CN107098964A

CN107098964A - A kind of tumour-specific lepirudin 023 ludon and its production and use

Info

Publication number: CN107098964A
Application number: CN201710362102.1A
Authority: CN
Inventors: 何向锋; 施文
Original assignee: Individual
Current assignee: New Key (nanjing) Biotechnology Co Ltd
Priority date: 2017-05-19
Filing date: 2017-05-19
Publication date: 2017-08-29

Abstract

The invention discloses a kind of tumour-specific lepirudin 023 ludon, the N-terminal of the lepirudin 023 ludon contains the oligopeptide sequence PLGI that can be recognized and be cracked by GELB (MMP9), and the hirudin for restructuring is hirudin isomers, the hirudin or hirudin fusion protein of hirudin mutant, the HIRULOG truncated, genetic modification.Correspondingly, present invention also offers the preparation method of tumour-specific lepirudin 023 ludon and its application in the medicine for preparing anti-curing oncoma, the preparation method and purposes of the adoptive immunity cell of expression tumour-specific lepirudin 023 ludon are additionally provided.The lepirudin 023 ludon of the present invention, hirudin anticoagulant activity is set to carry out tumor-targeting release, construction is difficult thrombosed microenvironment, established emboli even is dissolved to reach anti-freezing, thrombolysis, prevent the function of Nasopharyngeal neoplasms, and this just overcomes the risk that wild type hirudin systemic administration causes systemic bleeding.

Description

A kind of tumour-specific lepirudin 023 ludon and its production and use

Technical field

The present invention relates to biological therapy and biomedicine field, more particularly to a kind of tumour-specific lepirudin 023 ludon and its Preparation method and purposes.

Background technology

Hirudin is the activity most strong natural thrombin inhibitor found so far, initially from Hementaria officianalis saliva It is isolated in gland, it is made up of 65~66 amino acid, can be directly with fibrin ferment with l：L (mol ratio) mode combines to form non- Covalent complex, so that fibrin ferment loses the ability of cracking fibrinogen, suppresses the formation of thrombus.Natural hirudin is present More than ten kinds variants, mainly there is referred to as tri- kinds of homologys of HV1, HV2, HV3 very high isomers (Hirudin Variant).

At present, external existing two lepirudin 023 ludon products go through to list：1.Desirudin (trade names：Revasc, Switzerland's Novartis products), 2.Lepirudin (trade names：Refludan, Britain Pharmion and the U.S. BerlexLaboratories products).Only there is fine difference at N- ends in both structures, Desirudin is Val1-Val2, And Lepirudin is Leu1-Tyr2.The prevention and treatment of DVT (DVT) when Desirudin is used to perform the operation, Lepirudin is used for the anticoagulant therapy of heparin-induced thrombocytopenic Disease.In addition, hirudin is preventing and treating unstable Property angina pectoris, disseminated intravascular coagulation, cerebral thrombus, thrombophlebitis and coronary artery thrombosis in terms of all have it is huge latent In clinical value.

High blood coagulation state, such as lung cancer, breast cancer, stomach cancer, cancer of pancreas are there is in Several Kinds of Malignancy patient.Dislike Property tumor patient blood in hypercoagulative state, can not only promote internal thrombosis, and with tumour growth, invasion and attack and shifting It is substantially related.Coagulation function change can cause tumour cell phenotype and activity change, promote tumour cell and blood platelet, endothelium thin Born of the same parents, fibronectin (Fibrinectin) and Von Willebrand factor (vWF) stick, so that tumour is thin Born of the same parents even shift in local multiplication, infiltration to other positions.Thrombotic diseases are the second largest lethal factors of malignant tumor patient, only Inferior to metastases.Drug intervention is carried out early to the high-risk tumor patient of thrombosis, for extension patient survival, reduction The death rate is significant.Research shows that hirudin can also play a role in oncotherapy.Lepirudin 023 ludon passes through its anti-freezing Effect, suppress it is fibrinous formed, tumour cell and fibrin or platelet aggregation can be prevented, make NK cells or other The activity of cytotoxic effector cell is played.It is proved leech extract for treating and can play the tumour of curative effect to have fibrosarcoma, bone and flesh Knurl, angiosarcoma, melanoma and leukaemia etc..Hirudin can also coordinate chemotherapy and radiation, by promoting blood flow in tumour, changing It is apt to anoxia state and heightens the effect of a treatment.

Animal experiment and clinical research show that vein or hypodermic injection hirudin are without obvious toxic-side effects, semilethal agent Amount is easily accepted by much larger than dosage needed for treatment, body, and immunity is weak, and blood platelet, fibrinogen level and blood red egg are not influenceed Bai Hanliang, not easily passs through blood-CSF barrier.No matter acute, subacute toxicity test, hirudin is to breathing, blood pressure, heart rate Do not influence.Hirudin is in vivo without degraded, with active component through kidney excretion.

Hirudin as a kind of efficient, direct, special thrombin inhibitor, with very strong anti-freezing, antithrombotic and A variety of pharmacological activity such as anti-inflammatory.Compared with traditional anticoagulant such as heparin, its therapeutic dose is small, curative effect is high, will not cause Quick reaction.Leech have antigenicity, but its antibody does not do harm to, and prolongs long elimination half-life on the contrary, is that a kind of rare plus effect resists Body.Therefore the application of hirudin will be continuously available developing and promote, and lepirudin 023 ludon will turn into the anti-freezing of a new generation, anti-bolt And anti-cancer agent.But, hirudin can cause blood coagulation relevant parameter again, such as activated partial thromboplastin time (APTT), solidifying Hemase time (TT) and prothrombin time (PT) etc. are significantly raised, so as to cause whole body or systemic bleeding risk.Face On bed, as a preferable anticoagulant, under the conditions of systemic administration, should have clear and definite anti-freezing, anti-bolt effect, but Do not cause bleeding side effect, especially in the clinical practice for the purpose for the treatment of tumour, and its preferable working condition is targeting Property tumor by local play anti-freezing, anti-bolt, antitumor action so that clinical application security improve.In oncotherapy neck How domain, further reduce the side effect of hirudin, and it is the problem of being worth inquiring into increase its oncotherapy targeting.

The content of the invention

It is an object of the invention to provide a kind of tumour-specific lepirudin 023 ludon and expression tumour-specific lepirudin 023 ludon Adoptive immunity cell, at least can solve the problem that one of above mentioned problem.

The key of the guideline of inventive concept is to build hirudin derivative, hirudin anticoagulant activity is carried out tumour Targeting NO release, i.e., under normal operation, the hirudin derivative is without anticoagulating active, and when positioned at knub position, the leech The anticoagulating active of plain derivative is just in tumor by local release, and construction is difficult thrombosed microenvironment, or even dissolves established Emboli is to reach anti-freezing, thrombolysis, prevent the function of Nasopharyngeal neoplasms.This just overcomes wild type hirudin systemic administration Cause the risk of systemic bleeding.

Matrix metalloproteinase (MMPs) is the Zn that a class is secreted with inactive zymogen forms²⁺Dependence endopeptidase man Race, is to constitute the most important proteolytic system of extracellular matrix degraded.MMP9 is GELB, is the weight of its family Want member.The multiple protein collagen that can be degraded in tumor microenvironment, promotes tumor invasion.Substantial amounts of experimental study shows, MMP9 plays critically important effect during the fast breeding of cancer cell, invasion and attack and transfer.MMP9 promotees in the following manner Enter tumor-infiltrated and transfer：Degraded ECM molecular substance；Adjust the adhesion of cell；Coordinate other enzymes degraded blood vessel basement Film and blood vessel surrounding substrate.This is the essential condition of vascular tree growth, and only by degradation process, endothelial cell can just be migrated into Surrounding tissue, and vascular tree is gradually formed, promote the transfer and invasion and attack of tumour cell.It can make gelatin after the effect of MMP9 enzyme-to-substrates Peptide bond fracture between middle Gly-Ile.

The single chain polypeptide that hirudin is made up of 65~66 amino acid, its N-terminal can be with catalyzed by thrombin active sites Point is combined, and with anticoagulating active, C-terminal is combined with the substrate recognition site of fibrin ferment, there is very strong specificity parent to fibrin ferment And effect.The present invention devises the measure of a closing hirudin N-terminal, to reach that the anti-freezing for reducing the hirudin derivative is lived Property purpose, while also have the target-seeking function of cancer target.When the hirudin derivative makes positioned at tumor microenvironment, using swollen MMP9 enzyme features are rich in knurl microenvironment, the hirudin that the hirudin derivative N-terminal is closed is replied again as hirudin Original shape, selectively targeted anticoagulation is played in tumor by local, so that as a class is new, safe and effective tumor-targeting Anti-freezing, anticancer.

To achieve the above object, according to an aspect of the invention, there is provided a kind of tumour-specific lepirudin 023 ludon, weight The N-terminal of group hirudin contains can be by the oligopeptide sequence that MMP9 is recognized and is cracked, and the hirudin for restructuring is hirudin isomery Body, hirudin mutant, the HIRULOG truncated, the hirudin or hirudin fusion protein of genetic modification.

Further, oligopeptide sequence is can be by the small peptide that MMP9 recognize and is cracked, and its architectural feature is XnGI, and Xn is represented times The arbitrary amino acid for quantity of anticipating.

Lepirudin 023 ludon is that have oligopeptides PLG in hirudin isomers HV2 or HV3 N-terminal modification, with hirudin isomers HV2 or HV3 N-terminal sequence I collectively constitutes the substrate peptide sequences PLGI, lepirudin 023 ludon PLG-HV2 of MMP9 identifications and digestion Amino acid sequence is：SEQ ID NO.1, gene order is：SEQ ID NO.2, lepirudin 023 ludon PLG-HV3 amino acid sequences For：SEQ ID NO.3, gene order is：SEQ ID NO.4；

Or, it is I hirudin isomers HV2 or HV3 that the lepirudin 023 ludon, which is human albumin, with N-terminal amino acid The fusion protein HPH2 being formed by connecting by oligopeptides PLG, or HPH3, HPH2 amino acid sequence is：SEQ ID NO.17, gene sequence It is classified as：SEQ ID NO.18, HPH3 amino acid sequences are：SEQ ID NO.19, gene order is：SEQ ID NO.20；

Or, the lepirudin 023 ludon is Fc sections of people's gomphosis immunoglobulin with N-terminal amino acid be I hirudin it is different The fusion protein F PH2 that structure body HV2 or HV3 is formed by connecting by oligopeptides PLG, or FPH3, Fc sections of people's gomphosis immunoglobulin are included The hinge area of human IgG 4, Fc sections of sequences and IgM cauda sequences, FPH2 amino acid sequences are：SEQ ID NO.21, gene order is： SEQ ID NO.22, FPH3 amino acid sequences are：SEQ ID NO.23, gene order is：SEQ ID NO.24.

Tumour cell can build tumor microenvironment, the characteristics of by analyzing tumor microenvironment, can find cancer target The target of property.Research shows α v β 3 in kinds of tumor cells such as lung cancer, breast cancer, prostate cancer, carcinoma of urinary bladder, glioblastoma And the height expression of the surface such as wellability melanoma.The strong expression in tumor tissues neovascular endothelium cell, in ripe blood α v β 3 expression is very low in endothelial cell and most normal structures.In tumor tissues, tumor cell secretion Growth factor activates epithelial cell, and the expression of wherein integrin alpha v beta 3 is very high, and this causes α v β 3 to be controlled as cancer target The preferable site treated.Compared with Normal tissue vascular system, tumor tissues great expression α v β 3, α v β 3 are new in tumor tissues Expression in raw capillary system is higher by much than normal structure.Integrin alpha v beta 3 can recognize extracellular matrix protein, Specifically bound with it, the adhesion and migration of mediate tumor cell, in the growth of tumour, infiltration, shift especially angiogenesis In play a significant role.α v β 3 can be with a variety of protein bindings containing RGD sequence, such as vitronectin, fiber knot Hop protein, fibrin element are former, fibrin ferment is sensitive plain and some simulation RGD sequences endogenous proteins.The antibody of some designs Or the antagonists of α v β 3 of small-molecular-weight, it can suppress viscous between vascular endothelial cell and vitronectin in model in vivo Attached, the angiogenesis of tumour is significantly reduced.

In conventional research, RGD sequence is often by the carrier as targeted drug, by drug targeting to expression integrin alpha v beta 3 Tumor vasculature go.RGD sequence is found as the binding site of fibronectin and cell, and it is recognized substantially Site is made up of tri- amino acid of Arg-Gly-Asp.In RGD derivative, c (RGDfK) often by the carrier as medicine because Lysine residue K be it is optimal can with other chemical substances occur coupling reaction group.People use phage technology It is found that another RGD part that can be specifically bound with α v β 3：RGD sequence (ACDCRGDCFCG) containing two disulfide bond, People are called RGD4C.Its binding ability is 20 times containing a disulfide bond RGD sequence, is the 200 of generally linear RGD sequence Times.RGD4C shortcoming is can to form different cyclic structures, if foring additional monocyclic and bicyclic ring structures, and it is tied Conjunction ability reduces by 10 times.The two is compared, and c (RGDfK) stability is preferable and is easily coupled with other chemical groups, more suitable Cooperate the carrier for chemical coupling.And RGD4C is then suitable as the target medicine carrier designed with recombinant technique.

Further, lepirudin 023 ludon, be targeted integration element α v β 3 polypeptide RGD4C and N-terminal amino acid be I leech The fusion protein RPH2 that plain isomers HV2 or HV3 is formed by connecting by Linker and oligopeptides PLG, or RPH3, RPH2 amino acid sequence It is classified as：SEQ ID NO.13, gene order is：SEQ ID NO.14, RPH3 amino acid sequences are：SEQ ID NO.15, gene Sequence is：SEQ ID NO.16.

Understood at present, the N-terminal of hirudin molecule is its core, containing three pairs of disulfide bond (Cys6-Cys14, Cys16-Cys28, Cys32-Cys39), make N-terminal peptide chain around dense core cyclic peptide structures are changed into, the catalysis with fibrin ferment is lived Property site combine；And C-terminal is the chain structure of a random stretching, extension, wherein Gln49~Gly54 has space and hydrophobic effect, And Asp55~Pro60 is rich in acidic amino acid, with negative electrical charge, the easily substrate recognition site with fibrin ferment alkalescence is combined.HV1/ HV2 Ser32-Asp33-Gly34-Glu35 (SDGE) or HV3 Ser32-Asn33-Gly34-Lys35 (SNGK) are one prominent For the Loop structures between beta sheet, conformation is free, to activity without obvious effect., will be wild according to the above characteristic of hirudin The Ser32-Asn33-Gly34-Glu35 of type hirudin (HV2/HV3) peptide chain replaces with Arg32-Gly33-Asp34-Met35 (RGDM).RGD sequence is made up of arginine, glycine and aspartic acid, is present in various kinds of cell epimatrix, can be with integrin α v β 3 are specifically bound.The lepirudin 023 ludon modified by RGDS or RGDM can more preferable target tumor microenvironment integrin α v β 3, are discharged hirudin by MMP9 protease, play anti-freezing, anti-bolt and antitumor action.

Further, the lepirudin 023 ludon, is by the Ser32-Asn33- of wild type hirudin HV2 or HV3 peptide chain Gly34-Glu35 replaces with Arg32-Gly33-Asp34-Ser35, then in its N-terminal modification PLG oligopeptides, the restructuring leech Plain PLG-HV2-RGDS amino acid sequences are：SEQ ID NO.5, gene order is：SEQ ID NO.6, lepirudin 023 ludon PLG- HV3-RGDS amino acid sequences are：SEQ ID NO.7, gene order is：SEQ ID NO.8；

Or, the lepirudin 023 ludon is by the Ser32-Asn33-Gly34- of wild type hirudin HV2 or HV3 peptide chain Glu35 replaces with Arg32-Gly33-Asp34-Met35, then in its N-terminal modification PLGL oligopeptides, the lepirudin 023 ludon PLG- HV2-RGDM amino acid sequences are：SEQ ID NO.9, gene order is：SEQ ID NO.10, lepirudin 023 ludon PLG-HV3- RGDM amino acid sequences are：SEQ ID NO.11, gene order is：SEQ ID NO.12.

Correspondingly, present invention also offers the preparation method of above-mentioned tumour-specific lepirudin 023 ludon, comprise the following steps： The above-mentioned tumour-specific lepirudin 023 ludon genetic fragment of chemical synthesis, signal peptide sequence is connected in its N-terminal, and in restructuring coding The two ends addition restriction enzyme site of gene, Prepare restructuring carrier in genophore is inserted by restructuring encoding gene, heavy with this The conversion of group carrier or transfection host cell, cultivate host cell, and therefrom reclaim and purify the tumour-specific lepirudin 023 ludon, Host cell is Escherichia coli, lactic acid bacteria, saccharomycete, insect cell or mammalian cell.

Correspondingly, anti-freezing, antithrombotic, preventing and treating are being prepared present invention also offers above-mentioned tumour-specific lepirudin 023 ludon Application in the medicine of tumour, especially anti-curing oncoma occurs together hypercoagulative state and medicine, health products and the function of Thrombotic lesion Application in property food.

Correspondingly, present invention also offers the adoptive immunity cell preparation side for expressing above-mentioned tumour-specific lepirudin 023 ludon People source coding sequence of secretory signal peptide encoding gene is recombinated leech by method, vitro culture of human source immunocyte with above-mentioned tumour-specific The encoding gene of element is connected, and adds restriction enzyme site at the two ends of restructuring encoding gene, by encoding gene insertion vector In be prepared into recombination carrier, transfect immunocyte, obtain the adoptive immunity cell of expression tumour-specific lepirudin 023 ludon.

Further, people source coding sequence of secretory signal peptide behaviour amidating enzyme signal peptide, amino acid sequence is：SEQ ID NO.25, gene order is：SEQ ID NO.26.

Further, immunocyte is one kind in α β T, gamma delta T, NKT, NK, DC, CIK, CAR-T, CAR-NK, TCR-T Or a variety of, the immunocyte behaviour source immunocyte.

Further, the Membrane surface expression of adoptive immunity cell has target molecules, and target molecules are positioned by cross-film sequence In cell membrane surface.

Further, target molecules are CD19 total length or its truncated segment, or CD20 total length or its truncated segment.

Further, the structure of target molecules and cross-film section is：GM-CSFR signal peptides-Δ CD20-Linker2-CD28 across Film section, its amino acid sequence is：SEQ ID NO.27, gene order is SEQ ID NO.28.

Using adoptive immunity cell as carrier, the continual and steady expression tumour-specific lepirudin 023 ludon in tumor patient body, The effect in " minicell pharmaceutical factory " is played, both by adoptive cellular immunotherapy technology and the anti-bolt of tumor-targeting lepirudin 023 ludon Anti-cancer function is integrated, and preferably plays antitumor action, can recombinate water by expression tumour-specific steady in a long-term in vivo again Leech element, it is to avoid inject tumour-specific lepirudin 023 ludon repeatedly and cause potential allergic reaction.

Correspondingly, answering present invention also offers the adoptive immunity cell of above-mentioned expression tumour-specific lepirudin 023 ludon With, for anti-freezing, antithrombotic, anti-curing oncoma biological therapy, especially prevent and treat with hypercoagulative state and/or the tumour of thrombus trouble The biological therapy of person.

Beneficial effects of the present invention are：The N-terminal of the tumour-specific lepirudin 023 ludon of the present invention contains and can known by MMP9 Not simultaneously the small peptide of digestion, can not close the catalyzed by thrombin avtive spot of hirudin N-terminal, to reach that reducing the hirudin derives The anticoagulating active of thing, the purpose for reducing hemorrhage side effect, while also having the target-seeking function of cancer target.When the hirudin derivative When making positioned at tumor microenvironment, MMP9 enzyme features, specific excision hirudin derivative N are rich in using in tumor microenvironment The blocking peptide of end, makes the hirudin being closed reply again for hirudin original shape, and playing specific anti-freezing in tumor by local makees With so as to turn into a new class, safe and effective tumor-targeting anti-freezing, anti-bolt and anticancer.The present invention is further carried Supply to express the immunocyte of tumour-specific lepirudin 023 ludon, by using adoptive immunity cell as carrier, lasting table in vivo Up to tumour-specific lepirudin 023 ludon, it is possible to resolve the drawbacks of hirudin half-life short, without repetitively administered in the short time, it can avoid Repetitively administered causes allergy or induction of antibodies to produce, and is more suitable for needing long-term anti-freezing, the tumor patient of anti-bolt.Express tumour special The adoptive immunity cell of different in nature lepirudin 023 ludon can target tumor local expression, improvement tumor patient hypercoagulative state, the micro- blood of dissolving Bolt, prevents metastases, promotes immunocyte to enter inside tumor and plays killing ability, can be used alone or it is clinical with combination/ Radiotherapy is used.

Embodiment

The invention will be further described below.

The N-terminal of the tumour-specific lepirudin 023 ludon of the present invention contains can be by MMP9 is recognized and is cracked oligopeptides sequence Row, the hirudin for restructuring is hirudin isomers, hirudin mutant, the HIRULOG or hirudin fusion protein truncated. Oligopeptide sequence is can be recognized the small peptide of simultaneously digestion by MMP9, and its architectural feature is XnGI, and Xn represents any number of any amino Acid.

Natural hirudin has a variety of isomers, wherein three kinds of main isomers are respectively Hirudin variant 1 (HV1)、Hirudin variant 2(HV2)、Hirudin variant 3(HV3)。

HV2 and HV3 N-terminal amino acid is I, and MMP9 physiologic substrate sequence is GI, and breakaway poing position is G ↓ I after digestion. The PLG small peptides in HV2 or HV3 N-terminal connection, or other XXXG types have the small peptide of equivalent efficacy, the N ends with HV2 or HV3 The I at end collectively constitutes MMP9 substrate sequence, after being cracked by MMP9 between G and I, and the anti-freezing that can completely discharge hirudin is lived Property.

Further, the end of lepirudin 023 ludon is also associated with the polypeptide for targeted integration element α v β 3, and the polypeptide is included RGD sequence.The integrin alpha v beta 3 of the more preferable target tumor microenvironment of above-mentioned hirudin derivative can then be made.Specific RGD sequence For RGD4C sequences.After RGD4C oriented carriers are combined with tumor cell surface α v β 3, hirudin original shape is discharged by MMP9 Out, anti-freezing, anti-bolt and antitumor action are played.

Correspondingly, present invention also offers the preparation method of above-mentioned tumour-specific lepirudin 023 ludon, comprise the following steps： The above-mentioned tumour-specific lepirudin 023 ludon genetic fragment of chemical synthesis, signal peptide is connected in its N-terminal, and in restructuring encoding gene Two ends addition restriction enzyme site, will restructuring encoding gene insertion genophore in Prepare restructuring carrier, with the restructuring carry Body is converted or transfection host cell, cultivates host cell, and therefrom reclaim and purify the tumour-specific lepirudin 023 ludon, host Cell is Escherichia coli, lactic acid bacteria, yeast, insect cell or mammalian cell.

Carrier for restructuring is preferably carrier for expression of eukaryon, preferably pPIC9K.Host cell is preferably first Alcohol auxotype yeast strain GS115.

Correspondingly, present invention also offers the adoptive immunity cell preparation side for expressing above-mentioned tumour-specific lepirudin 023 ludon Method, people source coding sequence of secretory signal peptide encoding gene is connected with the encoding gene of above-mentioned tumour-specific lepirudin 023 ludon, is prepared Into recombination carrier, immunocyte is transfected, the adoptive immunity cell of expression tumour-specific lepirudin 023 ludon is obtained.

The Membrane surface expression of the adoptive immunity cell has target molecules, and target molecules are positioned at cell membrane by cross-film sequence Surface, tumour-specific lepirudin 023 ludon is directly expressed or carries out induced expression by being coupled inducible expression.Target molecules For CD19 total length or its truncated segment, or CD20 total length or its truncated segment.

The adoptive immunity cell of present invention expression tumour-specific lepirudin 023 ludon, tumour-specific lepirudin 023 ludon can be with Constructive expression, can also be coupled inducible expression and carry out induced expression.It can be that tetracycline is lured to be coupled inducible expression Lead expression system, moulting hormone (ecdysone) inducible expression, tacrolimus (tacrolimus, FK506)/rapamycin (rapamycin) inducible system or RU486 inducible systems.Immunocyte can be α β T, gamma delta T, NKT, NK, DC, CIK, CAR- One or more in T, CAR-NK, TCR-T, immunocyte behaviour source immunocyte.

The adoptive immunity cell of expression tumour-specific lepirudin 023 ludon also expresses suicide gene/prodrug system.Suicide base Cause/prodrug system is cyclic guanosine (HSV-tk/GCV) system of herpes simplex virus type 1 thymidine kinase/penta, herpes zoster disease Flucytosine (the CD/ of malicious thymidine kinase/arabinose methoxypurine (VZV-tk/Ara-M) system, cytosine deaminase/5 5FC) system, Cytochrome P450 microsomal enzyme CYP2B1/ endoxan (CYP2B1/CPA) system；Cytochrome P450 CYP4B1/ amino anthracenes (CYP4B1/2AA) system, deoxycytidine kinase/cytarabine (dCK/Ara-C) system, black glycosides-Huang are fast Purine phosphoribosyl transferase/6- Sulfurs purine (gpt/6TX) system, nitroreductase/CB1954 (NTR/CB1954) system, Purine nucleoside phosphorylase/6- methyl purines deoxyribonucleoside (PNP/6-MeP-dR) system, thymidine phosphorylase/5 '-de- Oxygen -5 FU 5 fluorouracil (TP/5 '-DFUR) system, carboxypeptidase G2/CMDA systems (CPG2/CMDA), carboxy-lesterase/CPT-11 (CE/CPT-11) one kind in system.

The preparation method of the adoptive immunity cell of above-mentioned expression tumour-specific lepirudin 023 ludon, comprises the following steps： The genophore of the specific lepirudin 023 ludon of codes for tumor and membrane-type molecules target is prepared, genophore includes (signal peptide sequence Row-tumour-specific lepirudin 023 ludon sequence) and two sections of (signal peptide sequence-target molecules sequence-Linker- cross-film section sequence) Gene order；The genophore of the specific lepirudin 023 ludon of above-mentioned codes for tumor and target molecules is turned by technique for gene engineering Contaminate immunocyte.

Technique for gene engineering is known technology, and gene transfection system used includes slow-virus transfection system, retrovirus Transfection system, Adenovirus Transfection system, adeno-associated virus transfection system, sleeping beauty transposon stand transfection system, plasmid electrotransfection system System.It is preferred that, using sleeping beauty transposon stand transfection system or electrotransfection system.With transient expression vector, by tumour-specific weight Group hirudin gene imports adoptive immunity cell, its exogenous gene expression amount can be made controllable, it is to avoid the risk of overexpression.

The structure of genophore of codes for tumor specificity lepirudin 023 ludon and membrane-type molecules target is：Signal peptide-tumour Specific lepirudin 023 ludon-IRES- signal peptides-target molecules-Linker- cross-films section, or, signal peptide-target molecules- Linker- cross-films section-P2A- signal peptides-tumour-specific lepirudin 023 ludon.

With reference to embodiment, the present invention is further detailed explanation.

Embodiment 1

Lepirudin 023 ludon is that have oligopeptides PLG in hirudin isomers HV2 N-terminal modification in the present embodiment, with hirudin Isomers HV2 N-terminal sequence I collectively constitutes the substrate peptide sequences PLGI that MMP9 is recognized and cracked, tumour-specific restructuring water Leech element PLG-HV2, amino acid sequence is：SEQ ID NO.1, gene order is：SEQ ID NO.2.

Wherein, PLGI can be to include GI sequences by other MMP9 identifications and the replacement of digestion substrate peptide sequences, its common feature Row, to ensure after MMP9 identifications and digestion, the amino acid of the hirudin N-terminal of release is I.

The application of the tumour-specific lepirudin 023 ludon in the medicine for preparing anti-curing oncoma, especially anti-curing oncoma occurs together Application in the medicine of hypercoagulative state and Thrombotic lesion.

Embodiment 2

Lepirudin 023 ludon is that have oligopeptides PLG in hirudin isomers HV3 N-terminal modification in the present embodiment, with hirudin Isomers HV3 N-terminal sequence I collectively constitutes the substrate peptide sequences PLGI, lepirudin 023 ludon PLG- of MMP9 identifications and digestion HV3, amino acid sequence is：SEQ ID NO.3, gene order is：SEQ ID NO.4.

Wherein, PLGI can be to include GI sequences by other MMP9 identifications and the replacement of digestion substrate peptide sequences, its common feature Row, to ensure after MMP9 digestions, the amino acid of the hirudin N-terminal of release is I.

Embodiment 3

Lepirudin 023 ludon in the present embodiment, is by the Ser32-Asn33-Gly34-Glu35 of wild type hirudin HV2 peptide chains Arg32-Gly33-Asp34-Ser35 is replaced with, then in its N-terminal modification PLG oligopeptides, lepirudin 023 ludon PLG-HV2-RGDS ammonia Base acid sequence is：SEQ ID NO.5, gene order is：SEQ ID NO.6.

Embodiment 4

The hirudin for being used to recombinate in the present embodiment is hirudin mutant, is by wild type hirudin HV3 peptide chains Ser32-Asn33-Gly34-Lys35 replaces with Arg32-Gly33-Asp34-Ser35, then in its N-terminal modification PLG oligopeptides, The structure of lepirudin 023 ludon is：PLG-HV3-RGDS, amino acid sequence is：SEQ ID NO.7, gene order is：SEQ ID NO.8。

Embodiment 5

Lepirudin 023 ludon in the present embodiment, is by the Ser32-Asn33-Gly34-Glu35 of wild type hirudin HV2 peptide chains Arg32-Gly33-Asp34-Met35 is replaced with, then in its N-terminal modification PLGL oligopeptides, lepirudin 023 ludon PLG-HV2-RGDM Amino acid sequence is：SEQ ID NO.9, gene order is：SEQ ID NO.10.

Embodiment 6

The hirudin for being used to recombinate in the present embodiment is hirudin mutant, is by wild type hirudin HV3 peptide chains Ser32-Asn33-Gly34-Lys35 replaces with Arg32-Gly33-Asp34-Met35 (RGDM), lepirudin 023 ludon PLG-HV3- RGDM, amino acid sequence is：SEQ ID NO.11, gene order is：SEQ ID NO.12.

Embodiment 7

The lepirudin 023 ludon of the present embodiment, be targeted integration element α v β 3 polypeptide RGD4C and N-terminal amino acid be I water The fusion protein RPH2 (RGD4C-PLG-HV2) that leech element isomers HV2 is formed by connecting by Linker and oligopeptides PLG, recombinates water Leech element structure be：RGD4C-Linker1-MMP9 restriction enzyme site-HV2, amino acid sequence is：SEQ ID NO.13 gene orders For：SEQ ID NO.14.

Wherein, RGD4C can be by other short peptide sequence replacements targetted with reference to α v β 3 comprising RGD sequence.PLGI can quilt Other MMP9 identifications and the replacement of digestion substrate peptide sequences, its common feature is includes GI sequences, to ensure after MMP9 digestions, The amino acid of the hirudin N-terminal of release is I；Linker1 length is 2~15 amino acid.

Embodiment 8

The lepirudin 023 ludon of the present embodiment, be targeted integration element α v β 3 polypeptide RGD4C and N-terminal amino acid be I water The fusion protein RPH3 (RGD4C-PLG-HV3) that leech element isomers HV3 is formed by connecting by Linker and oligopeptides PLG, recombinates water Leech element structure be：RGD4C-Linker1-MMP9 restriction enzyme site-HV3, amino acid sequence is：SEQ ID NO.15, gene sequence It is classified as：SEQ ID NO.16.

Wherein, RGD4C can be by other short peptide sequence replacements targetted with reference to α v β 3 comprising RGD sequence.PLGI can quilt Other MMP9 identifications and the replacement of digestion substrate peptide sequences, its common feature is includes GI sequences, to ensure after MMP9 digestions, The amino acid of the hirudin N-terminal of release is I.Linker1 length is 2~15 amino acid.

Embodiment 9

The hirudin isomers HV2 that the lepirudin 023 ludon of present embodiment is human albumin with N-terminal amino acid is I passes through The fusion protein HPH2 (HSA-PLG-HV2) that oligopeptides PLG is formed by connecting, HPH2 amino acid sequences are：SEQ ID NO.17, gene Sequence is：SEQ ID NO.18

Embodiment 10

The hirudin isomers HV3 that the lepirudin 023 ludon of present embodiment is human albumin with N-terminal amino acid is I passes through The fusion protein HPH3 (HSA-PLG-HV3) that oligopeptides PLG is formed by connecting, HPH3 amino acid sequences are：SEQ ID NO.19, gene Sequence is：SEQ ID NO.20.

Embodiment 11

The hirudin that the lepirudin 023 ludon of present embodiment is Fc sections of people's gomphosis immunoglobulin with N-terminal amino acid is I The fusion protein F PH2 (Fc-PLG-HV2) that isomers HV2 is formed by connecting by oligopeptides PLG, Fc sections of bags of people's gomphosis immunoglobulin Containing the hinge area of human IgG 4, Fc sections of sequences and IgM cauda sequences, FPH2 amino acid sequences are：SEQ ID NO.21, gene order For：SEQ ID NO.22.

Embodiment 12

The hirudin that the lepirudin 023 ludon of present embodiment is Fc sections of people's gomphosis immunoglobulin with N-terminal amino acid is I The fusion protein F PH3 (Fc-PLG-HV3) that isomers HV3 is formed by connecting by oligopeptides PLG, Fc sections of bags of people's gomphosis immunoglobulin Containing the hinge area of human IgG 4, Fc sections of sequences and IgM cauda sequences, FPH3 amino acid sequences are：SEQ ID NO.23, gene order For：SEQ ID NO.24.

Embodiment 13

The preparation method of the tumour-specific lepirudin 023 ludon of the present embodiment, comprises the following steps：The above-mentioned reality of chemical synthesis Any tumour-specific lepirudin 023 ludon genetic fragment of example 1~12 is applied, and connects into encoding gene, encoding gene is inserted Enter Prepare restructuring carrier in carrier, with recombinant vector conversion or transfection host cell, host cell is Escherichia coli, lactic acid Bacterium, yeast, insect cell or mammalian cell, cultivate host cell, supernatant are collected by centrifugation afterwards, through being concentrated by ultrafiltration, coagulating Glue filtering, the purifying of ion exchange three-step approach obtain tumour-specific lepirudin 023 ludon.

The carrier that the present embodiment is used to recombinate is preferably carrier for expression of eukaryon, using pPIC9K.Host cell is methanol Auxotype yeast strain GS115.

Host cell GS115 fermentation process is：Engineering bacteria is expanded with less salt culture medium, with containing trace element before induction Glycerite is carried out after feed supplement, culture propagation, carries out induced expression with the methanol solution containing trace element, fermentation parameter is：Temperature 30 DEG C of degree, oxygen capacity control are interlocked in 35 ± 5%, pH=5, mixing speed and DO.

Embodiment 14

The adoptive immunity cell preparation method of expression tumour-specific lepirudin 023 ludon is to compile people source coding sequence of secretory signal peptide The encoding gene of the code gene tumour-specific lepirudin 023 ludon any with above-described embodiment 1~12 is connected, and is prepared into restructuring base Because of carrier, immunocyte is transfected, the adoptive immunity cell of expression tumour-specific lepirudin 023 ludon is obtained.

The Membrane surface expression of adoptive immunity cell has target molecules, and target molecules are positioned at cell membrane table by cross-film sequence Face.The Membrane surface expression of the adoptive immunity cell has target molecules, and target molecules are positioned at cell membrane surface by cross-film sequence. Total length or its truncated segment that target molecules are CD19, or CD20 total length or its truncated segment.

The present embodiment expression tumour-specific lepirudin 023 ludon adoptive immunity cell preparation method include step S1~ S3, it is specific as follows.

S1, vitro culture of human source CIK cell, including step A PMNC (PBMC) are gathered with step B's CIK cell culture.

A, the collection of PMNC have steps of：

A1, the extraction peripheral blood 50-100ml from patient's body；

PBMC is further purified in A2, lymphocyte separation medium density-gradient centrifugation method；

A3, serum-free medium are washed 2 times, obtain PBMC of the purity more than 90%.

B, CIK cell culture：

B1, by PBMC press 1~2 × 10⁶/ ml concentration is suspended in serum-free medium, adds 1,000U/ml restructuring People's IFN-γ, 37 DEG C, 5%CO₂Cultivated in incubator；

50ng/ml CD3 monoclonal antibodies and 300U/ml recombinant human il-2 are added after B2,24h, CIK cell is stimulated Growth and propagation；

Note：100U/ml recombined human IL-1 α now can be also added simultaneously.

B3, every 3 days half amounts change liquid or expand bottle once, and add recombinant human il-2 300U/ml；

B4, the 14d in culture, harvest CIK cell.

Wherein, 5%~20% autoserum can be also added in cell culture medium.

S2, Prepare restructuring genophore

This implementation row use tumour-specific lepirudin 023 ludon RPH2 (RGD4C-PGR-HV2) gene order, fusion protein RPH2 amino acid sequence is：SEQ ID NO.13, gene order is：SEQ ID NO.14.Water is recombinated according to tumour-specific Leech element RPH2 gene order, people's amidating enzyme signal peptide sequence is added in its N-terminal, and its amino acid sequence is：SEQ ID NO.25, gene order is SEQ ID NO.26.Chemical synthesis genetic fragment, connects into restructuring encoding gene, N-terminal addition The restriction enzyme sites of Nhe I, the C-terminal addition restriction enzyme sites of EcoR I.

This implementation row use target molecules structure for：GM-CSFR signal peptides-Δ CD20-Linker2-CD28 cross-films section, its Amino acid sequence is：SEQ ID NO.27, gene order is SEQ ID NO.28.Chemical synthesis genetic fragment, N-terminal addition BamH I restriction enzyme site, the C-terminal addition restriction enzyme sites of Not I.

The genophore of the specific lepirudin 023 ludon of codes for tumor and membrane-type molecules target is prepared using technique for gene engineering, By signal peptide sequence-tumour-specific lepirudin 023 ludon sequence and signal peptide sequence-target molecules sequence-Linker- cross-films section Two sections of genes of sequence are cloned into two cloning sites of pIRES plasmids respectively.Technique for gene engineering used is known technology, summary It is as follows：

The digestion with restriction enzyme system of standard is set up according to existing method, is then handled using Nhe I and EcoR I PIRES plasmids, reclaim large fragment；Handled using identical restriction enzyme Nhe I and EcoR I and to reclaim recombinant tumor special Property hirudin RPH genetic fragments, then the carrier pIRES large fragment good with above-mentioned digestion be connected, construct recombinant tumor special Property hirudin expression Plasmid pIRES-RPH；Convert after bacillus coli DH 5 alpha, filter out positive colony and expand.

PIRES-RPH2 plasmids are purified, then using BamH I and Not I processing pIRES-RPH2 plasmids, large fragment are reclaimed； Handled using identical restriction enzyme BamH I and Not I and reclaim leukine R signal peptide-Δ CD20-Linker2- CD28 cross-films section genetic fragment, then the pIRES-RPH2 carrier large fragment good with above-mentioned digestion be connected, construct expression simultaneously The double expression plasmid pIRES-RPH2/CD20 of recombinant tumor specificity hirudin and membrane-type molecules target CD20.Convert large intestine bar After bacterium DH5 α, filter out positive colony and expand, purify pIRES-RPH2/CD20 plasmids.

The pIRES-RPH2/CD20 recombinant plasmids that S3, purifying are obtained through step S2, with the recombinant vector with plasmid electrotransfection The CIK cell that method transfection procedure S1 is obtained, carries out more than cell culture 36h afterwards, and the CIK for obtaining expressing lepirudin 023 ludon is thin Born of the same parents, transfection efficiency is obtained through flow cytomery, is needed positive cell quantity needed for calculating according to clinic, is fed back tumor patient.

The CIK cell of the expression tumour-specific lepirudin 023 ludon, the biology for anti-freezing, antithrombotic, anti-curing oncoma is controlled Treat, especially prevent and treat the biological therapy of the tumor patient with hypercoagulative state and/or thrombus.

Above-described is only some embodiments of the present invention.For the person of ordinary skill of the art, not On the premise of departing from the invention design, various modifications and improvements can be made, these belong to the protection model of the present invention Enclose.

Sequence table

<110>He Xiangfeng

<120>A kind of tumour-specific lepirudin 023 ludon and its production and use

<160>28

<210>1

<211>68

<212>PRT

<213>Artificial sequence

<400>1

Pro Leu Gly Ile Thr Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn

5 10 15

Leu Cys Leu Cys Glu Gly Ser Asn Val Cys Gly Lys Gly Asn Lys

20 25 30

Cys Ile Leu Gly Ser Asn Gly Glu Glu Asn Gln Cys Val Thr Gly

35 40 45

Glu Gly Thr Pro Lys Pro Gln Ser His Asn Asn Gly Asp Phe Glu

50 55 60

Glu Ile Pro Glu Glu Tyr Leu Gln

65

<210>2

<211>207

<212>DNA

<213>Artificial sequence

<400>2

cccttaggta ttacttacac tgattgtaca gaatcgggtc aaaatttgtg cctctgcgag 60

ggaagcaatg tttgcggtaa aggcaataag tgcatattgg gttctaatgg agaggaaaac 120

caatgtgtca ctggcgaagg tacaccgaag cctcaaagcc ataataacgg cgatttcgaa 180

gaaattccag aagaatattt acaatga 207

<210>3

<211>70

<212>PRT

<213>Artificial sequence

<400>3

Pro Leu Gly Ile Thr Tyr Thr Asp Cys Ile Glu Ser Gly Gln Asn

5 10 15

Leu Cys Leu Cys Glu Gly Ser Asn Val Cys Gly Lys Gly Asn Lys

20 25 30

Cys Ile Leu Gly Ser Asn Gly Lys Asp Asn Gln Cys Val Thr Gly

35 40 45

Glu Gly Thr Pro Lys Pro Gln Ser His Asn Gln Gly Asp Phe Glu

50 55 60

Pro Ile Pro Glu Asp Ala Tyr Asp Glu Lys

65 70

<210>4

<211>213

<212>DNA

<213>Artificial sequence

<400>4

cccttaggta ttacttacac tgattgtata gaatcgggtc aaaatttgtg cctctgcgag 60

ggaagcaatg tttgtggtaa aggcaataag tgcatattgg gttctaatgg aaaggacaac 120

caatgtgtca ctggcgaagg tacaccgaag cctcaaagcc ataatcaagg cgatttcgaa 180

ccaattccag aagacgctta tgatgaaaaa tga 213

<210>5

<211>68

<212> PRT

<213>Artificial sequence

<400>5

Pro Leu Gly Ile Thr Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn

5 10 15

Leu Cys Leu Cys Glu Gly Ser Asn Val Cys Gly Lys Gly Asn Lys

20 25 30

Cys Ile Leu Gly Arg Gly Asp Ser Glu Asn Gln Cys Val Thr Gly

35 40 45

Glu Gly Thr Pro Lys Pro Gln Ser His Asn Asn Gly Asp Phe Glu

50 55 60

Glu Ile Pro Glu Glu Tyr Leu Gln

65

<210>6

<211>207

<212> DNA

<213>Artificial sequence

<400>6

cccttaggta ttacttacac tgattgtaca gaatcgggtc aaaatttgtg cctctgcgag 60

ggaagcaatg tttgcggtaa aggcaataag tgcatattgg gtcgcggaga ttctgaaaac 120

caatgtgtca ctggcgaagg tacaccgaag cctcaaagcc ataataacgg cgatttcgaa 180

gaaattccag aagaatattt acaatga 207

<210>7

<211>70

<212> PRT

<213>Artificial sequence

<400>7

Pro Leu Gly Ile Thr Tyr Thr Asp Cys Ile Glu Ser Gly Gln Asn

5 10 15

Leu Cys Leu Cys Glu Gly Ser Asn Val Cys Gly Lys Gly Asn Lys

20 25 30

Cys Ile Leu GlyArg Gly Asp Ser Asp Asn Gln Cys Val Thr Gly

35 40 45

Glu Gly Thr Pro Lys Pro Gln Ser His Asn Gln Gly Asp Phe Glu

50 55 60

Pro Ile Pro Glu Asp Ala Tyr Asp Glu Lys

65 70

<210>8

<211>213

<212> DNA

<213>Artificial sequence

<400>8

cccttaggta ttacttacac tgattgtata gaatcgggtc aaaatttgtg cctctgcgag 60

ggaagcaatg tttgtggtaa aggcaataag tgcatattgg gtcgcggaga ttctgacaac 120

caatgtgtca ctggcgaagg tacaccgaag cctcaaagcc ataatcaagg cgatttcgaa 180

ccaattccag aagacgctta tgatgaaaaa tga 213

<210>9

<211>68

<212> PRT

<213>Artificial sequence

<400>9

Pro Leu Gly Ile Thr Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn

5 10 15

Leu Cys Leu Cys Glu Gly Ser Asn Val Cys Gly Lys Gly Asn Lys

20 25 30

Cys Ile Leu Gly Arg Gly Asp Met Glu Asn Gln Cys Val Thr Gly

35 40 45

Glu Gly Thr Pro Lys Pro Gln Ser His Asn Asn Gly Asp Phe Glu

50 55 60

Glu Ile Pro Glu Glu Tyr Leu Gln

65

<210>10

<211>207

<212> DNA

<213>Artificial sequence

<400>10

cccttaggta ttacttacac tgattgtaca gaatcgggtc aaaatttgtg cctctgcgag 60

ggaagcaatg tttgcggtaa aggcaataag tgcatattgg gtcgcggaga tatggaaaac 120

caatgtgtca ctggcgaagg tacaccgaag cctcaaagcc ataataacgg cgatttcgaa 180

gaaattccag aagaatattt acaatga 207

<210>11

<211>70

<212> PRT

<213>Artificial sequence

<400>11

Pro Leu Gly Ile Thr Tyr Thr Asp Cys Ile Glu Ser Gly Gln Asn

5 10 15

Leu Cys Leu Cys Glu Gly Ser Asn Val Cys Gly Lys Gly Asn Lys

20 25 30

Cys Ile Leu Gly Arg Gly Asp Met Asp Asn Gln Cys Val Thr Gly

35 40 45

Glu Gly Thr Pro Lys Pro Gln Ser His Asn Gln Gly Asp Phe Glu

50 55 60

Pro Ile Pro Glu Asp Ala Tyr Asp Glu Lys

65 70

<210>12

<211>213

<212> DNA

<213>Artificial sequence

<400>12

cccttaggta ttacttacac tgattgtata gaatcgggtc aaaatttgtg cctctgcgag 60

ggaagcaatg tttgtggtaa aggcaataag tgcatattgg gtcgcggaga tatggacaac 120

caatgtgtca ctggcgaagg tacaccgaag cctcaaagcc ataatcaagg cgatttcgaa 180

ccaattccag aagacgctta tgatgaaaaa tga 213

<210>13

<211>109

<212> PRT

<213>Artificial sequence

<400>13

Met Glu Phe Trp Leu Ser Trp Val Phe Leu Val Ala Ile Leu Lys

5 10 15

Gly Val Gln Cys Ala Cys Asp CysArg Gly Asp Cys Phe Cys Gly

20 25 30

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Pro Leu Gly Ile

35 40 45

Thr Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn Leu Cys Leu Cys

50 55 60

Glu Gly Ser Asn Val Cys Gly Lys Gly Asn Lys Cys Ile Leu Gly

65 70 75

Ser Asn Gly Glu Glu Asn Gln Cys Val Thr Gly Glu Gly Thr Pro

80 85 90

Lys Pro Gln Ser His Asn Asn Gly Asp Phe Glu Glu Ile Pro Glu

95 100 105

Glu Tyr Leu Gln

<210>14

<211>330

<212> DNA

<213>Artificial sequence

<400>14

atggagtttt ggctgagctg ggttttcctt gttgctattt taaaaggtgt ccagtgtgca 60

tgtgattgta ggggagattg tttttgtggt agcggtggcg gtggctctgg tggtggtggc 120

agccccttag gtattactta cactgattgt acagaatcgg gtcaaaattt gtgcctctgc 180

gagggaagca atgtttgcgg taaaggcaat aagtgcatat tgggttctaa tggagaggaa 240

aaccaatgtg tcactggcga aggtacaccg aagcctcaaa gccataataa cggcgatttc 300

gaagaaattc cagaagaata tttacaatga 330

<210>15

<211>111

<212> PRT

<213>Artificial sequence

<400>15

Met Glu Phe Trp Leu Ser Trp Val Phe Leu Val Ala Ile Leu Lys

5 10 15

Gly Val Gln Cys Ala Cys Asp CysArg Gly Asp Cys Phe Cys Gly

20 25 30

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Pro Leu Gly Ile

35 40 45

Thr Tyr Thr Asp Cys Ile Glu Ser Gly Gln Asn Leu Cys Leu Cys

50 55 60

Glu Gly Ser Asn Val Cys Gly Lys Gly Asn Lys Cys Ile Leu Gly

65 70 75

Ser Asn Gly Lys Asp Asn Gln Cys Val Thr Gly Glu Gly Thr Pro

80 85 90

Lys Pro Gln Ser His Asn Gln Gly Asp Phe Glu Pro Ile Pro Glu

95 100 105

Asp Ala Tyr Asp Glu Lys

110

<210>16

<211>336

<212> DNA

<213>Artificial sequence

<400>16

atggagtttt ggctgagctg ggttttcctt gttgctattt taaaaggtgt ccagtgtgca 60

tgtgattgta ggggagattg tttttgtggt agcggtggcg gtggctctgg tggtggtggc 120

agccccttag gtattactta cactgattgt atagaatcgg gtcaaaattt gtgcctctgc 180

gagggaagca atgtttgtgg taaaggcaat aagtgcatat tgggttctaa tggaaaggac 240

aaccaatgtg tcactggcga aggtacaccg aagcctcaaa gccataatca aggcgatttc 300

gaaccaatc cagaagacgc ttatgatgaa aaatga 336

<210>17

<211>653

<212> PRT

<213>Artificial sequence

<400>17

Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly

5 10 15

Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr

20 25 30

Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu

35 40 45

Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu

50 55 60

Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys

65 70 75

Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys

80 85 90

Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His

95 100 105

Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val

110 115 120

Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu

125 130 135

Lys Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr

140 145 150

Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe

155 160 175

Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro

180 185 190

Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys

195 200 205

Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala

210 215 220

Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys

225 230 235

Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys

240 245 250

Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp

255 260 265

Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser

270 275 280

Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu

285 290 295

Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala

300 305 310

Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val

315 320 325

Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe

330 335 340

Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu

345 350 355

Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys

360 365 370

Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp

375 380 385

Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln

390 395 400

Asn Cys Glu Leu Phe Lys Gln Leu Gly Glu Tyr Lys Phe Gln Asn

405 410 415

Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr

420 425 430

Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser

435 440 445

Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu

450 455 450

Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu

455 460 465

Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser

470 475 480

Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu

485 490 495

Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His

500 505 510

Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys

515 520 525

Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr

530 535 540

Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val

545 550 555

Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu

560 565 570

Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu

575 580 585

Pro Leu Gly Ile Thr Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn

590 595 600

Leu Cys Leu Cys Glu Gly Ser Asn Val Cys Gly Lys Gly Asn Lys

605 610 615

Cys Ile Leu Gly Ser Asn Gly Glu Glu Asn Gln Cys Val Thr Gly

620 625 630

Glu Gly Thr Pro Lys Pro Gln Ser His Asn Asn Gly Asp Phe Glu

635 640 645

Glu Ile Pro Glu Glu Tyr Leu Gln

650

<210>18

<211>336

<212> DNA

<213>Artificial sequence

<400>18

gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60

gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120

aaattagtga atgaagtaac tgaatttgca aaaacatgtg tagctgatga gtcagctgaa 180

aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240

cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300

tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360

gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420

gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480

tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540

aagctcgatg aacttcggga tgaagggaag gcttcgtctg ccaaacagag actcaaatgt 600

gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtggc tcgcctgagc 660

cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720

gtccacacgg aatgctgcca tggagatctg cttgaatgtg ctgatgacag ggcggacctt 780

gccaagtata tctgtgaaaa tcaggattcg atctccagta aactgaagga atgctgtgaa 840

aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900

gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960

gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020

tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactcta gagaagtgc 1080

tgtgccgctg cagatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140

gtggaagagc ctcagaattt aatcaaacaa aactgtgagc tttttaagca gcttggagag 1200

tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260

ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gcagcaaatg ttgtaaacat 1320

cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380

tgtgtgttgc atgagaaaac gccagtaagt gacagagtca caaaatgctg cacagagtcc 1440

ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg atgaaacata cgttcccaaa 1500

gagtttaatg ctgaaacatt caccttccat gcagatatatg cacactttct gagaaggag 1560

agacaaatca agaaacaaac tgcacttgtt gagcttgtga aacacaagcc caaggcaaca 1620

aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680

gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740

gctgccttag gcttaccctt aggtattact tacactgatt gtacagaatc gggtcaaaat 1800

ttgtgcctctg cgagggaa gcaatgtttgc ggtaaaggca ataagtgcat attgggttct 1860

aatggagagg aaaaccaatg tgtcactggc gaaggtacac cgaagcctcaa agccataat 1920

aacggcgatt tcgaagaaat tccagaagaa tatttacaat ga 1962

<210>19

<211>655

<212> PRT

<213>Artificial sequence

<400>19

Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly

5 10 15

Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr

20 25 30

Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu

35 40 45

Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu

50 55 60

Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys

65 70 75

Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys

80 85 90

Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His

95 100 105

Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val

110 115 120

Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu

125 130 135

Lys Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr

140 145 150

Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe

155 160 165

Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro

170 175 180

Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys

185 190 195

Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala

200 205 210

Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys

215 220 225

Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys

230 235 240

Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp

245 250 255

Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser

260 265 270

Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu

275 280 285

Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala

290 295 300

Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val

305 310 315

Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe

320 325 330

Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu

335 340 345

Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys

350 355 360

Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp

365 370 375

Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln

380 385 390

Asn Cys Glu Leu Phe Lys Gln Leu Gly Glu Tyr Lys Phe Gln Asn

405 410 415

Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr

420 425 430

Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser

435 440 445

Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu

450 455 450

Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu

455 460 465

Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser

470 475 480

Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu

485 490 495

Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His

500 505 510

Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys

515 520 525

Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr

530 535 540

Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val

545 550 555

Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu

560 565 570

Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu

575 580 585

Pro Leu Gly Ile Thr Tyr Thr Asp Cys Ile Glu Ser Gly Gln Asn

590 595 600

Leu Cys Leu Cys Glu Gly Ser Asn Val Cys Gly Lys Gly Asn Lys

605 610 615

Cys Ile Leu Gly Ser Asn Gly Lys Asp Asn Gln Cys Val Thr Gly

620 625 630

Glu Gly Thr Pro Lys Pro Gln Ser His Asn Gln Gly Asp Phe Glu

635 640 645

Pro Ile Pro Glu Asp Ala Tyr Asp Glu Lys

650

<210>20

<211>336

<212> DNA

<213>Artificial sequence

<400>20

gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60

gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120

aaattagtga atgaagtaac tgaatttgca aaaacatgtg tagctgatga gtcagctgaa 180

aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240

cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300

tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360

gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420

gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480

tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540

aagctcgatg aacttcggga tgaagggaag gcttcgtctg ccaaacagag actcaaatgt 600

gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtggc tcgcctgagc 660

cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720

gtccacacgg aatgctgcca tggagatctg cttgaatgtg ctgatgacag ggcggacctt 780

gccaagtata tctgtgaaaa tcaggattcg atctccagta aactgaagga atgctgtgaa 840

aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900

gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960

gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020

tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactcta gagaagtgc 1080

tgtgccgctg cagatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140

gtggaagagc ctcagaattt aatcaaacaa aactgtgagc tttttaagca gcttggagag 1200

tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260

ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gcagcaaatg ttgtaaacat 1320

cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380

tgtgtgttgc atgagaaaac gccagtaagt gacagagtca caaaatgctg cacagagtcc 1440

ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg atgaaacata cgttcccaaa 1500

gagtttaatg ctgaaacatt caccttccat gcagatatatg cacactttct gagaaggag 1560

agacaaatca agaaacaaac tgcacttgtt gagcttgtga aacacaagcc caaggcaaca 1620

aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680

gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740

gctgccttag gcttaccctt aggtattact tacactgatt gtatagaatc gggtcaaaat 1800

ttgtgcctct gcgagggaag caatgtttgt ggtaaaggca ataagtgcat attgggttct 1860

aatggaaagg acaaccaatg tgtcactggc gaaggtacac cgaagcctca aagccataat 1920

caaggcgatt tcgaaccaat tccagaagac gcttatgatg aaaaatga 1968

<210>21

<211>313

<212> PRT

<213>Artificial sequence

<400>21

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu

5 10 15

Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

20 25 30

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr

50 55 60

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

65 70 75

Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

80 85 90

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

95 100 105

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

110 115 120

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

125 130 135

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

140 145 150

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

155 160 165

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

170 175 180

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp

185 190 195

Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met

200 205 200

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

215 220 225

Ser Leu Gly Lys Pro Thr Leu Tyr Asn Val Ser Leu Val Met Ser

230 235 240

Asp Thr Ala Gly Thr Pro Leu Gly Ile Thr Tyr Thr Asp Cys Thr

245 250 255

Glu Ser Gly Gln Asn Leu Cys Leu Cys Glu Gly Ser Asn Val Cys

260 265 270

Gly Lys Gly Asn Lys Cys Ile Leu Gly Ser Asn Gly Glu Glu Asn

275 280 285

Gln Cys Val Thr Gly Glu Gly Thr Pro Lys Pro Gln Ser His Asn

290 295 300

Asn Gly Asp Phe Glu Glu Ile Pro Glu Glu Tyr Leu Gln

300 310

<210>22

<211>942

<212> DNA

<213>Artificial sequence

<400>22

gaatctaagt acggccctcc ctgcccaccc tgtcctgctc cagagtttct gggcggaccc 60

tccgtgttcc tgttcccccc aaagcccaag gacaccctga tgatctcccg gacccccgaa 120

gtgacctgcg tggtggtgga cgtgtcccag gaagatcccg aggtccagtt caattggtac 180

gtggacggcg tggaagtgca caacgccaag accaagccca gagaggaaca gttcaactcc 240

acctaccggg tggtgtctgt gctgaccgtg ctgcaccagg actggctgaa cggcaaagag 300

tacaagtgca aggtctccaa caagggcctg ccctccagca tcgaaaagac catctccaag 360

gccaagggcc agccccgcga gcctcaggtg tacacactgc cccctagcca agaagagatg 420

accaagaacc aggtgtccct gacatgcctg gtgaagggct tctacccctc cgatatcgcc 480

gtggaatggg agtccaacgg ccagcccgag aacaactaca agaccacccc ccctgtgctg 540

gactccgacg gctccttctt cctgtactct cggctgaccg tggacaagtc ccggtggcag 600

gaaggcaacg tcttctcctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag 660

aagtctctga gcctgtccct gggcaagccc accctgtaca acgtgtccct ggtcatgtcc 720

gacacagctg gcaccccctt aggtattact tacactgatt gtacagaatc gggtcaaaat 780

ttgtgcctct gcgagggaag caatgtttgc ggtaaaggca ataagtgcat attgggttct 840

aatggagagg aaaaccaatg tgtcactggc gaaggtacac cgaagcctca aagccataat 900

aacggcgatt tcgaagaaat tccagaaga atatttacaat ga 942

<210>23

<211>315

<212> PRT

<213>Artificial sequence

<400>23

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu

5 10 15

Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

20 25 30

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr

50 55 60

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

65 70 75

Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

80 85 90

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

95 100 105

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

110 115 120

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

125 130 135

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

140 145 150

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

155 160 165

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

170 175 180

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp

185 190 195

Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met

200 205 200

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

215 220 225

Ser Leu Gly Lys Pro Thr Leu Tyr Asn Val Ser Leu Val Met Ser

230 235 240

Asp Thr Ala Gly Thr Pro Leu Gly Ile Thr Tyr Thr Asp Cys Ile

245 250 255

Glu Ser Gly Gln Asn Leu Cys Leu Cys Glu Gly Ser Asn Val Cys

260 265 270

Gly Lys Gly Asn Lys Cys Ile Leu Gly Ser Asn Gly Lys Asp Asn

275 280 285

Gln Cys Val Thr Gly Glu Gly Thr Pro Lys Pro Gln Ser His Asn

290 295 300

Gln Gly Asp Phe Glu Pro Ile Pro Glu Asp Ala Tyr Asp Glu Lys

305 310 315

<210>24

<211>948

<212> DNA

<213>Artificial sequence

<400>24

gaatctaagt acggccctcc ctgcccaccc tgtcctgctc cagagtttct gggcggaccc 60

tccgtgttcc tgttcccccc aaagcccaag gacaccctga tgatctcccg gacccccgaa 120

gtgacctgcg tggtggtgga cgtgtcccag gaagatcccg aggtccagtt caattggtac 180

gtggacggcg tggaagtgca caacgccaag accaagccca gagaggaaca gttcaactcc 240

acctaccggg tggtgtctgt gctgaccgtg ctgcaccagg actggctgaa cggcaaagag 300

tacaagtgca aggtctccaa caagggcctg ccctccagca tcgaaaagac catctccaag 360

gccaagggcc agccccgcga gcctcaggtg tacacactgc cccctagcca agaagagatg 420

accaagaacc aggtgtccct gacatgcctg gtgaagggct tctacccctc cgatatcgcc 480

gtggaatggg agtccaacgg ccagcccgag aacaactaca agaccacccc ccctgtgctg 540

gactccgacg gctccttctt cctgtactct cggctgaccg tggacaagtc ccggtggcag 600

gaaggcaacg tcttctcctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag 660

aagtctctga gcctgtccct gggcaagccc accctgtaca acgtgtccct ggtcatgtcc 720

gacacagctg gcaccccctt aggtattact tacactgatt gtatagaatc gggtcaaaat 780

ttgtgcctct gcgagggaag caatgtttgt ggtaaaggca ataagtgcat attgggttct 840

aatggaaagg acaaccaatg tgtcactggc gaaggtacac cgaagcctca aagccataat 900

caaggcgatt tcgaaccaat tccagaagac gcttatgatg aaaaatga 948

<210>25

<211>20

<212> PRT

<213>Artificial sequence

<400>25

Met Ala Gly Arg Val Pro Ser Leu Leu Val Leu Leu Val Phe Pro

5 10 15

Ser Ser Cys Leu Ala

20

<210>26

<211>60

<212> DNA

<213>Artificial sequence

<400>26

atggctggcc gcgtccctag cctgctagtt ctccttgttt ttccaagcag ctgtttggct 60

<210>27

<211>83

<212> PRT

<213>Artificial sequence

<400>27

Met Val Leu Ala Gln Gly Leu Leu Ser Met Ala Leu Leu Ala Leu

5 10 15

Cys Trp Glu Arg Ser Leu Ala Asn Ile Tyr Asn Cys Glu Pro Ala

20 25 30

Asn Pro Ser Glu Lys Asn Ser Pro Ser Thr Gln Tyr Cys Tyr Ser

35 40 45

Ile Gln Ser Gly Gly Ser Gly Gly Pro Phe Trp Val Leu Val Val

50 55 60

Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala

65 70 75

Phe Ile Ile Phe Trp Val Arg Ser

80

<210>28

<211>252

<212> DNA

<213>Artificial sequence

<400>28

atggtgctgg cccaggggct gctctccatg gccctgctgg ccctgtgctg ggagcgcagc 60

ctggcaaaca tatacaactg tgaaccagct aatccctctg agaaaaactc cccatctacc 120

caatactgtt acagcataca atctggcgga agcggaggcc ccttttgggt gctggtggtg 180

gttggtggag tcctggcttg ctatagcttg ctagtaacag tggcctttat tattttctgg 240

gtgaggagtt aa 252

Claims

1. a kind of tumour-specific lepirudin 023 ludon, it is characterised in that the N-terminal of the lepirudin 023 ludon, which contains, to be known by MMP9 Not and crack oligopeptide sequence, for restructuring hirudin for hirudin isomers, hirudin mutant, hirudin chimera, The HIRULOG of truncation, the hirudin of genetic modification or hirudin fusion protein.

2. tumour-specific lepirudin 023 ludon according to claim 1, it is characterised in that the oligopeptide sequence is can quilt The small peptide that MMP9 is recognized and cracked, its architectural feature is XnGI, and Xn represents any number of arbitrary amino acid.

3. tumour-specific lepirudin 023 ludon according to claim 1, it is characterised in that the lepirudin 023 ludon is in water Leech element isomers HV2 or HV3 N-terminal modification have oligopeptides PLG, the different bright ammonia with hirudin isomers HV2 or HV3 N-terminal Acid collectively constitutes the substrate peptide sequences PLGI of MMP9 identifications and digestion, and lepirudin 023 ludon PLG-HV2 amino acid sequences are：SEQ ID NO.1, gene order is：SEQ ID NO.2, lepirudin 023 ludon PLG-HV3 amino acid sequences are：SEQ ID NO.3, gene sequence It is classified as：SEQ ID NO.4；

Or, the lepirudin 023 ludon is by the Ser32-Asn33-Gly34-Glu35 of wild type hirudin HV2 or HV3 peptide chain Arg32-Gly33-Asp34-Ser35 is replaced with, then in its N-terminal modification PLG oligopeptides, the lepirudin 023 ludon PLG-HV2- RGDS amino acid sequences are：SEQ ID NO.5, gene order is：SEQ ID NO.6, lepirudin 023 ludon PLG-HV3-RGDS ammonia Base acid sequence is：SEQ ID NO.7, gene order is：SEQ ID NO.8；

Or, the lepirudin 023 ludon is by the Ser32-Asn33-Gly34-Glu35 of wild type hirudin HV2 or HV3 peptide chain Arg32-Gly33-Asp34-Met35 is replaced with, then in its N-terminal modification PLG oligopeptides, the lepirudin 023 ludon PLG-HV2- RGDM amino acid sequences are：SEQ ID NO.9, gene order is：SEQ ID NO.10, lepirudin 023 ludon PLG-HV3-RGDM ammonia Base acid sequence is：SEQ ID NO.11, gene order is：SEQ ID NO.12；

Or, the lepirudin 023 ludon, be targeted integration element α v β 3 polypeptide RGD4C and N-terminal amino acid be I hirudin it is different The fusion protein RPH2 that structure body HV2 or HV3 is formed by connecting by Linker and oligopeptides PLG, or RPH3, RPH2 amino acid sequence For：SEQ ID NO.13, gene order is：SEQ ID NO.14, RPH3 amino acid sequences are：SEQ ID NO.15, gene sequence It is classified as：SEQ ID NO.16；

Or, hirudin the isomers HV2 or HV3 that the lepirudin 023 ludon is human albumin with N-terminal amino acid is I pass through The fusion protein HPH2 that oligopeptides PLG is formed by connecting, or HPH3, HPH2 amino acid sequence is：SEQ ID NO.17, gene order For：SEQ ID NO.18, HPH3 amino acid sequences are：SEQ ID NO.19, gene order is：SEQ ID NO.20；

Or, the hirudin isomers that the lepirudin 023 ludon is Fc sections of people's gomphosis immunoglobulin with N-terminal amino acid is I The fusion protein F PH2 that HV2 or HV3 is formed by connecting by oligopeptides PLG, or FPH3, Fc sections of people's gomphosis immunoglobulin include people IgG4 hinge areas, Fc section sequence and IgM cauda sequences, FPH2 amino acid sequences are：SEQ ID NO.21, gene order is：SEQ ID NO.22, FPH3 amino acid sequences are：SEQ ID NO.23, gene order is：SEQ ID NO.24.

4. the preparation method of tumour-specific lepirudin 023 ludon, it is characterised in that comprise the following steps：Chemical synthesis claim Any one of 1~3 tumour-specific lepirudin 023 ludon genetic fragment, signal peptide sequence is connected in its N-terminal, and in restructuring coding The two ends addition restriction enzyme site of gene, Prepare restructuring carrier in encoding gene insertion vector is turned with the recombinant vector Change or transfection host cell, cultivate host cell, and therefrom reclaim and purify the tumour-specific lepirudin 023 ludon, host cell For Escherichia coli, lactic acid bacteria, saccharomycete, insect cell or mammalian cell.

5. the tumour-specific lepirudin 023 ludon described in any one of claims 1 to 3 is preparing anti-freezing, antithrombotic, anti-curing oncoma Medicine in application, especially anti-curing oncoma occurs together medicine, health products and the feature food of hypercoagulative state and Thrombotic lesion Application in product.

6. express the adoptive immunity cell preparation method of tumour-specific lepirudin 023 ludon, it is characterised in that vitro culture of human source Immunocyte, by the tumour-specific lepirudin 023 ludon of any one of people source coding sequence of secretory signal peptide encoding gene and claims 1 to 3 Encoding gene be connected, and restructuring encoding gene two ends add restriction enzyme site, by encoding gene insertion vector Recombination carrier is prepared into, immunocyte is transfected, the adoptive immunity cell of expression tumour-specific lepirudin 023 ludon is obtained.

7. the adoptive immunity cell preparation method of expression tumour-specific lepirudin 023 ludon according to claim 6, it is special Levy and be, the people source coding sequence of secretory signal peptide behaviour amidating enzyme signal peptide, amino acid sequence is：SEQ ID NO.25, gene Sequence is：SEQ ID NO.26.

8. the adoptive immunity cell preparation method of expression tumour-specific lepirudin 023 ludon according to claim 6, it is special Levy and be, the immunocyte is the one or more in α β T, gamma delta T, NKT, NK, DC, CIK, CAR-T, CAR-NK, TCR-T, The immunocyte behaviour source immunocyte.

9. the adoptive immunity cell preparation method of expression tumour-specific lepirudin 023 ludon according to claim 6, it is special Levy and be, the Membrane surface expression of the adoptive immunity cell there are target molecules, and target molecules are positioned at cell by cross-film sequence Film surface.

10. the adoptive immunity cell of expression hirudin according to claim 9, it is characterised in that the target molecules are CD19 total length or its truncated segment, or CD20 total length or its truncated segment.

11. the adoptive immunity cell preparation method of expression tumour-specific lepirudin 023 ludon according to claim 10, its It is characterised by, the structure of target molecules and the cross-film section is：GM-CSFR signal peptides-Δ CD20-Linker2-CD28 cross-films Section, its amino acid sequence is：SEQ ID NO.27, gene order is SEQ ID NO.28.

12. described in claim 6 expression tumour-specific lepirudin 023 ludon adoptive immunity cell, for anti-freezing, antithrombotic, The biological therapy of anti-curing oncoma, especially prevents and treats the biological therapy of the tumor patient with hypercoagulative state and/or thrombus.