptosis ligand individually or and film Connection albumen V compares when being mixed, and tumour putrescence gene related apoptosis ligand fusion protein exists with higher polymer Form, for example, the trimeric form of tumour putrescence gene related apoptosis ligand fusion protein TP8 is than wild type tumor necrosin Related apoptosis ligand is 11 times high.Since the apoptosis-induced first step of tumour putrescence gene related apoptosis ligand is tripolymer TRAIL is combined with its receptor, and therefore, trimeric form is then necessarily so that tumour putrescence gene related apoptosis ligand fusion protein tool There is the bioactivity of higher inducing cell apoptosis.

（2）The effect of highly efficient inducing apoptosis of tumour cell：Tumour putrescence gene related apoptosis ligand fusion protein Show the effect of inducing apoptosis of tumour cell more higher than tumour putrescence gene related apoptosis ligand.Tumor necrosis factor is related Apoptosis ligand fusion protein can significantly induce TRAIL sensitivities, medium sensitivity even insensitive TRAIL tumour cell to wither It dies, and for Colo-205 and MDA-MB-231 tumour cells, when being measured EC50 experiments, tumor necrosis factor correlation is withered Die ligand need only to function cells 4 it is small when rather than as needed during other tumour cells of conventional determining effect 24 it is small when, thus Visual tumors putrescence gene related apoptosis ligand fusion protein has the ability of the inducing apoptosis of tumour cell strongly enhanced.

In addition to inducing apoptosis of tumour cell, tumour putrescence gene related apoptosis ligand fusion protein also has long-term work With effect, it can significantly inhibit the Clone formation of tumour cell, therefore with the ability for inhibiting tumour growth.

（3）Reliable selectively acting：Although tumour putrescence gene related apoptosis ligand fusion protein induces tumour cell The ability of apoptosis significantly increases, but tumour putrescence gene related apoptosis ligand fusion protein to tumour cell specificity not Change, normal cell system（Such as LO2 and 293T）And it is not responding to the thin of tumour putrescence gene related apoptosis ligand fusion protein induction Born of the same parents' apoptosis, i other words compared with TRAIL, tumour putrescence gene related apoptosis ligand fusion protein will not bring additional poison secondary Effect.The mode of tumour putrescence gene related apoptosis ligand fusion protein inducing apoptosis of tumour cell is identical with TRAIL, Dependent on Caspase-8.TRAIL, annexin V and tumour putrescence gene related apoptosis ligand mixture（Annexin V+ TRAIL）And the receptor DR4/DR5 of tumour putrescence gene related apoptosis ligand fusion protein and tumour cell A549 cell surfaces Binding ability there is no difference.The signaling molecule in downstream（Such as DR4, DR5, DcR2 and with the relevant albumen of the mitochondrial pathways such as Bcl-XL, Bax, Bim, Noxa, Puma）Expression after being handled through different pharmaceutical also indifference.But Caspase-8, Caspase-3, PARP are only just substantially being activated after the processing of tumour putrescence gene related apoptosis ligand fusion protein.And And Caspase-8 inhibitor Z-IETD-FMK can obviously reduce the induction of tumour putrescence gene related apoptosis ligand fusion protein Apoptosis, and what Caspase-9 inhibitor Z-LEHK-FMK induced tumour putrescence gene related apoptosis ligand fusion protein Apoptosis has no significant effect.This absolutely proves that tumour putrescence gene related apoptosis ligand fusion protein has just the same with TRAIL Mechanism of action, security is identical with TRAIL.

（4）Highly efficient antitumous effect：Since the tumour putrescence gene related apoptosis ligand of invention merges The cell that albumen has sensitive, medium sensitivity the even insensitive TRAIL tumour cells of the induction TRAIL dramatically increased withers The ability died, with wild type TRAIL, the integrinα Vβ 3 for targeting tumor cell surface, α V β 5 TRAIL variants RGD-L- TRAIL（Chinese invention patent application number：200710133862.1）, target tumor cell surface CD13 TRAIL variants NGR-L-TRAIL（Chinese invention patent application number：200710133862.1）Compare, no matter be used alone or with it is existing Chemotherapy, radiotherapy, Chinese medicine treatment, biological therapy the methods of all show that there is better antitumous effect when being used in combination.

（5）Lower dosage：Since tumour putrescence gene related apoptosis ligand fusion protein is more swollen than same dose Tumor necrosis factor related apoptosis ligand has better antitumous effect, therefore when in use, it can be identical antitumor in guarantee In the case of curative effect, the dosage of tumour putrescence gene related apoptosis ligand fusion protein is substantially reduced.Tumor necrosis factor is related The reduction of apoptosis ligand fusion protein dosage will overcome tumour putrescence gene related apoptosis ligand in oncotherapy Potential side effect, while the medical expense of tumor patient can be reduced, reach efficient, less toxic side effect, inexpensive tumour is controlled The effect for the treatment of.

（6）It soluble expression and easily isolates and purifies：Although current tumour putrescence gene related apoptosis ligand is big Easily form the inclusion body products of inactive when being expressed in enterobacteria, and the tumor necrosin relative death of invention The molecular structure and molecular weight of ligand fusion protein are more increasingly complex than wild type tumor putrescence gene related apoptosis ligand, therefore according to In expression in escherichia coli fusion protein, inclusion body is formed even more serious, purifying difficulty bigger more solito condition.This hair It is bright to employ the expression of culture and induced expression tumour putrescence gene related apoptosis ligand fusion protein under low temperature so that table The formation of inclusion body is effectively prevented up to product；The present invention make use of tumour putrescence gene related apoptosis ligand to have chelating simultaneously Metal affinity chromatography is combined to ion-exchange chromatography method so that tumor necrosis factor is related by the biological function of metal ion Apoptosis ligand fusion protein has obtained effective purifying, the protein for obtaining high-purity.The present invention passes through soluble table Purified product is ultimately resulted in a high proportion of dimer form up to the optimization with simple isolation and purification method, is obtained so as to ensure that Product have extraordinary bioactivity.Produce the tumour putrescence gene related apoptosis ligand fusion egg of dimer form at high proportion In vain, this is of the invention with similar research significant difference.Obtain the present invention provides a kind of effective expression and efficiently high aggressiveness The expression and purifying process of the tumour putrescence gene related apoptosis ligand fusion protein of content.

Description of the drawings

In order that the present invention can be more clearly and readily understood, it is right below according to specific embodiment and with reference to attached drawing The present invention is described in further detail, wherein

Fig. 1 .SDS-PAGE analyze tumour putrescence gene related apoptosis ligand fusion protein TP8 purification results and aggressiveness is formed Situation.

Fig. 1 .1.Zn²⁺Ionic metal affinity chromatography is related to the tumor necrosis factor that DEAE cation exchange chromatographies obtain Apoptosis ligand fusion protein TP8：1. protein molecular weight standard, 2. tumour putrescence gene related apoptosis ligand fusion after purification Albumen TP8；

In Fig. 1 .2. non-reduced SDS-PAGE analysis TRAIL and Annexin V+TRAIL TRAIL monomers, dimer with And tripolymer formational situation：1. protein molecular weight standard, 2.Annexin V, 3.TRAIL, 4.Annexin V+TRAIL； 21.TRAIL tripolymers, 22.TRAIL dimers, 23.TRAIL monomers；

The non-reduced SDA-PAGE analysis tumour putrescence gene related apoptosis ligand fusion protein TP8 monomers of Fig. 1 .3., dimerization Body and tripolymer formational situation：31.TP8 tripolymers, 32.TP8 dimers, 33.TP8 monomers.

Fig. 2 differences cell lines are to tumour putrescence gene related apoptosis ligand fusion protein TP8 sensitivity analyses.

The tumour putrescence gene related apoptosis ligand fusion protein TP8 of Fig. 2 .1. various concentrations is to normal human archeocyte The impact analysis of 293T；

The tumour putrescence gene related apoptosis ligand fusion protein TP8 of Fig. 2 .2. various concentrations is to normal human archeocyte The impact analysis of LO2.

Fig. 3 equimolar numbers different pharmaceutical processing A549 same times after A549 cellular morphologies, Apoptosis assay and Clone formation is analyzed.

Annexin V, TRAIL, Annexin V+TRAIL and the tumour putrescence gene related apoptosis ligand of equimolar number After fusion protein TP8 processing A549 same times, using PBS as negative control：

Fig. 3 .1. observe the variation of cellular morphology under the microscope:1.PBS；2.Annexin V；3.TRAIL； 4.Annexin V+TRAIL；5.TP8；

Fig. 3 .2. transmission electron microscope observings cellular morphology after the processing of different albumen changes：1.PBS；2.Annexin V； 3.TRAIL；4.Annexin V+TRAIL；5.TP8；

A549 apoptosis situations after the processing of Fig. 3 .3. flow cytometry analysis different pharmaceutical：1.PBS；2.Annexin V； 3.TRAIL；4.Annexin V+TRAIL；5.TP8；

Influence of Fig. 3 .4. different pharmaceuticals processing to A549 Clone formations：1.PBS；2.Annexin V；3.TRAIL； 4.Annexin V+TRAIL；5.TP8.

Fig. 4 tumour putrescence gene related apoptosis ligand fusion proteins TP8 induces Non-small cell lung carcinoma cell A549 cells The dosage of apoptosis and the relationship analysis of action time.

Fig. 4 .1. flow cytometries analyze the relation of A549 apoptosis and TP8 dosage：1.PBS；2.10ng/ml TP8； 3.50ng/ml TP8；4.100ng/ml TP8；5.200ng/ml TP8；6.400ng/ml TP8；7.600ng/ml TP8； 8.800ng/ml TP8；

Fig. 4 .2.TP8 processing times and the relation of A549 Apoptosis.

Fig. 5 tumour putrescence gene related apoptosis ligands TRAIL, tumour putrescence gene related apoptosis ligand variant RGD- TRAIL, tumour putrescence gene related apoptosis ligand fusion protein TP8 and TP8 variant induction Non-small cell lung carcinoma cell The effect of A549 apoptosis compares.

Fig. 5 .1. tumour putrescence gene related apoptosis ligands TRAIL, tumour putrescence gene related apoptosis ligand variant RGD- TRAIL, tumour putrescence gene related apoptosis ligand fusion protein TP8 induce the effect of Non-small cell lung carcinoma cell A549 apoptosis Compare.

Fig. 5 .2. tumour putrescence gene related apoptosis ligand fusion protein TP8 melt with tumour putrescence gene related apoptosis ligand The effect of hop protein TP8 variants TP8-C230G induction Non-small cell lung carcinoma cell A549 apoptosis compares：1. non-administration compares Group；2-6.100,200,400,600,800ng/ml tumour putrescence gene related apoptosis ligand fusion proteins TP8（Black column） And tumour putrescence gene related apoptosis ligand fusion protein TP8 variants TP8-C230G（White column）.

Fig. 6 .TP8 induction A549 Apoptosis mechanism analyses

The situation of change of important albumen in Fig. 6 .1.Western blotting analysis apoptosis pathway：1.PBS groups； 2.Annexin V groups；3.TRAIL groups；4.Annexin V+TRAIL groups；5.TP8 groups；

The situation of change of key protein in Fig. 6 .2.RT-PCR analysis apoptosis pathways：1.PBS groups； 2.Annexin V groups；3.TRAIL groups；4.Annexin V+TRAIL groups；5.TP8 groups；

Fig. 6 .3. flow cytometry analysis Annexin V, TRAIL, Annexin V+TRAIL and TP8 and A549 cells With reference to situation.Albumen is incubated with after fluorochrome label with A549, then through flow cytometer detection；

TRAIL receptors DR4 and the DR5 expression of A549 cell surfaces after the processing of Fig. 6 .4. flow cytometry analysis different pharmaceutical Situation：1.DR5；2.DR4.

Fig. 7 .Caspase8 and Caspase3 activity analysis：

Fig. 7 .1.Caspase8 activity analysis：1.PBS groups；2.Annexin V groups；3.TRAIL groups；4.Annexin V+ TRAIL groups；5.TP8 groups；**p<0.01, TP8 group is compared with other groups；

Fig. 7 .2.Caspase3 activity analysis：1.PBS groups；2.Annexin V groups；3.TRAIL groups；4.Annexin V+ TRAIL groups；5.TP8 groups；**p<0.01, TP8 group is compared with other groups；

Fig. 7 .3.Caspase8 and Caspase3 inhibitor wither to the Non-small cell lung carcinoma cell A549 cells that TP8 is induced The impact analysis died；**p<0.01, TP8 with Caspase-8 inhibitor simultaneously processing group compared with TP8 processing groups.

Fig. 8 tumour putrescence gene related apoptosis ligand fusion protein TP8 analyze the tumor killing effect of different tumours.

Fig. 8 .1. differences albumen compares A549 tumor inhibition effects：1.PBS；2.Annexin V；3.TRAIL； 4.Annexin V+TRAIL；5.TP86.25mg/kg；6.TP812.5mg/kg；7.TP825mg/kg；* P ＜ 0.01；

Fig. 8 .2. differences albumen is to A549 tumor doubling times（I.e. gross tumor volume doubles required number of days）It is given birth to tumour High delay time（I.e. gross tumor volume grows to 1000mm³When compared with number of days needed for PBS groups）Comparison：

Fig. 8 .2.1. tumor doubling times：1.PBS；2.Annexin V；3.TRAIL；4.Annexin V+TRAIL； 5.TP86.25mg/kg；6.TP812.5mg/kg；7.TP825mg/kg；* P ＜ 0.01, TP812.5mg/kg groups and PBS groups ratio Compared with；++<0.01, TP825mg/kg group is compared with TP86.25mg/kg groups；

Fig. 8 .2.2. tumor growth delay times：1.PBS；2.Annexin V；3.TRAIL；4.Annexin V+TRAIL； 5.TP86.25mg/kg；6.TP812.5mg/kg；7.TP825mg/kg；* P ＜ 0.01, TP812.5mg/kg groups and PBS groups ratio Compared with；++<0.01, TP825mg/kg group is compared with TP86.25mg/kg groups；

Fig. 8 .3. differences albumen compares Bel7402 tumor inhibition effects：1.PBS；2.Annexin V；3.TRAIL； 4.Annexin V+TRAIL；5.TP86.25mg/kg；6.TP812.5mg/kg；7.TP825mg/kg；* P ＜ 0.01；

Comparison of Fig. 8 .4. differences albumen to Bel7402 tumor doubling times and tumor growth delay time：

Fig. 8 .4.1. tumor doubling times：1.PBS；2.Annexin V；3.TRAIL；4.Annexin V+TRAIL； 5.TP86.25mg/kg；6.TP812.5mg/kg；7.TP825mg/kg；* P ＜ 0.01, TP812.5mg/kg groups and PBS groups ratio Compared with；++<0.01, TP825mg/kg group is compared with TP86.25mg/kg groups；

Fig. 8 .4.2. tumor growth delay times：1.PBS；2.Annexin V；3.TRAIL；4.Annexin V+TRAIL； 5.TP86.25mg/kg；6.TP812.5mg/kg；7.TP825mg/kg；* P ＜ 0.01, TP812.5mg/kg groups and PBS groups ratio Compared with；++<0.01, TP825mg/kg group is compared with TP86.25mg/kg groups；

Fig. 8 .5. differences albumen compares Colo205 tumor inhibition effects：1.PBS；2.Annexin V；3.TRAIL； 4.Annexin V+TRAIL；5.TP86.25mg/kg；6.TP812.5mg/kg；7.TP825mg/kg；*p<0.05;**p< 0.01；

Comparison of Fig. 8 .6. differences albumen to Colo-205 tumor doubling times and tumor growth delay time：

Fig. 8 .6.1. tumor doubling times：1.PBS；2.Annexin V；3.TRAIL；4.Annexin V+TRAIL； 5.TP86.25mg/kg；6.TP812.5mg/kg；7.TP825mg/kg；* P ＜ 0.01, TP812.5mg/kg groups and PBS groups ratio Compared with；++<0.01, TP825mg/kg group is compared with TP86.25mg/kg groups；

Fig. 8 .6.2. tumor growth delay times：1.PBS；2.Annexin V；3.TRAIL；4.Annexin V+TRAIL； 5.TP86.25mg/kg；6.TP812.5mg/kg；7.TP825mg/kg；* P ＜ 0.01, TP812.5mg/kg groups and PBS groups ratio Compared with；++<0.01, TP825mg/kg group is compared with TP86.25mg/kg groups.

Fig. 9 tumour putrescence gene related apoptosis ligand fusion proteins TP8 treats the pathological analysis of A549 tumours.

The different protein induced tumor death situations of TUNEL methods analysis：1.PBS；2.Annexin V；3.TRAIL； 4.Annexin V+TRAIL；5.TP8.

Figure 10 tumour putrescence gene related apoptosis ligand fusion protein TP8 inhibit Non-small cell lung carcinoma cell with combining A549 tumor effects are evaluated.

1.PBS；2. cis-platinum；3.TP8；4.TP8+ cis-platinums；**p<0.01.

Specific embodiment

Embodiment 1

Design, expression, preparation and the identification of tumour putrescence gene related apoptosis ligand fusion protein

Using area of computer aided structural simulation and MOLECULE DESIGN, on SGI computer workstations, point of MSI companies is utilized Sub- design software（The modules such as InsightII, Discover）, it is brilliant in tumour putrescence gene related apoptosis ligand and annexin V On the basis of body structure, to tumour putrescence gene related apoptosis ligand and annexin V fusion protein, carry out Molecular modeling and divide Son design determines the amino acid length of connection peptide.

In the computer aided molecular design of tumour putrescence gene related apoptosis ligand fusion protein, inventor's utilization pair Tumour putrescence gene related apoptosis ligand fusion protein has carried out structural simulation and MOLECULE DESIGN, withers in tumor necrosis factor correlation On the basis of dying ligand and annexin V crystal structure, between tumour putrescence gene related apoptosis ligand and annexin V It connects peptide amino acid sequence and length carries out Molecular modeling and MOLECULE DESIGN, determine the amino acid length of connection peptide.It turns out that： Since the N-terminal and C-terminal of tumour putrescence gene related apoptosis ligand and annexin V are all exposed to the outer surface of molecule, nothing Respective space structure can be kept by which molecule of 2 molecules is placed on N-terminal, does not influence 2 molecules and respective target The combination of point or receptor.In computer MOLECULE DESIGN, inventor has found：It is adding in 1-30 amino acid short peptide length, As long as small peptide is by being free of branch, flexible larger amino acid residue, such as：The compositions such as glycine, alanine, serine, all It will not be to the tumour putrescence gene related apoptosis ligand and annexin V in tumour putrescence gene related apoptosis ligand fusion protein Structure has an impact, and will not influence 2 function；And it is calculated in molecular dynamics larger without branch, flexibility In 30 amino acid short peptide length ranges, small peptide length is longer, to tumour putrescence gene related apoptosis ligand and annexin V The disturbance that structure generates is smaller；Therefore, connection peptides more than 30 amino acid will not affecting tumor necrosis factor related apoptosis The function of ligand fusion protein.In addition, two protein are merged even if not adding connection peptide between 2 molecules, two A protein can also preferably keep respective space structure, and the combination of 2 molecules and respective target spot or receptor can also It is substantially unaffected, but the activity of fusion protein still can be subject to a degree of influence.

On the basis of MOLECULE DESIGN, it is glycine that we, which attempt to express connection peptide,（SEQ ID NO：2）, alanine- Gly-Gly-Ser-Ser-Gly-Gly-Gly（SEQ ID NO：3）Small peptide neoplasm necrosis Factor related apoptosis ligand fusion protein, the glycine repeated containing three above-（Ala-Gly-glycine-silk ammonia Acid-Serine-Glycine-Gly-Gly）-（Ala-Gly-glycine-serine-Serine-Glycine-sweet Propylhomoserin-glycine）-（Ala-Gly-glycine-serine-Serine-Glycine-Gly-Gly）25 The tumour putrescence gene related apoptosis ligand fusion protein of amino acid residue（SEQ ID NO：4）And contain glycine-sweet ammonia Acid-Gly-Gly-Serine-Glycine-Gly-Gly-Gly-Serine-Glycine-glycine-sweet ammonia The tumour putrescence gene related apoptosis ligand fusion protein of the small peptide of acid-glycine（SEQ ID NO：1）, and in each connection In the case of peptide, all attempt tumour putrescence gene related apoptosis ligand and annexin V being respectively placed in N-terminal（Such as neoplasm necrosis Factor related apoptosis ligand fusion protein PT8, SEQ ID NO：5）, similar result and activity are all obtained, so as to demonstrate The correctness of computer MOLECULE DESIGN.Certainly the expression quantity of difference fused protein has some difference, by contrast, is with the addition of The tumour putrescence gene related apoptosis ligand fusion protein of 14 amino acid connecting peptides（Annexin V- Gly-Glies-sweet Propylhomoserin-glycine-serine-Gly-Gly-Gly-glycine-serine-Gly-Gly-Gly-sweet Propylhomoserin-TRAIL, code name TP8, i.e. the 8th kind of fusion protein）（SEQ ID NO：1）Expression highest, and its expression product Soluble expression levels are best, therefore convenient for preparing and isolating and purifying on a large scale, other fused proteins are not that expression omits Solubility that is low, being exactly expression product is poor, is unfavorable for being isolated and purified on a large scale for zoopery.Therefore, this patent is focused on The 8th kind of fusion protein is presented（That is TP8）Experimental data.

This patent is also constructed by another apoptosis protein tumor necrosis factor of tnf family cytokines（TNF）It forms Tumour putrescence gene related apoptosis ligand fusion protein（Annexin V- Gly-Gly-Gly-glycine-silk ammonia Acid-Gly-Gly-Gly-glycine-serine-Gly-Gly-Gly-glycine-TNF）（SEQ ID NO：6）, also obtain the result similar to TRAIL fusion proteins.

The structure of tumour putrescence gene related apoptosis ligand fusion protein TP8 expression vectors：It is obtained from ncbi database The Annexin V in people source and the mRNA sequence of people source TRAIL soluble fractions, design corresponding PCR primer, and chemical synthesis is corresponding Primer, wherein, Annexin V sense primers：5 '-GTTCCATGGGCGCACAGGTTCT CAGAGGCA-3 ', anti-sense primer： 5’-ACCACCACCAGAACCACCACCACCAGAACCACCACCACCGTCATCTTCTCCACAGAGCA-3’；TRAIL draws upstream Object is：5 '-GGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGT GTG AGAGAA-3 ', anti-sense primer are： 5’-TCCGCTCGAGTTAGCCAACTAAAAA GGCCC CG-3’。

Using Annexin V expression plasmids and TRAIL expression plasmids as template, expand to obtain respectively with the method for PCR AnnexinV and TRAIL sequences.PCR reaction condition be：Annexin V genes：94 DEG C of thermal denaturations 1 minute, 60 DEG C are annealed 1 point Clock, 72C extend 1.5 minutes, carry out 25 Xun Huans altogether.Trail dna：94 DEG C of thermal denaturations 1 minute, 60 DEG C are annealed 1 minute, 72 DEG C Extension 1 minute carries out 25 Xun Huans altogether.Using the AnnexinV of PCR amplification and TRAIL as template, with Annexin V sense primers It is upstream and downstream primer with TRAIL anti-sense primers, PCR amplification, obtains TP8 sequences again.PCR reaction conditions are：94 DEG C of thermal denaturations 1 Minute, 65 DEG C are annealed 1 minute, and 72 DEG C extend 2 minutes, carry out 26 Xun Huans altogether.PCR product is after agarose gel electrophoresis recycles Nco I/XhoI double digestions are carried out, expression vector pET28a is also through Nco I/XhoI double digestions, using T4DNA ligases by two Double digestion recycling segment is attached, and connection product conversion Escherichia coli TOP10 bacterial strain competent cells use restriction enzyme Digestion and PCR method screening positive clones, obtain plasmid pET28a-TP8, by the correct of DNA sequence dna sequencing analysis verification sequence Property.

The expression and purification of tumour putrescence gene related apoptosis ligand fusion protein TP8:Expression plasmid pET28a-TP8 is converted Into Escherichia coli BL21 (DE3) host strain, and induced expression is carried out in host strain.By the fresh conversion in tablet Bacterium be inoculated into LB（Kanamycins）In culture medium, then 37 DEG C of overnight incubations on shaking table are transferred to fresh LB（It is mould to block that Element）When 37 DEG C of cultures 2.5 are small in culture medium or so, cultivation temperature is down to 28 DEG C of continuation when OD600nm is 0.6~0.8 or so Cultivate 0.5 it is small when after, add in final concentration of 1mM IPTG induction TP8 expression 4 it is small when.The thalline of induced expression is through 50mM After Tris, pH8.0 are resuspended, the lysozyme of final concentration of 1mg/ml, 20mM Cacl2 are added in, 65% sucrose stirs 1 at room temperature Hour.With 50mM Tris, bacterium solution is diluted 20 times and when stirring 1 is small at room temperature by pH8.0,20mM Cacl2, is centrifuged and is gone Clearly, for precipitation with after 50mM Tris, pH8.0,20mM EDTA dissociation, ultrasound makes nucleic acid break.Centrifugation recycling soluble protein, and The albumen to be got off with ammonium sulfate precipitation collection by the ammonium sulfate precipitation of 10%-50%, through phosphate buffer（50mM phosphoric acid hydrogen two Sodium, 10mM sodium dihydrogen phosphates, 300mM sodium chloride, pH7.6）Purified after dissolving with Zn-NTA affinity columns.Level pad is 50mM disodium hydrogen phosphates, 10mM sodium dihydrogen phosphates, 300mM sodium chloride, pH7.6, elution buffer are 50mM disodium hydrogen phosphates, 10mM sodium dihydrogen phosphates, 300mM sodium chloride, 60mM imidazoles, pH7.6 collect eluting peak.The protein solution being collected into is to 50mM After Tris, pH8.0 dialysis remove salt ion, it is further purified with DEAE-Sepharose anion exchange resin.Equalizing and buffering Liquid is 50mM Tris, pH8.0,10mM sodium chloride, and lavation buffer solution is 50mM Tris, pH8.0,70mM sodium chloride, and elution is slow Fliud flushing is 50mM Tris, pH8.0,220mM sodium chloride, collects eluting peak.Protein solution removes Tris, filtering to PBS dialysis Degerming, packing, is stored in -80 DEG C.

The purified TP8 albumen distribution reproducibility SDS-PAGE electrophoresis of 12% reproducibility SDS-PAGE and 8%, is used in combination Coomassie brilliant blue staining detects.Reproducibility SDS-PAGE is used to analyze the purity of albumen, and irreducibility SDS-PAGE is used to analyze Dimer and tripolymer formational situation.

In previous report, wild type tumor putrescence gene related apoptosis ligand is in expression in escherichia coli, usually Can exist in the form of the inclusion body products of inactive；The tumour putrescence gene related apoptosis ligand fusion egg of invention 318 amino acid are added again on the basis of TRAIL in vain, which should compare wild type in expression in escherichia coli Tumour putrescence gene related apoptosis ligand is more prone to be formed the inclusion body products of inactive, purifying would be more difficult.This Invention passes through Optimal Expression condition and isolates and purifies process, successfully realizes tumour putrescence gene related apoptosis ligand fusion egg It is expressed with soluble form, and is obtained by ammonium sulfate precipitation, nickel metal affinity chromatography and ion exchange resin chromatography, purifying in vain Biologically active, high-purity tumour putrescence gene related apoptosis ligand fusion protein, high purity more than 98%（Figure 1.1）.And under the expression condition and purifying process of this patent, tumour putrescence gene related apoptosis ligand TRAIL and TP8 tool There is very high aggressiveness ratio（Fig. 1 .3）, this point is in tumour putrescence gene related apoptosis ligand expression and the job family purified In be rare to see.Tnfsf protein has to form the characteristic of monomer, dimer and tripolymer, and it Biological activity dependent on dimer and trimeric form, tumour putrescence gene related apoptosis ligand is no exception.In order to Whether detection forms aggressiveness after being merged with Annexin V to tumor-targeting tumour putrescence gene related apoptosis ligand fusion protein Ability have an impact, non denatured non-reducing polyacrylamide gel electrophoretic analysis is shown：Wild type tumor necrosin is related When apoptosis ligand is independent or is mixed with annexin V, have 70.14% for monomeric form, 27.25% is dimeric forms, 2.62% is trimeric form.And the monomeric form of tumour putrescence gene related apoptosis ligand fusion protein TP8 is only 31.69%, 38.70% is dimeric forms, and trimeric form is then up to 29.61%, and trimeric form is more related than wild type tumor necrosin Apoptosis ligand is 11 times high.Annexin V enhance the dimer of fusion protein TP8 and tripolymer Forming ability, this with TP8 compared with TRAIL has stronger apoptosis-induced ability correlation.Recombinant tumor necrosis factor related apoptosis ligand fusion protein, which has, to be formed The ability of aggressiveness.Result above proves：This patent expression, the tumour putrescence gene related apoptosis ligand fusion protein of purifying are certain The TRAIL of Bacillus coli expression with correct space structure, than previously reported is with better bioactivity.

Embodiment 2

The different tumor cell lines in people source are to the sensitivity Detection of tumour putrescence gene related apoptosis ligand fusion protein TP8

By different people source tumor cell lines：It is colon cancer Colo-205, breast cancer MDA-MB-231, cervical cancer Hela cells, small Cell lung cancer H446, liver cancer PLC, non-small cell lung cancer H1229, liver cancer Bel7402, non-small cell lung cancer A549, breast cancer MCF-7, lymthoma U937, liver cancer HepG2, colon cancer HT29, breast cancer high-transfer cell MDA-MB-435, Dendritic cell TCA8113, colon cancer HCT116, cancer of pancreas SW1990, K562 cell, chronic marrow original leukemia K 562 etc. are containing 10% new born bovine The DMEM of serum（Penicillin containing 100U/ml and 100 μ g/ml streptomysins）In, and 37 DEG C are placed, it is raw in the incubator of 5%CO2 It is long.K562 and Colo205 is in the RPMI1640 containing 10% newborn bovine serum（Penicillin containing 100U/ml and 100 μ g/ml streptomysins） In, and 37 DEG C are placed, it is grown in the incubator of 5%CO2.All cells are purchased from ATCC.The cell in exponential phase is taken, It after pancreatin digestion, is inoculated in 12 orifice plates, adds in 1.5ml culture solutions, culture plate is moved into incubator and is cultivated.Treat cell Dosing when density grows to 70%, concentration 10ng/ml, 30ng/ml, 50ng/ml, 100ng/ml, 200ng/ml, 400ng/ml, 600ng/ml, 800ng/ml, three holes of each concentration repeat, when drug-treated 18 is small after, suctioned out after pancreatin digests from hole, PBS is washed twice, and 4 DEG C of 800rpm are centrifuged 5 minutes, remove supernatant, and cell is resuspended with 200 μ l combination buffers, adds in final concentration of 2 μ The EGFP-Annexin V incubations at room temperature of g/ml after twenty minutes, add in 300 μ l combination buffers and are transferred in streaming pipe, often pipe adds Enter 1 μ g Propidium iodides（PI）, flow cytometry analysis was used in 30 minutes.Since the human tumor cell line of research is large number of, Below only by taking non-small cell lung cancer A549 as an example, research tumour putrescence gene related apoptosis ligand fusion protein TP8 is briefly described In vitro, the method for in vivo antitumor activity and its mechanism of action：

Cellular morphology is analyzed：The cell in exponential phase is taken, after pancreatin digestion, is inoculated in 12 orifice plates, adds in Culture plate is moved into incubator and cultivated by 1.5ml culture solutions.With different drug-treateds when cell density grows to 70%, Drug and its concentration are as follows：Annexin V（360ng/ml）,TRAIL（180ng/ml）,Annexin V（360ng/ml）+ TRAIL（180ng/ml）And TP8（600ng/ml）, using PBS as control, processing 6 it is small when after, washed twice with PBS, micro- Microscopic observation.

Analysis of the different pharmaceutical to induction A549 apoptosis：The cell in exponential phase is taken, after pancreatin digestion, is inoculated in In 12 orifice plates, 1.5ml culture solutions are added in, culture plate is moved into incubator and is cultivated.It is used not when cell density grows to 70% Same drug-treated, drug and its concentration are as follows：Annexin V（360ng/ml）,TRAIL（180ng/ml）,Annexin V （360ng/ml）+TRAIL（180ng/ml）And TP8（600ng/ml）, using PBS as control, processing 6 it is small when after, through pancreatin It is suctioned out after digestion from hole, PBS is washed twice, and 4 DEG C of 800rpm are centrifuged 5 minutes, remove supernatant, is resuspended with 200 μ l combination buffers thin Born of the same parents, the EGFP-Annexin V incubations at room temperature for adding in final concentration of 2 μ g/ml after twenty minutes, add in 300 μ l combination buffers and turn Enter in streaming pipe, often pipe adds in 1 μ g Propidium iodides（PI）, flow cytometry analysis was used in 30 minutes.

Analysis of the drug effect dosage to induction A549 apoptosis：The cell in exponential phase is taken, after pancreatin digestion, is connect Kind adds in 1.5ml culture solutions in 12 orifice plates, and culture plate is moved into incubator and is cultivated.When cell density grows to 70% I.e. 10ng/ml, 30ng/ml, 50ng/ml are handled with the tumour putrescence gene related apoptosis ligand fusion protein TP8 of various concentration, 100ng/ml, 200ng/ml, 400ng/ml, 600ng/ml, 800ng/ml, processing 6 it is small when after, inhaled after pancreatin digests from hole Go out, PBS is washed twice, and 4 DEG C of 800rpm are centrifuged 5 minutes, remove supernatant, and cell is resuspended with 200 μ l combination buffers, adds in final concentration of The EGFP-Annexin V incubations at room temperature of 2 μ g/ml after twenty minutes, add in 300 μ l combination buffers and are transferred in streaming pipe, often manage Add in 1 μ g Propidium iodides（PI）, flow cytometry analysis was used in 30 minutes.

Analysis of the drug exposure times to induction A549 apoptosis：The cell in exponential phase is taken, after pancreatin digestion, is connect Kind adds in 1.5ml culture solutions in 12 orifice plates, and culture plate is moved into incubator and is cultivated.When cell density grows to 70% Handled respectively with 60,0ng,/ml,TP8 1 it is small when, 2 it is small when, 4 it is small when, 6 it is small when, 8 it is small when, 10 digested when small by pancreatin after from hole It suctions out, PBS is washed twice, and 4 DEG C of 800rpm are centrifuged 5 minutes, remove supernatant, and cell is resuspended with 200 μ l combination buffers, adds in final concentration It is incubated at room temperature after twenty minutes for the EGFP-Annexin V of 2 μ g/ml, adds in 300 μ l combination buffers and be simultaneously transferred in streaming pipe, often Pipe adds in 1 μ g Propidium iodides（PI）, flow cytometry analysis was used in 30 minutes.

The effect of TRAIL, RGD-TRAIL and TP8 induction A549 apoptosis compares：The cell in exponential phase is taken, It after pancreatin digestion, is inoculated in 12 orifice plates, adds in 1.5ml culture solutions, culture plate is moved into incubator and is cultivated.Treat cell With TRAIL, RGD-TRAIL and the TP8 processing of equimolar number when density grows to 70%, when processing 6 is small after, after pancreatin digests It is suctioned out from hole, PBS is washed twice, and 4 DEG C of 800rpm are centrifuged 5 minutes, remove supernatant, and cell is resuspended with 200 μ l combination buffers, adds in The EGFP-Annexin V incubations at room temperature of final concentration of 2 μ g/ml after twenty minutes, add in 300 μ l combination buffers and are transferred to streaming Guan Zhong, 1 μ g Propidium iodides of often pipe addition（PI）, flow cytometry analysis was used in 30 minutes.

The present invention compares a series of difference of tumour cells to TRAIL and TP8 sensibility in vitro.TP8 has compared with TRAIL The ability of stronger inducing apoptosis of tumour cell, even TRAIL that can induce TRAIL sensitivities, medium sensitivity are insensitive Apoptosis of tumor cells.Tumour putrescence gene related apoptosis ligand is for the EC50 of colon cancer cell Colo-205（Median effective dose） It is 1.67nM, the EC50 of tumour putrescence gene related apoptosis ligand fusion protein TP8 is then 0.61nM, and activity improves 2.74 times； Tumour putrescence gene related apoptosis ligand is 2.96nM for the EC50 of breast cancer cell MDA-MB-231, tumor necrosis factor phase The EC50 for closing apoptosis ligand fusion protein TP8 is then 0.57nM, and activity improves 5.19 times；Tumour putrescence gene related apoptosis ligand EC50 for cervical cancer cell Hela is 33.75nM, and the EC50 of tumour putrescence gene related apoptosis ligand fusion protein TP8 is then For 0.84nM, activity improves 40.18 times；Tumour putrescence gene related apoptosis ligand is for the EC50 of small cell lung cancer cell H446 It is 48.63nM, the EC50 of tumour putrescence gene related apoptosis ligand fusion protein TP8 is then 0.85nM, and activity improves 57.21 Times；Tumour putrescence gene related apoptosis ligand is 78.97nM for the EC50 of liver cancer cells PLC, and tumor necrosis factor correlation is withered The EC50 for dying ligand fusion protein TP8 is then 0.46nM, and activity improves 171.67 times；Tumour putrescence gene related apoptosis ligand pair In the EC50 of non-small cell lung cancer cell H1229 be 67.71nM, tumour putrescence gene related apoptosis ligand fusion protein TP8's EC50 is then 1.01nM, and activity improves 67.04 times；Tumour putrescence gene related apoptosis ligand is for liver cancer cells Bel7402's EC50 is 389.5nM, and the EC50 of tumour putrescence gene related apoptosis ligand fusion protein TP8 is then 1.58nM, and activity improves 246.52 again；Tumour putrescence gene related apoptosis ligand is more than 1000.0nM for the EC50 of non-small cell lung cancer cell A549 （It can not be detected under experiment condition set by the present invention）, the EC50 of tumour putrescence gene related apoptosis ligand fusion protein TP8 is then For 5.82nM, activity at least improves 171.82 times or more；Tumour putrescence gene related apoptosis ligand is for breast cancer cell MCF-7 EC50 be more than 1000.0nM（It can not be detected under experiment condition set by the present invention）, tumour putrescence gene related apoptosis ligand melts The EC50 of hop protein TP8 is then 0.57nM, and activity at least improves 1754.39 times or more；Tumour putrescence gene related apoptosis ligand EC50 for lymphoma cell U937 EC50 be more than 1000.0nM（It can not be detected under experiment condition set by the present invention）, swell The EC50 of tumor necrosis factor related apoptosis ligand fusion protein TP8 is then 2.96nM, and activity at least improves 337.84 times or more；It is swollen Tumor necrosis factor related apoptosis ligand is more than 1000.0nM for the EC50 of hepatoma Hep G 2 cells（That is the set experiment item of the present invention It can not be detected under part）, the EC50 of tumour putrescence gene related apoptosis ligand fusion protein TP8 is then 3.82nM, and activity at least carries High 261.87 times or more.

By the above results as it can be seen that tumour putrescence gene related apoptosis ligand fusion protein can induce TRAIL sensitivities, moderate Sensitive even insensitive TRAIL apoptosis of tumor cells, and for Colo-205 and MDA-MB-231 tumour cells, carry out Measure EC50 experiment when, tumour putrescence gene related apoptosis ligand need only to function cells 4 it is small when rather than as conventional determining When needing effect 24 small during other tumour cells, it can be seen that tumour putrescence gene related apoptosis ligand fusion protein has strong The ability of the inducing apoptosis of tumour cell of enhancing.Moreover, either to the extremely sensitive cells of TRAIL, TRAIL medium sensitivities are thin Born of the same parents or TRAIL resist cell, and the EC50 of tumour putrescence gene related apoptosis ligand fusion protein is no more than 6nM, this is fully Illustrate the powerful activity inducing apoptosis of tumour putrescence gene related apoptosis ligand fusion protein.

Although apoptosis-induced ability enhances, TP8 does not change the specificity of tumour, because normal cell system LO2 and 293T from TP8 influence, i other words TP8 will not bring additional toxic side effect（Fig. 2 .1,2.2）.In order to determine TP8 Induction of apoptosis of tumor cells, Annexin V, TRAIL, Annexin V+TRAIL and the TP8 processing through equimolar number A549 cells are distinguished flow cytomery apoptosis situation and are changed with microscope and transmission electron microscope observing cellular morphology.Streaming The result shows that only TP8 is induction of apparent apoptosis（Fig. 3 .3）.It is consistent with streaming result, A549 cells ability after TP8 processing There is apparent apoptosis form（Fig. 3 .1,3.2）.The A549 apoptosis of TP8 inductions has time and dose dependent（Fig. 4 .1,4.2）. In addition to inducing apoptosis of tumour cell, tumour putrescence gene related apoptosis ligand fusion protein also has long-term action effect, It can significantly inhibit the Clone formation of tumour cell A549（Fig. 3 .4）, therefore with the ability for inhibiting tumour growth.

RGD-TRAIL is the tumour putrescence gene related apoptosis ligand variant of a tumor-targeting of this seminar research and development Albumen, the activity of inducing apoptosis of tumour cell are considerably better than wild type TRAIL（Chinese invention patent number： ZL200710133862.1）.TRAIL, RGD-TRAIL, TP8 of equimolar number when inducing A549 apoptosis, RGD-TRAIL compared with TRAIL effects are good, but the effect of TP8 is best, is the 2 times or more of RGD-TRAIL.RGD-TRAIL and TP8 induction apoptosis with It the rise of concentration and increases, TRAIL does not have significant change with the rise of concentration in apoptosis-induced effect（Fig. 5 .1.）.

The tumour putrescence gene related apoptosis ligand fusion protein being made of Annexin V-TNF（SEQ ID NO：6） The activity inducing apoptosis that shows similar enhancing is surveyed in the TNF of standard on living cells L929.

Embodiment 3

The mechanism of action analysis of tumour putrescence gene related apoptosis ligand fusion protein

Western Blot are detected and the relevant important protein expression situation of apoptosis pathway from protein level：By A549 cells Be placed in 6 porocyte culture plates and grow to 80% density, as requested plus the drug of equimolar number is handled, continue culture 6 it is small when Afterwards, cell is collected, after cell precipitation washed once with the PBS of precooling, adds in appropriate cell pyrolysis liquid（Inhibit containing protease Agent, Roche companies）Suspension mixing, after placing 30 minutes on ice, 4 DEG C, 12000rpm is centrifuged 15 minutes, supernatant is drawn, using examining Mas bright blue method（Bradford methods）Protein concentration is measured, adjusts protein concentration, adds in 4 × loading buffer and in boiling water Sample is boiled in bath 5 minutes, sample is placed in -20 DEG C and saves backup.

12%SDS-PAGE gel are prepared according to the formula in molecular cloning, by the sample protein content calculation of measure, It is consistent each group sample applied sample amount, applied sample amount is 50 μ g.Concentration glue is compressed using 80 volts of constant pressures, and separation gel uses 120 volts of voltage separation, are then transferred to PVDF using semidry method by albumen in SDS-PAGE glue（polyvinylidene fluoride）On film, 10 volts of constant pressure electricity turn 2-3 it is small when.Transfer efficiency is determined according to pre-dyed Marker after transfer.Then will Film after transfer is placed on 5% skimmed milk power of Fresh（PBST is prepared）In closed, incubation at room temperature 1 it is small when.Closing knot Shu Hou illustrates the PBST solution containing 5% skimmed milk power is used to dilute primary antibody according to antibody（primary antibody）To optimal work Make concentration, film is coated in primary antibody and is placed in 4 DEG C of effects overnight.Then film is placed in PBST on shaking table and gently sways washing 3 It is secondary, it is 5-10 minutes each.Coating secondary antibody after the completion of washing, by HRP（Horseradish peroxidase）The secondary antibody of mark is used containing 5% degreasing The PBST solution of milk powder presses 1：2000 dilution, be incubated jointly with film 1 it is small when, equally with PBST wash 3 times, it is 5-10 minutes each. Finally, luminous substrate solution is prepared according to the description of product（ECL chemical luminous substrates）It is added dropwise on pvdf membrane, being placed at room temperature for 1 point Clock is blotted extra luminescent solution with filter paper, and piece pressing clip is put into after film is coated with preservative film and moves into dark place, X-ray is taken to be placed in film It after upper exposure 1-5 minutes, develops photographic film, the expression of results of albumen is judged according to exposure band.

RT-PCR is detected and the relevant important protein expression situation of apoptosis pathway from mRNA level in-site：

（1）Cell mRNA is extracted：All items are handled and sterilized with DEPC in advance（Or the RNase free productions of purchase Product）.Six porocyte culture plates are sub-packed in after cell dissociation is counted, put cell incubator culture, treat cell growth to 80% density Afterwards, dosing is handled, continue culture 6 it is small when after, PBS washed once.1ml Trizol are added in, are blown and beaten with liquid-transfering gun to cell It is completely dissolved.It is placed at room temperature for 5 minutes.Chloroform/ml Trizol of 200 μ l are added in, vibration is placed at room temperature for 2-3 minutes after 15 seconds, so After 4 DEG C, 12000g is centrifuged 15 minutes.Draw upper strata（About 400 μ l）, it is transferred in another new eppendorf pipes, adds in 500 μ L isopropanols, mixing are placed 10 minutes, and 4 DEG C, 12000g is centrifuged 10 minutes.It can be seen that RNA precipitate.Supernatant discarding adds in 1ml75% Ethyl alcohol washing precipitation, 7500g are centrifuged 5 minutes, dry RNA precipitate in air, are added in the DDW dissolvings of 30 μ l DEPC processing, are surveyed Determine RNA concentration and purity, a part is taken to carry out reverse transcription immediately, remaining RNA is frozen in -70 DEG C, spare.

（2）RNA reverse transcriptions：Using 20 μ l reverse transcription systems, by the template ribonucleic acid of extraction about 2 μ g and 5 × buffer4 μ l, 2 μ L dNTP (10mM), 1 μ l oligo (dT) 18 (10 μM), RNase Inhibitor1 μ l, reverse transcriptase Rever-Tra Ace- α -1 μ l, 37 DEG C 30 minutes after mixing, 85 DEG C of water-baths 5 seconds.

（3）Semiquantitive PCR：Take the cDNA of reverse transcription that using GAPDH as internal standard, PCR is carried out using its primer as template, The usage amount of template is adjusted, then using the template while amplifying target genes and GAPDH genes after adjustment, using GAPDH as ginseng According to observing different agent-feeding treatments pair and the influence of apoptosis pathway related protein gene rna transcription level.PCR uses 20 μ l PCR Reaction system：2 μ l10 × Taq buffer, 2 μ l dNTPs, 2 μ l Mg2+, 1 μ l sense primers, 1 μ l anti-sense primers, 1 μ l CDNA templates, 0.5 μ l rTaq polymerase, are supplied with DDW to 20 μ l.PCR conditions：94 DEG C of thermal denaturation 4min, 94 DEG C of changes Property 40s, 55 DEG C of annealing 40s, 72 DEG C of extension 40s, period is 20-32, last 72 DEG C of extension minutes.PCR product carries out 1% Agarose gel electrophoresis, EB are dyed, and are observed, are photographed to record under ultraviolet visualizer.

Specific primer used is：DR4 sense primers：5 '-CAGAACGTCCTGGAGCCTGTAAC-3 ', anti-sense primer： 5’-ATGTCCATTGCCTGATTCTTTGTG-3’；DR5 sense primers：5 '-GGGAAGAAGATTCTCCTGAGATGTG-3 ', under Swim primer：5’-ACATTGTCCTC AGCCCCAGGTCG-3’；DcR2 sense primers：5’-CTTCAGGAAACCAGAGCTTCCCT C-3 ', anti-sense primer 5 '-TTCTCCCGTTTGCTTA-TCACACGC-3 '；Bcl-xl sense primers：5’- TTGGACAATGGACTGGTTGA-3 ', anti-sense primer：5’-GTAGAGTGGATGGTC AGTG-3’；Bax sense primers：5’- AAGAAGCTGAGCGAGTGT-3 ', anti-sense primer：5’-GGAGGAAGTCCAATGTC-3’；Bim sense primers:5’- ATGAGAAGATCCTCCCTGC T-3 ', anti-sense primer：5’-AATGCATTCTCCACACCAGG-3’；Noxa sense primers：5’- GTGCCCTTGGAAACGGAGA-3 ', anti-sense primer are：5’-CCAGCCGCCCAGTCTAATC A-3’；Puma sense primers： 5 '-CAGACTGTGAATCCTGTGCT-3 ', anti-sense primer are：5’-ACAGTATCTTACAGGCTGGG-3’；GAPDH draws upstream Object:5 '-ACCACAGTCCAT GCCATCAC-3 ', anti-sense primer：5’-TCCACCACCCTGTTGCTGTA-3’；

Different albumen are analyzed with A549 binding abilities：The albumen FAM-AnnexinV, FAM- of fluorescent dye FAM marks TRAIL, FAM-Annexin V+FAM-TRAIL, FAM-TP8 and A549 are incubated 30 minutes in 37 DEG C, and pancreatin digestion is collected thin Born of the same parents after PBS washes 3 times, use flow cytometry analysis.Using FAM-BSA as negative control.

The flow cytometer showed of DR4 and DR5 expressions：A549 cells are through PBS, Annexin V, TRAIL, Annexin V+ After when TRAIL, TP8 processing 6 is small, after receiving cell, washed one time with the PBS containing 0.2%FBS.Cell with containing 0.2%FBS's PBS dilution after fluorescent marker antibody 4 DEG C be protected from light incubation 1 it is small when.With the PBS containing 0.2%FBS will with after antibody incubation Cell wash 3 times after, cell is fixed by 4% paraformaldehyde and is detected and analyzed with flow cytometer.With with containing 0.2% The cell that the PBS of FBS is incubated is as negative control.

Caspase8 and Caspase3 activity analysis：A549 cells are through PBS, Annexin V, TRAIL, Annexin V+ After when TRAIL, TP8 processing 6 is small, receive after cell Caspase8 and Caspase3 activity respectively with the Caspase8 in the green skies with Caspase3 activity detection kits are analyzed.The substrate and standard items provided with kit draws standard curve.Activity inspection The concrete operations of survey are as follows：

Pancreatin digests and collects cell, and 600g4 DEG C centrifuges 5 minutes, and washed once with PBS.Lysate is added in, is cracked on ice 15 minutes.4 DEG C of 16,000-20,000g are centrifuged 10-15 minutes, and supernatant is transferred in the centrifuge tube of precooling, are measured cspase immediately and are lived Property.By protein lysate add in substrate in, 37 DEG C be incubated 1 hour to 2 it is small when, when color change is more apparent, measure A405, with mark Directrix curve comparison can draw caspase activity.

The mode of tumour putrescence gene related apoptosis ligand fusion protein inducing apoptosis of tumour cell and the complete phases of TRAIL Together, Caspase-8 is all relied on.TRAIL, Annexin V+TRAIL and tumour putrescence gene related apoptosis ligand fusion egg The binding ability with the receptor DR4/DR5 of tumour cell A549 cell surfaces does not have difference in vain（Fig. 6 .3）.The signaling molecule in downstream （Such as DR4, DR5, DcR2 and with the relevant albumen of the mitochondrial pathways such as Bcl-XL, Bax, Bim, Noxa, Puma）Expression exist Also indifference after different pharmaceutical processing（Fig. 6 .1,6.2,6.4）.But Caspase-8, Caspase-3, PARP are only through swollen It is just substantially activated after the processing of tumor necrosis factor related apoptosis ligand fusion protein（Fig. 6 .1,7.1,7.2）.Moreover, Caspase- 8 inhibitor Z-IETD-FMK can obviously reduce the Apoptosis of tumour putrescence gene related apoptosis ligand fusion protein induction, and Caspase-9 inhibitor Z-LEHK-FMK is to the apoptosis that tumour putrescence gene related apoptosis ligand fusion protein induces without apparent shadow It rings（Fig. 7 .3）.This absolutely proves that tumour putrescence gene related apoptosis ligand fusion protein has duplicate work with TRAIL With mechanism, security is identical with TRAIL.

In addition, we also build, express and are prepared for the variant of tumour putrescence gene related apoptosis ligand fusion protein, The cysteine of the activated centre-TRAIL of tumour putrescence gene related apoptosis ligand the 230th in the variant（Cys230）Become prominent Become glycine.As shown in Fig. 5 .2, tumour putrescence gene related apoptosis ligand fusion protein variant just no longer has inducing cell The activity of apoptosis, this absolutely proves that tumour putrescence gene related apoptosis ligand fusion protein is by increasing tumour cell to tumour Putrescence gene related apoptosis ligand sensibility is led to by the signal of tumour putrescence gene related apoptosis ligand inducing cell apoptosis Road plays antitumor action.Tumour putrescence gene related apoptosis ligand fusion protein does not add other inducing cell apoptosis The mode of action, therefore its have the security identical with tumour putrescence gene related apoptosis ligand.

Embodiment 4

Tumour putrescence gene related apoptosis ligand fusion protein is tested in the antitumor curative effect of animal model for tumour

It is prepared by mouse back subcutaneous tumor model：The Female nude mice of 5-6 week old is purchased from Beijing military field engineering institute.Fetching number is given birth to Long-term A549 or Bel7402 or Colo205 cells, after pancreatin digests, with PBS in 800 × g centrifuge washings 2 times.After counting PBS is resuspended and adjusts cell concentration to 10⁷Cell/ml and then every 100 μ L of right side of mice dorsal sc subcutaneous injection（10⁶Carefully Born of the same parents）, one week or so, tumor size grew to 50mm³Left and right, is divided into 8 groups, every group 8 by mouse.PBS, Annexin are used respectively V, TRAIL, Annexin V+TRAIL, TP8 and chemotherapeutics administration, protein medicaments and PBS are through intraperitoneal injection, often Its injection once, is treated 14 days altogether.Chemotherapeutics is administered through tail vein, and injection in every four days once, is treated 14 days altogether.From starting to control Every two days measurement tumor sizes after treatment, and calculate gross tumor volume by following equation：0.5×L1×(L2)², wherein L1 is that tumour is long Axis, L2 are tumour short axle.It treats the 15th day, puts to death mouse.

Tumor tissue section's CD31 staining analysis：After administration, mouse is put to death, takes out tumor tissues OTC immediately It after embedding, is cut into slices with freezing microtome, slice thickness is 5 μm.After the completion of section, air-dry, 10 minutes are fixed in ice acetone, room Temperature is dried.It is developed a film three times with PBST, dries edge, drawn a circle with oil pike along histotomy outer rim, sealed with sheep blood serum confining liquid room temperature Close 30min.Primary antibody is diluted with antibody diluent（CD311:50）.PBST is coated with 4 DEG C of primary antibody overnight after rinsing slice, thin piece.PBST is rinsed 3-5 times, dry edge.Coating secondary antibody, 37 DEG C, 45min.PBST is washed three times.Dry edge.DAPI contaminates nucleus, is shown with fluorescence Micro mirror microscopy.

Tumor tissue section TUNEL is analyzed：After administration, mouse is put to death, tumor tissues is taken out immediately and is embedded with OTC Afterwards, cut into slices with freezing microtome, slice thickness is 5 μm.After the completion of section, air-dry, 10 minutes are fixed in ice acetone, room temperature is dried in the air It is dry.It is developed a film three times with PBST, dries edge, drawn a circle with oil pike along histotomy outer rim, is incubated at room temperature 5 minutes with Proteinase K, TBS is washed once.3%H₂O₂After five minutes, TBS is washed once for incubation at room temperature.TdT Equilibration Buffer are incubated at room temperature 15 points Clock blots TdT buffer, TdT labeling reaction mixture is added dropwise, when 37 DEG C of incubations 1.5 are small, TBS is washed three times And blot, it adds in stop buffer and is incubated at room temperature 10 minutes, blot stop buffer, add in conjugate buffer, room Temperature is incubated 30 minutes, and TBS is washed once and blotted, and DAB is added dropwise in tissue surface, and after incubation at room temperature 15 minutes, ddH2O is washed once simultaneously It blots, methyl green, which is added dropwise, in tissue surface is incubated at room temperature 3 minutes, sucks methyl green, and 100% ethyl alcohol is added dropwise three times in tissue surface To tissue surface redgreen.Dimethylbenzene is added dropwise rapidly in tissue surface, and blots rapidly, is repeated once, with glycerine mounting, glimmering Viewed under light microscopy.

Present invention employs three kinds of tumor models, i.e., A549 tumor models insensitive to TRAIL are quick to TRAIL moderates It the Bel7402 tumor models of sense and tumor necrosis factor correlation is had evaluated to the Colo-205 tumor models of TRAIL sensitivities withers Die the antitumous effect of ligand fusion protein TP8.In A549 tumor models, TRAIL is injected intraperitoneally（5mg/kg/ days）, Annexin V（7.5mg/kg/ my god）, TRAIL（5mg/kg/ days）+Annexin V（7.5mg/kg/ my god）, tumor necrosis factor phase Close apoptosis ligand fusion protein TP8（6.25mg/kg/ days, 12.5mg/kg/ days, 25mg/kg/ days）.With negative control group（Phosphoric acid Buffer solution group）It compares（Tumor doubling time is 5.3 days）, Annexin V, TRAIL, Annexin V+TRAIL fails effectively to press down Tumour growth processed, three groups of tumor doubling time are respectively：6.1 days, 6.1 days, 6.0 days；Compared with negative control group, three groups The tumor growth delay time（Volume reaches 1000mm³When, compared with the time required to PBS groups）Respectively：0 day, 0.6 day, 1.1 My god.And the tumour putrescence gene related apoptosis ligand fusion protein TP8 of various dose can substantially inhibit tumour growth, and it is this Inhibitory action enhances with the increase of tumour putrescence gene related apoptosis ligand fusion protein TP8 dosage, three dosage groups Tumor doubling time is respectively：6.5 days, 7.4 days, 7.9 days；Compared with negative control group, three groups of the tumor growth delay time Respectively：2.8 days, 6.9 days, 12.8 days（Fig. 8 .1,8.2）.

In Bel7402 tumor models, TRAIL is injected intraperitoneally（5mg/kg/ days）, Annexin V（7.5mg/kg/ my god）, TRAIL（5mg/kg/ days）+Annexin V（7.5mg/kg/ my god）, tumour putrescence gene related apoptosis ligand fusion protein TP8 （6.25mg/kg/ days, 12.5mg/kg/ days, 25mg/kg/ days）.Tumour putrescence gene related apoptosis ligand fusion protein TP8 （12.5mg/kg groups）Inhibit the effect of tumour growth compared with negative control group（Phosphate buffer group）, Annexin V groups, TRAIL groups And Annexin V+TRAIL groups are apparent, and this inhibitory action is with tumour putrescence gene related apoptosis ligand fusion egg The increase of white TP8 dosage and enhance.With negative control group（Phosphate buffer group）It compares（Tumor doubling time is 7.3 days）, Annexin V, TRAIL, Annexin V+TRAIL fail effectively to inhibit tumour growth, and three groups of tumor doubling time is respectively： 7.8 days, 8.0 days, 7.6 days；Compared with negative control group, three groups of tumor growth delay time is respectively：4 days, 5.4 days, 5.0 My god.And the tumour putrescence gene related apoptosis ligand fusion protein TP8 of various dose can substantially inhibit tumour growth, and it is this Inhibitory action enhances with the increase of tumour putrescence gene related apoptosis ligand fusion protein TP8 concentration, three dosage groups Tumor doubling time is respectively：9.0 days, 9.3 days, 10.1 days；Compared with negative control group, three groups of the tumor growth delay time Respectively：9.3 days, 10.0 days, 18.5 days（Fig. 8 .3,8.4）.

In Colo-205 tumor models, TRAIL is injected intraperitoneally（5mg/kg/ days）, Annexin V（7.5mg/kg/ my god）, TRAIL（5mg/kg/ days）+Annexin V（7.5mg/kg/ my god）, tumour putrescence gene related apoptosis ligand fusion protein TP8 （6.25mg/kg/ days, 12.5mg/kg/ days, 25mg/kg/ days）.Tumour putrescence gene related apoptosis ligand fusion protein TP8 （12.5mg/kg groups）Inhibit the effect of tumour growth compared with negative control group（Phosphate buffer group）, Annexin V groups, TRAIL groups And Annexin V+TRAIL groups are apparent, and this inhibitory action is with tumour putrescence gene related apoptosis ligand fusion egg The increase of white TP8 dosage and enhance.With negative control group（Phosphate buffer group）It compares（Tumor doubling time is 10.0 days）, Annexin V, TRAIL, Annexin V+TRAIL fail effectively to inhibit tumour growth, and three groups of tumor doubling time is respectively： 10.2 days, 12.5 days, 10.4 days；Compared with negative control group, three groups of tumor growth delay time is respectively：0.9 day, 4.6 My god, 3.1 days.And the tumour putrescence gene related apoptosis ligand fusion protein TP8 of various dose can substantially inhibit tumour growth, And this inhibitory action enhances, three agent with the increase of tumour putrescence gene related apoptosis ligand fusion protein TP8 concentration The tumor doubling time of amount group is respectively：14.5 days, 15.0 days, 14.0 days；Compared with negative control group, three groups of tumour growth Lag time is respectively：12.2 days, 18.3 days, 22 days（Fig. 8 .5,8.6）.

The apoptosis of tumor cells in tumor tissue section is assessed with TUNEL.It is consistent with Vitro Experimental Results, only TP8 groups can significantly induce the apoptosis of tumour cell（Fig. 9）.

Embodiment 5

The effect of tumour putrescence gene related apoptosis ligand fusion protein TP8 inhibits tumour growth with Cisplatin evaluation is real It tests

It is seeded in subcutaneous A549 tumor sizes and grows to 50-100mm3 or so, mouse is divided into 8 groups, every group 8.Point It Yong not PBS, TP8（6.25mg/kg/day）And cis-platinum (5mg/kg/day) is treated.TP8 and PBS is administered to through abdominal cavity Medicine, daily injection once, are treated 14 days altogether.Cis-platinum is administered through tail vein, and injection in every four days once, is treated 14 days altogether.From the beginning of Every two days measurement tumor sizes after treatment, and calculate gross tumor volume by following equation：0.5×L1×(L2)², wherein L1 is tumour Long axis, L2 are tumour short axle.

Compared with PBS negative control groups, tumour putrescence gene related apoptosis ligand fusion protein TP8, cis-platinum and TP8- Cisplatin can inhibit the growth of A549 tumours.TP8 and during Cisplatin, inhibits the with obvious effects of A549 tumour growths and is better than Therapeutic effect when TP8 and cis-platinum are used alone.PBS groups tumor doubling time is 16.1 days, cis-platinum group tumor doubling time For 31.35 days, TP8 groups tumor doubling time was 30.96 days, and TP8 is 40.92 days with Cisplatin group tumor doubling time.It is swollen The knurl growth delay time（I.e. gross tumor volume reaches 1000mm³When with respect to the time required to PBS groups）Cis-platinum group is 15.25 days, TP8 groups For 14.87 days, TP8 was 24.83 days with Cisplatin group（Figure 10）.

The present invention is also attempted tumour putrescence gene related apoptosis ligand fusion protein and anti-tumor biological medicine, such as Recombinant Endostatin（Endostatin）, angiostatin（Vasostatin）Etc. being used in combination, also achieve similar collaboration and control Therapeutic effect.

The present invention is also attempted tumour putrescence gene related apoptosis ligand fusion protein and anti-tumor Chinese medicine ingredient, such as Thunder God Rattan lactone alcohol etc. is used in combination, and also achieves similar synergistic therapeutic effect.

The present invention also attempts to have studied tumour putrescence gene related apoptosis ligand fusion protein and this laboratory early period Antitumor attenuation salmonella VNP20009 for many years is used in combination, and also achieves similar synergistic therapeutic effect.

It is gene therapy vector that the present invention, which is also attempted using the attenuation salmonella VNP20009 of tumor-targeting, utilizes this The technology of the foregoing exploitation in laboratory（《Therapeutic gene stablizes expression and application in the delivering of anaerobism tissue-targeting and selectivity》 Chinese invention patent application number：201010565011.6）Carry out the gene of tumour putrescence gene related apoptosis ligand fusion protein Treatment.Using attenuation salmonella VNP20009 by the expression plasmid of tumour putrescence gene related apoptosis ligand antigen-4 fusion protein gene Gene therapy is carried out in bearing animals, is achieved than individually using attenuation salmonella VNP20009 treatments preferably treatment effect Fruit.

The tumor necrosis factor phase formed using annexin and tumour putrescence gene related apoptosis ligand as main component Closing apoptosis ligand fusion protein has the bioactivity of significantly powerful anti-kinds of tumors, can overcome kinds of tumor cells to thin Born of the same parents' apoptosis insensitive is resisted, and can be with chemotherapeutics, bio-pharmaceutical, gene therapy, microbial medicine, Chinese medicine etc. Therapy use in conjunction generates better therapeutic effect.

Therefore, by combining the annexin family member protein of activity with phosphatide or the film of activity being combined with phosphatide Join protein family member protein matter derivative with having the member protein of activity inducing apoptosis in tnf family cytokines Matter or the tumor necrosis factor that there is the tnf family cytokines apoptosis protein derivative of activity inducing apoptosis to form Sub- related apoptosis ligand fusion protein has the bioactivity of the inducing cell apoptosis significantly increased, can overcome and deposit extensively at present A variety of pathological cells including tumour cell to the insensitive of Apoptosis or resist, and can with it is existing The drug or therapy of inducing cell apoptosis（Chemotherapeutics, bio-pharmaceutical, gene therapy, microbial medicine, Chinese medicine etc.） Use in conjunction generates better therapeutic effect.

All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to the same.In addition, it should also be understood that, after content is lectured in the appeal for having read the present invention, those skilled in the art can be with To the present invention, various modifications may be made or change, such equivalent forms should all be included in the protection scope of the present invention.

<210>7

<211>1506

<212>DNA

<213>Artificial synthesized sequence

<220>

<223>Tumour putrescence gene related apoptosis ligand fusion protein TP8 gene orders

<400>7

GCACAGGTTC TCAGAGGCAC TGTGACTGAC TTCCCTGGAT TTGATGAGCG 50

GGCTGATGCA GAAACTCTTC GGAAGGCTAT GAAAGGCTTG GGCACAGATG 100

AGGAGAGCAT CCTGACTCTG TTGACATCCC GAAGTAATGC TCAGCGCCAG 150

GAAATCTCTG CAGCTTTTAA GACTCTGTTT GGCAGGGATC TTCTGGATGA 200

CCTGAAATCA GAACTAACTG GAAAATTTGA AAAATTAATT GTGGCTCTGA 250

TGAAACCCTC TCGGCTTTAT GATGCTTATG AACTGAAACA TGCCTTGAAG 300

GGAGCTGGAA CAAATGAAAA AGTACTGACA GAAATTATTG CTTCAAGGAC 350

ACCTGAAGAA CTGAGAGCCA TCAAACAAGT TTATGAAGAA GAATATGGCT 400

CAAGCCTGGA AGATGACGTG GTGGGGGACA CTTCAGGGTA CTACCAGCGG 450

ATGTTGGTGG TTCTCCTTCA GGCTAACAGA GACCCTGATG CTGGAATTGA 500

TGAAGCTCAA GTTGAACAAG ATGCTCAGGC TTTATTTCAG GCTGGAGAAC 550

TTAAATGGGG GACAGATGAA GAAAAGTTTA TCACCATCTT TGGAACACGA 600

AGTGTGTCTC ATTTGAGAAA GGTGTTTGAC AAGTACATGA CTATATCAGG 650

ATTTCAAATT GAGGAAACCA TTGACCGCGA GACTTCTGGC AATTTAGAGC 700

AACTACTCCT TGCTGTTGTG AAATCTATTC GAAGTATACC TGCCTACCTT 750

GCAGAGACCC TCTATTATGC TATGAAGGGA GCTGGGACAG ATGATCATAC 800

CCTCATCAGA GTCATGGTTT CCAGGAGTGA GATTGATCTG TTTAACATCA 850

GGAAGGAGTT TAGGAAGAAT TTTGCCACCT CTCTTTATTC CATGATTAAG 900

GGAGATACAT CTGGGGACTA TAAGAAAGCT CTTCTGCTGC TCTGTGGAGA 950

AGATGACGGT GGTGGTGGTT CTGGTGGTGG TGGTTCTGGT GGTGGTGGTG 1000

TGAGAGAAAG AGGTCCTCAG AGAGTAGCAG CTCACATAAC TGGGACCAGA 1050

GGAAGAAGCA ACACATTGTC TTCTCCAAAC TCCAAGAATG AAAAGGCTCT 1100

GGGCCGCAAA ATAAACTCCT GGGAATCATC AAGGAGTGGG CATTCATTCC 1150

TGAGCAACTT GCACTTGAGG AATGGTGAAC TGGTCATCCA TGAAAAAGGG 1200

TTTTACTACA TCTATTCCCA AACATACTTT CGATTTCAGG AGGAAATAAA 1250

AGAAAACACA AAGAACGACA AACAAATGGT CCAATATATT TACAAATACA 1300

CAAGTTATCC TGACCCTATA TTGTTGATGA AAAGTGCTAG AAATAGTTGT 1350

TGGTCTAAAG ATGCAGAATA TGGACTCTAT TCCATCTATC AAGGGGGAAT 1400

ATTTGAGCTT AAGGAAAATG ACAGAATTTT TGTTTCTGTA ACAAATGAGC 1450

ACTTGATAGA CATGGACCAT GAAGCCAGTT TTTTTGGGGC CTTTTTAGTT 1500

GGCTAA 1506

<210>8

<211>1467

<212>DNA

<213>Artificial synthesized sequence

<220>

<223>Tumour putrescence gene related apoptosis ligand antigen-4 fusion protein gene sequence

<400>8

GCACAGGTTC TCAGAGGCAC TGTGACTGAC TTCCCTGGAT TTGATGAGCG 50

GGCTGATGCA GAAACTCTTC GGAAGGCTAT GAAAGGCTTG GGCACAGATG 100

AGGAGAGCAT CCTGACTCTG TTGACATCCC GAAGTAATGC TCAGCGCCAG 150

GAAATCTCTG CAGCTTTTAA GACTCTGTTT GGCAGGGATC TTCTGGATGA 200

CCTGAAATCA GAACTAACTG GAAAATTTGA AAAATTAATT GTGGCTCTGA 250

TGAAACCCTC TCGGCTTTAT GATGCTTATG AACTGAAACA TGCCTTGAAG 300

GGAGCTGGAA CAAATGAAAA AGTACTGACA GAAATTATTG CTTCAAGGAC 350

ACCTGAAGAA CTGAGAGCCA TCAAACAAGT TTATGAAGAA GAATATGGCT 400

CAAGCCTGGA AGATGACGTG GTGGGGGACA CTTCAGGGTA CTACCAGCGG 450

ATGTTGGTGG TTCTCCTTCA GGCTAACAGA GACCCTGATG CTGGAATTGA 500

TGAAGCTCAA GTTGAACAAG ATGCTCAGGC TTTATTTCAG GCTGGAGAAC 550

TTAAATGGGG GACAGATGAA GAAAAGTTTA TCACCATCTT TGGAACACGA 600

AGTGTGTCTC ATTTGAGAAA GGTGTTTGAC AAGTACATGA CTATATCAGG 650

ATTTCAAATT GAGGAAACCA TTGACCGCGA GACTTCTGGC AATTTAGAGC 700

AACTACTCCT TGCTGTTGTG AAATCTATTC GAAGTATACC TGCCTACCTT 750

GCAGAGACCC TCTATTATGC TATGAAGGGA GCTGGGACAG ATGATCATAC 800

CCTCATCAGA GTCATGGTTT CCAGGAGTGA GATTGATCTG TTTAACATCA 850

GGAAGGAGTT TAGGAAGAAT TTTGCCACCT CTCTTTATTC CATGATTAAG 900

GGAGATACAT CTGGGGACTA TAAGAAAGCT CTTCTGCTGC TCTGTGGAGA 950

AGATGACGGT GTGAGAGAAA GAGGTCCTCA GAGAGTAGCA GCTCACATAA 1000

CTGGGACCAG AGGAAGAAGC AACACATTGT CTTCTCCAAA CTCCAAGAAT 1050

GAAAAGGCTC TGGGCCGCAA AATAAACTCC TGGGAATCAT CAAGGAGTGG 1100

GCATTCATTC CTGAGCAACT TGCACTTGAG GAATGGTGAA CTGGTCATCC 1150

ATGAAAAAGG GTTTTACTAC ATCTATTCCC AAACATACTT TCGATTTCAG 1200

GAGGAAATAA AAGAAAACAC AAAGAACGAC AAACAAATGG TCCAATATAT 1250

TTACAAATAC ACAAGTTATC CTGACCCTAT ATTGTTGATG AAAAGTGCTA 1300

GAAATAGTTG TTGGTCTAAA GATGCAGAAT ATGGACTCTA TTCCATCTAT 1350

CAAGGGGGAA TATTTGAGCT TAAGGAAAAT GACAGAATTT TTGTTTCTGT 1400

AACAAATGAG CACTTGATAG ACATGGACCA TGAAGCCAGT TTTTTTGGGG 1450

CCTTTTTAGT TGGCTAA 1467

<210>9

<211>1488

<212>DNA

<213>Artificial synthesized sequence

<220>

<223>Tumour putrescence gene related apoptosis ligand antigen-4 fusion protein gene sequence

<400>9

GCACAGGTTC TCAGAGGCAC TGTGACTGAC TTCCCTGGAT TTGATGAGCG 50

GGCTGATGCA GAAACTCTTC GGAAGGCTAT GAAAGGCTTG GGCACAGATG 100

AGGAGAGCAT CCTGACTCTG TTGACATCCC GAAGTAATGC TCAGCGCCAG 150

GAAATCTCTG CAGCTTTTAA GACTCTGTTT GGCAGGGATC TTCTGGATGA 200

CCTGAAATCA GAACTAACTG GAAAATTTGA AAAATTAATT GTGGCTCTGA 250

TGAAACCCTC TCGGCTTTAT GATGCTTATG AACTGAAACA TGCCTTGAAG 300

GGAGCTGGAA CAAATGAAAA AGTACTGACA GAAATTATTG CTTCAAGGAC 350

ACCTGAAGAA CTGAGAGCCA TCAAACAAGT TTATGAAGAA GAATATGGCT 400

CAAGCCTGGA AGATGACGTG GTGGGGGACA CTTCAGGGTA CTACCAGCGG 450

ATGTTGGTGG TTCTCCTTCA GGCTAACAGA GACCCTGATG CTGGAATTGA 500

TGAAGCTCAA GTTGAACAAG ATGCTCAGGC TTTATTTCAG GCTGGAGAAC 550

TTAAATGGGG GACAGATGAA GAAAAGTTTA TCACCATCTT TGGAACACGA 600

AGTGTGTCTC ATTTGAGAAA GGTGTTTGAC AAGTACATGA CTATATCAGG 650

ATTTCAAATT GAGGAAACCA TTGACCGCGA GACTTCTGGC AATTTAGAGC 700

AACTACTCCT TGCTGTTGTG AAATCTATTC GAAGTATACC TGCCTACCTT 750

GCAGAGACCC TCTATTATGC TATGAAGGGA GCTGGGACAG ATGATCATAC 800

CCTCATCAGA GTCATGGTTT CCAGGAGTGA GATTGATCTG TTTAACATCA 850

GGAAGGAGTT TAGGAAGAAT TTTGCCACCT CTCTTTATTC CATGATTAAG 900

GGAGATACAT CTGGGGACTA TAAGAAAGCT CTTCTGCTGC TCTGTGGAGA 950

AGATGACGCA GGTGGTTCTT CTGGTGGTGG TGTGAGAGAA AGAGGTCCTC 1000

AGAGAGTAGC AGCTCACATA ACTGGGACCA GAGGAAGAAG CAACACATTG 1050

TCTTCTCCAA ACTCCAAGAA TGAAAAGGCT CTGGGCCGCA AAATAAACTC 1100

CTGGGAATCA TCAAGGAGTG GGCATTCATT CCTGAGCAAC TTGCACTTGA 1150

GGAATGGTGA ACTGGTCATC CATGAAAAAG GGTTTTACTA CATCTATTCC 1200

CAAACATACT TTCGATTTCA GGAGGAAATA AAAGAAAACA CAAAGAACGA 1250

CAAACAAATG GTCCAATATA TTTACAAATA CACAAGTTAT CCTGACCCTA 1300

TATTGTTGAT GAAAAGTGCT AGAAATAGTT GTTGGTCTAA AGATGCAGAA 1350

TATGGACTCT ATTCCATCTA TCAAGGGGGA ATATTTGAGC TTAAGGAAAA 1400

TGACAGAATT TTTGTTTCTG TAACAAATGA GCACTTGATA GACATGGACC 1450

ATGAAGCCAG TTTTTTTGGG GCCTTTTTAG TTGGCTAA 1488

<210>10

<211>1539

<212>DNA

<213>Artificial synthesized sequence

<220>

<223>Tumour putrescence gene related apoptosis ligand antigen-4 fusion protein gene sequence

<400>10

GCACAGGTTC TCAGAGGCAC TGTGACTGAC TTCCCTGGAT TTGATGAGCG 50

GGCTGATGCA GAAACTCTTC GGAAGGCTAT GAAAGGCTTG GGCACAGATG 100

AGGAGAGCAT CCTGACTCTG TTGACATCCC GAAGTAATGC TCAGCGCCAG 150

GAAATCTCTG CAGCTTTTAA GACTCTGTTT GGCAGGGATC TTCTGGATGA 200

CCTGAAATCA GAACTAACTG GAAAATTTGA AAAATTAATT GTGGCTCTGA 250

TGAAACCCTC TCGGCTTTAT GATGCTTATG AACTGAAACA TGCCTTGAAG 300

GGAGCTGGAA CAAATGAAAA AGTACTGACA GAAATTATTG CTTCAAGGAC 350

ACCTGAAGAA CTGAGAGCCA TCAAACAAGT TTATGAAGAA GAATATGGCT 400

CAAGCCTGGA AGATGACGTG GTGGGGGACA CTTCAGGGTA CTACCAGCGG 450

ATGTTGGTGG TTCTCCTTCA GGCTAACAGA GACCCTGATG CTGGAATTGA 500

TGAAGCTCAA GTTGAACAAG ATGCTCAGGC TTTATTTCAG GCTGGAGAAC 550

TTAAATGGGG GACAGATGAA GAAAAGTTTA TCACCATCTT TGGAACACGA 600

AGTGTGTCTC ATTTGAGAAA GGTGTTTGAC AAGTACATGA CTATATCAGG 650

ATTTCAAATT GAGGAAACCA TTGACCGCGA GACTTCTGGC AATTTAGAGC 700

AACTACTCCT TGCTGTTGTG AAATCTATTC GAAGTATACC TGCCTACCTT 750

GCAGAGACCC TCTATTATGC TATGAAGGGA GCTGGGACAG ATGATCATAC 800

CCTCATCAGA GTCATGGTTT CCAGGAGTGA GATTGATCTG TTTAACATCA 850

GGAAGGAGTT TAGGAAGAAT TTTGCCACCT CTCTTTATTC CATGATTAAG 900

GGAGATACAT CTGGGGACTA TAAGAAAGCT CTTCTGCTGC TCTGTGGAGA 950

AGATGACGGT GCAGGTGGTT CTTCTGGTGG TGGTGCAGGT GGTTCTTCTG 1000

GTGGTGGTGC AGGTGGTTCT TCTGGTGGTG GTGTGAGAGA AAGAGGTCCT 1050

CAGAGAGTAG CAGCTCACAT AACTGGGACC AGAGGAAGAA GCAACACATT 1100

GTCTTCTCCA AACTCCAAGA ATGAAAAGGC TCTGGGCCGC AAAATAAACT 1150

CCTGGGAATC ATCAAGGAGT GGGCATTCAT TCCTGAGCAA CTTGCACTTG 1200

AGGAATGGTG AACTGGTCAT CCATGAAAAA GGGTTTTACT ACATCTATTC 1250

CCAAACATAC TTTCGATTTC AGGAGGAAAT AAAAGAAAAC ACAAAGAACG 1300

ACAAACAAAT GGTCCAATAT ATTTACAAAT ACACAAGTTA TCCTGACCCT 1350

ATATTGTTGA TGAAAAGTGC TAGAAATAGT TGTTGGTCTA AAGATGCAGA 1400

ATATGGACTC TATTCCATCT ATCAAGGGGG AATATTTGAG CTTAAGGAAA 1450

ATGACAGAAT TTTTGTTTCT GTAACAAATG AGCACTTGAT AGACATGGAC 1500

CATGAAGCCA GTTTTTTTGG GGCCTTTTTA GTTGGCTAA 1539

<210>11

<211>1506

<212>DNA

<213>Artificial synthesized sequence

<220>

<223>Tumour putrescence gene related apoptosis ligand fusion protein PT8 gene orders

<400>11

GTGAGAGAAA GAGGTCCTCA GAGAGTAGCA GCTCACATAA CTGGGACCAG 50

AGGAAGAAGC AACACATTGT CTTCTCCAAA CTCCAAGAAT GAAAAGGCTC 100

TGGGCCGCAA AATAAACTCC TGGGAATCAT CAAGGAGTGG GCATTCATTC 150

CTGAGCAACT TGCACTTGAG GAATGGTGAA CTGGTCATCC ATGAAAAAGG 200

GTTTTACTAC ATCTATTCCC AAACATACTT TCGATTTCAG GAGGAAATAA 250

AAGAAAACAC AAAGAACGAC AAACAAATGG TCCAATATAT TTACAAATAC 300

ACAAGTTATC CTGACCCTAT ATTGTTGATG AAAAGTGCTA GAAATAGTTG 350

TTGGTCTAAA GATGCAGAAT ATGGACTCTA TTCCATCTAT CAAGGGGGAA 400

TATTTGAGCT TAAGGAAAAT GACAGAATTT TTGTTTCTGT AACAAATGAG 450

CACTTGATAG ACATGGACCA TGAAGCCAGT TTTTTTGGGG CCTTTTTAGT 500

TGGCGGTGGT GGTGGTTCTG GTGGTGGTGG TTCTGGTGGT GGTGGTGCAC 550

AGGTTCTCAG AGGCACTGTG ACTGACTTCC CTGGATTTGA TGAGCGGGCT 600

GATGCAGAAA CTCTTCGGAA GGCTATGAAA GGCTTGGGCA CAGATGAGGA 650

GAGCATCCTG ACTCTGTTGA CATCCCGAAG TAATGCTCAG CGCCAGGAAA 700

TCTCTGCAGC TTTTAAGACT CTGTTTGGCA GGGATCTTCT GGATGACCTG 750

AAATCAGAAC TAACTGGAAA ATTTGAAAAA TTAATTGTGG CTCTGATGAA 800

ACCCTCTCGG CTTTATGATG CTTATGAACT GAAACATGCC TTGAAGGGAG 850

CTGGAACAAA TGAAAAAGTA CTGACAGAAA TTATTGCTTC AAGGACACCT 900

GAAGAACTGA GAGCCATCAA ACAAGTTTAT GAAGAAGAAT ATGGCTCAAG 950

CCTGGAAGAT GACGTGGTGG GGGACACTTC AGGGTACTAC CAGCGGATGT 1000

TGGTGGTTCT CCTTCAGGCT AACAGAGACC CTGATGCTGG AATTGATGAA 1050

GCTCAAGTTG AACAAGATGC TCAGGCTTTA TTTCAGGCTG GAGAACTTAA 1100

ATGGGGGACA GATGAAGAAA AGTTTATCAC CATCTTTGGA ACACGAAGTG 1150

TGTCTCATTT GAGAAAGGTG TTTGACAAGT ACATGACTAT ATCAGGATTT 1200

CAAATTGAGG AAACCATTGA CCGCGAGACT TCTGGCAATT TAGAGCAACT 1250

ACTCCTTGCT GTTGTGAAAT CTATTCGAAG TATACCTGCC TACCTTGCAG 1300

AGACCCTCTA TTATGCTATG AAGGGAGCTG GGACAGATGA TCATACCCTC 1350

ATCAGAGTCA TGGTTTCCAG GAGTGAGATT GATCTGTTTA ACATCAGGAA 1400

GGAGTTTAGG AAGAATTTTG CCACCTCTCT TTATTCCATG ATTAAGGGAG 1450

ATACATCTGG GGACTATAAG AAAGCTCTTC TGCTGCTCTG TGGAGAAGAT 1500

GACTAA 1506

<210>12

<211>1473

<212>DNA

<213>Artificial synthesized sequence

<220>

<223>Annexin-tumour putrescence factor alpha fusion protein gene order

<400>12

GCACAGGTTC TCAGAGGCAC TGTGACTGAC TTCCCTGGAT TTGATGAGCG 50

GGCTGATGCA GAAACTCTTC GGAAGGCTAT GAAAGGCTTG GGCACAGATG 100

AGGAGAGCAT CCTGACTCTG TTGACATCCC GAAGTAATGC TCAGCGCCAG 150

GAAATCTCTG CAGCTTTTAA GACTCTGTTT GGCAGGGATC TTCTGGATGA 200

CCTGAAATCA GAACTAACTG GAAAATTTGA AAAATTAATT GTGGCTCTGA 250

TGAAACCCTC TCGGCTTTAT GATGCTTATG AACTGAAACA TGCCTTGAAG 300

GGAGCTGGAA CAAATGAAAA AGTACTGACA GAAATTATTG CTTCAAGGAC 350

ACCTGAAGAA CTGAGAGCCA TCAAACAAGT TTATGAAGAA GAATATGGCT 400

CAAGCCTGGA AGATGACGTG GTGGGGGACA CTTCAGGGTA CTACCAGCGG 450

ATGTTGGTGG TTCTCCTTCA GGCTAACAGA GACCCTGATG CTGGAATTGA 500

TGAAGCTCAA GTTGAACAAG ATGCTCAGGC TTTATTTCAG GCTGGAGAAC 550

TTAAATGGGG GACAGATGAA GAAAAGTTTA TCACCATCTT TGGAACACGA 600

AGTGTGTCTC ATTTGAGAAA GGTGTTTGAC AAGTACATGA CTATATCAGG 650

ATTTCAAATT GAGGAAACCA TTGACCGCGA GACTTCTGGC AATTTAGAGC 700

AACTACTCCT TGCTGTTGTG AAATCTATTC GAAGTATACC TGCCTACCTT 750

GCAGAGACCC TCTATTATGC TATGAAGGGA GCTGGGACAG ATGATCATAC 800

CCTCATCAGA GTCATGGTTT CCAGGAGTGA GATTGATCTG TTTAACATCA 850

GGAAGGAGTT TAGGAAGAAT TTTGCCACCT CTCTTTATTC CATGATTAAG 900

GGAGATACAT CTGGGGACTA TAAGAAAGCT CTTCTGCTGC TCTGTGGAGA 950

AGATGACGGT GGTGGTGGTT CTGGTGGTGG TGGTTCTGGT GGTGGTGGTG 1000

TCAGATCATC TTCTCGAACC CCGAGTGACA AGCCTGTAGC CCATGTTGTA 1050

GCAAACCCTC AAGCTGAGGG GCAGCTCCAG TGGCTGAACC GCCGGGCCAA 1100

TGCCCTCCTG GCCAATGGCG TGGAGCTGAG AGATAACCAG CTGGTGGTGC 1150

CATCAGAGGG CCTGTACCTC ATCTACTCCC AGGTCCTCTT CAAGGGCCAA 1200

GGCTGCCCCT CCACCCATGT GCTCCTCACC CACACCATCA GCCGCATCGC 1250

CGTCTCCTAC CAGACCAAGG TCAACCTCCT CTCTGCCATC AAGAGCCCCT 1300

GCCAGAGGGA GACCCCAGAG GGGGCTGAGG CCAAGCCCTG GTATGAGCCC 1350

ATCTATCTGG GAGGGGTCTT CCAGCTGGAG AAGGGTGACC GACTCAGCGC 1400

TGAGATCAAT CGGCCCGACT ATCTCGACTT TGCCGAGTCT GGGCAGGTCT 1450

ACTTTGGGAT CATTGCCCTG TGA 1473

Claims (13)

1. a kind of tumour putrescence gene related apoptosis ligand fusion protein, it is characterised in that：The amino acid sequence of the fusion protein Row such as SEQ ID NO：Shown in 1, it includes amino acid sequence, the tnf family cytokines Apoptosis egg of annexin V White amino acid sequence and positioned at annexin amino acid sequence and tnf family cytokines apoptosis protein amino acid 3 part of connection peptide sequence between sequence, wherein the tnf family cytokines apoptosis protein refers to neoplasm necrosis There is the member protein matter of activity inducing apoptosis in factor family.

2. a kind of separated DNA molecular, it is characterised in that：It encodes fusion protein described in claim 1.

3. DNA molecular according to claim 2, it is characterised in that：Its nucleotide sequence such as SEQ ID NO：Shown in 7.

4. a kind of carrier, it is characterised in that：It contains the DNA molecular described in Claims 2 or 3.

5. a kind of host cell, it is characterised in that：It contains the carrier described in claim 4.

6. host cell according to claim 5, it is characterised in that：It includes recombinant E. coli BL21.

A kind of 7. method for preparing fusion protein described in claim 1, which is characterized in that it includes the following steps：It is being suitble to Under conditions of expressing the fusion protein, the host cell described in claim 5 is cultivated, so as to give expression to the fusion egg In vain；The fusion protein with separation.

8. preparation method according to claim 7, it is characterised in that：The host cell is cultivated and lured at low temperature Expression is led, the low temperature refers to 10-35 DEG C.

9. preparation method according to claim 8, it is characterised in that：The separation fusion protein uses ion-exchange chromatography And metal affinity chromatography.

10. a kind of tumour putrescence gene related apoptosis ligand fusion protein according to claim 1 is preparing inducing cell Application in the medicine of apoptosis.

11. a kind of tumour putrescence gene related apoptosis ligand fusion protein according to claim 1 is preparing oncotherapy Application in drug.

12. tumour putrescence gene related apoptosis ligand fusion protein according to claim 11, which is applied, is preparing oncotherapy Application in drug, it is characterised in that：The application include by the fusion protein be individually used for preparing anti-tumor medicine or With existing anti-tumor medicine drug combination or with existing anti-tumor medicine form composition or with existing chemotherapy, put It treats, Chinese medicine is treated, Biotherapy method use in conjunction in oncotherapy.

13. a kind of pharmaceutical composition, it is characterised in that：Including pharmaceutically acceptable carrier or excipient or diluent and A effective amount of fusion protein described in claim 1.