WO2016078549A1 - Tumor vascular infarction agent polypeptide, gene and expression vector and application thereof - Google Patents

Tumor vascular infarction agent polypeptide, gene and expression vector and application thereof Download PDF

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WO2016078549A1
WO2016078549A1 PCT/CN2015/094576 CN2015094576W WO2016078549A1 WO 2016078549 A1 WO2016078549 A1 WO 2016078549A1 CN 2015094576 W CN2015094576 W CN 2015094576W WO 2016078549 A1 WO2016078549 A1 WO 2016078549A1
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tumor
polypeptide
tumor vascular
gene
ttf
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聂广军
李素萍
张银龙
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北京华安科创生物技术有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

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  • the invention belongs to the technical field of tumor vascular targeted drugs, in particular to a tumor vascular infarction polypeptide, a gene, an expression vector and an application thereof.
  • Tumor vasculature is a channel for tumor cells to acquire nutrients and exclude metabolites. It is also one of the important ways for tumor cells to escape and metastasize. Its morphology and function are different from the normal vascular system of the body, so it is the key to tumor targeted therapy.
  • Tumor vascular targeted therapy mainly includes two modes: inhibiting neovascularization and blocking existing tumor blood vessels. Among them, vascular therapy for blocking tumors mainly aims to treat tumors by selectively destroying the existing blood vessels of the tumor, cutting off the blood supply of the tumor, and inducing ischemic necrosis of the tumor cells. Therefore, how to achieve specific blocking of blood vessels in the tumor site, but has no effect on the normal tissue of the body, has become a research hotspot.
  • Tissue factor is a transmembrane glycoprotein with a molecular weight of approximately 47 kDa and plays an important role in the thrombosis process. Normally, the tissue factor is located in the adventitial cells of the vessel wall and is not present in the circulation or in contact with circulating blood. When the integrity of the vessel wall is disrupted, the tissue factor is exposed to circulating blood, which exerts a hemostatic effect by activating the coagulation cascade. The tissue factor consists of 263 amino acid residues, of which the 219 amino acid residues at the amino terminus are located outside the cell membrane and are the active sites of tissue factor.
  • tTF truncated tissue factor
  • the present invention utilizes a specific tumor vascular targeting peptide to specifically localize a truncated tissue factor tTF having procoagulant activity to the surface of a tumor vascular endothelium, thereby inducing thrombosis and blocking tumor blood supply.
  • the invention provides a tumor vascular infarction polypeptide consisting of a polypeptide having a coagulation function and a targeting peptide having a tumor targeting function, the targeting peptide being linked to the C-terminus of the polypeptide having coagulation function.
  • the polypeptide having coagulation function is a truncated tissue factor tTF having an amino acid sequence as shown in SEQ ID NO: 2.
  • the sequence is the amino acid residue sequence of the procoagulant-tissue factor located outside the cell membrane. When it is in the free state, it has no clotting activity; when it is localized on the tumor vascular endothelial cell membrane through the targeting peptide, it will play the tissue factor. The function activates the coagulation pathway and induces thrombosis.
  • the amino acid sequence of the targeting peptide is set forth in SEQ ID NO: 3.
  • the tumor vascular targeting molecule used in the present invention is a polypeptide consisting of 5 amino acids (CREKA).
  • the polypeptide is obtained by phage antibody library screening technology, and can specifically recognize the fibrin-fibronectin complex on the surface of the tumor blood vessel wall, thereby localizing the surface of the tumor vascular endothelium.
  • the amino acid sequence of the tumor vascular infarct polypeptide of the present invention is shown in SEQ ID NO: 1. It is a fusion protein that recombines the above tTF with the tumor vascular targeting peptide CREKA, which can localize to the surface of tumor vascular endothelial cells through CREKA, thereby initiating the coagulation function of tTF in tumor blood vessels, inducing thrombosis, and blocking tumor sites. Blood supply, to achieve the purpose of treating tumors by "starving" tumors.
  • the invention also provides a gene encoding the above-described tumor vascular infarct polypeptide.
  • the nucleotide sequence encoding the tumor vascular infarct polypeptide of the present invention is not unique, and any nucleoside capable of encoding and expressing the tumor vasorant polypeptide can be encoded.
  • the acid sequences are all genes claimed in the present invention.
  • the present invention also provides a vector comprising a gene encoding the above-described tumor vascular infarct polypeptide.
  • the vector includes a cloning vector and an expression vector.
  • the vector plasmid used in the expression vector of the present invention is not particularly limited, and those skilled in the art can select a suitable vector plasmid for use on the basis of the gene sequence of the present invention in combination with common knowledge in the art. Gene expression of the invention.
  • the expression vector of the present invention is preferably an expression vector constructed using the pET30a vector plasmid.
  • the present invention also provides a transgenic cell line and an engineered bacterium comprising a gene encoding the above-described tumor vascular infarction polypeptide.
  • the present invention also provides a transgenic cell line and an engineered bacteria comprising the above vector.
  • the invention also provides a tumor vascular infarct composition comprising the tumor vascular infarct polypeptide.
  • the present invention also provides a medicament for treating a tumor, the active ingredient of which comprises the tumor vascular infarct polypeptide.
  • the present invention also provides the use of the tumor vascular infarct polypeptide, a gene encoding the same, a vector containing the gene encoding the same, or an engineered bacterium for preparing a medicament for treating a tumor.
  • the invention further provides the use of the tumor vascular infarct polypeptide encoding gene or the vector containing the gene encoding the same in tumor gene therapy.
  • Tumors described in the present invention include, but are not limited to, melanoma, breast cancer.
  • the tumor vascular infarction polypeptide of the present invention can specifically synthesize the corresponding gene sequence, construct a fusion protein expression plasmid, and transfer it into, for example, BL21 E. coli, induce expression by IPTG and purify, and obtain tumor targeting property and Coagulation-active tumor vascular infarct polypeptide.
  • the gene sequence of the fusion protein is first synthesized by whole genes, and Nde I and Xho I restriction sites are designed at both ends. Next, the gene sequence was ligated into the pET30a expression plasmid, and the expression plasmid was transformed into BL21 competent E. coli. Finally, expression and purification were induced by IPTG to obtain the desired protein. The target protein was tested for its therapeutic effect on tumors by C57BL/6 mouse melanoma model, tail vein The fusion protein was injected into tumor-bearing mice, and then the length and width of the tumor were measured using a vernier caliper, and the tumor volume was recorded.
  • the tumor vascular infarction polypeptide provided by the invention has the characteristics of high targeting, safety and low immunogenicity, and the main part thereof is a TF extracellular region derived from itself, and therefore can well evade the immune system. Identify and clear.
  • the tumor vascular infarction polypeptide of the present invention utilizes a specific tumor targeting peptide to localize tTF to the surface of the tumor vascular endothelium, and is more specific than other ligand-receptor localization methods of tTF.
  • the present invention can be applied to the treatment of other hemorrhagic diseases by changing the targeting molecule, and the extracellular functional region (tTF) of the procoagulant factor-tissue factor can be applied in a wide range of applications.
  • tTF extracellular functional region
  • Example 1 is a result of SDS-PAGE electrophoresis detection of the fusion protein CREKA-tTF in Example 2 of the present invention.
  • Example 2 is a result of evaluating the growth inhibitory effect of a tumor vascular infarction polypeptide on melanoma tumors in Example 3 of the present invention.
  • Fig. 3 is a graph showing the results of evaluating the growth inhibition effect of tumor vascular infarction polypeptide on breast cancer in Example 4 of the present invention.
  • Fig. 2 and Fig. 3 the arrows indicate the time of administration
  • Saline is the saline group
  • Free CREKA is the single targeting peptide group
  • CREKA/tTF is the mixture of the single targeting peptide and the tTF protein
  • CREKA-tTF is Fusion protein drug group.
  • the 219 amino acid sequence (SEQ ID NO: 2) and its gene sequence of the extracellular domain (tTF) of the procoagulant factor-tissue factor (TF) published by NCBI, and the ability to specifically recognize tumors by phage antibody library screening technology A fibrin-fibronectin complex on the surface of the vessel wall to localize to a targeting peptide on the surface of the vascular endothelium of the tumor.
  • the targeting peptide is a five amino acid polypeptide CREKA (SEQ ID NO) with tumor endothelial binding function. :3).
  • the targeting peptide CREKA is ligated to the C-terminus of the polypeptide tTF having coagulation function to obtain the fusion protein CREKA-tTF (ie, tumor vaso-infarct polypeptide, SEQ ID NO: 1), and then the fusion protein is synthesized by whole-gene synthesis.
  • the expression sequence of Nde I and Xho I restriction sites were designed at both ends; finally, the fusion protein gene was ligated into the pET30a vector through the above-mentioned restriction site, thereby constructing a fusion protein expression vector.
  • the above-mentioned IPTG induced expression of the bacterial liquid was centrifuged (6000 rpm, 5 min), the supernatant was discarded, and the bacterial cell pellet was blown off with 25 mL of 10 mM Tris-HCl (pH 8.0) solution, ultrasonically disrupted, centrifuged at 12,000 rpm for 10 min, and the supernatant was removed.
  • the precipitate obtained by ultrasonic centrifugation was resuspended in 25 mL of 10 mM Tris-HCl (pH 8.0) solution and allowed to stand for 10 min. The above operation was repeated once to obtain a precipitate.
  • the pellet was resuspended by adding a small amount of 10 mM Tris-HCl (pH 8.0) solution, and 8 mL of 10 mM Tris-HCl (pH 8.0) solution containing 8 M urea was added to dissolve the protein, and centrifuged at 12,000 rpm for 10 min. The collected supernatant contained Said fusion protein CREKA-tTF. The fusion protein was subjected to SDS-PAGE electrophoresis, and the results are shown in Fig. 1.
  • the C57BL/6 mouse melanoma model was constructed.
  • the experimental group was injected with 750 ⁇ g/kg body weight of fusion protein CREKA-tTF in the tail vein, and the control group was free CREKA (single targeting peptide).
  • Group CREKA/tTF group (mixed group of single-target peptide and tTF protein) were injected into the tail vein in the same amount as the experimental group, and the normal saline group was in the same volume as the experimental group.
  • the mice were injected into the tail vein.
  • the tumor volume was measured every 2-3 days and the tumor volume was measured after 10 days of injection.
  • the tumor volume is calculated according to the following formula:
  • Tumor volume (d 2 ⁇ D) / 2
  • d is the smallest diameter of the tumor and D is the largest diameter of the tumor.
  • the saline injection group was a negative control group.
  • the MDA-MB-231 mouse breast cancer tumor model was constructed.
  • the experimental group injected the mice with the dose of 750 ⁇ g/kg body weight fusion protein CREKA-tTF, and the control group was free CREKA ( The single-target peptide group), the CREKA/tTF group (mixed group of single-target peptide and tTF protein) were injected into the tail vein in the same amount as the experimental group, and the saline group was the same as the experimental group.
  • Volume of the tail was injected intravenously into mice. The tumor volume was measured every 3 days and the tumor volume was measured statistically after 12 days of injection. The tumor volume is calculated according to the following formula:
  • Tumor volume (d 2 ⁇ D) / 2
  • d is the smallest diameter of the tumor and D is the largest diameter of the tumor.
  • the saline injection group was a negative control group.
  • the invention uses CREKA polypeptide as a targeting factor and tTF as an effector to construct a coagulant (infarcting agent) which can selectively induce tumor vascular embolization, and induces tumor by blocking nutrient supply and excretion of metabolites at the tumor site.
  • Ischemic death of cells is a novel tumor therapy targeting agent based on blocking blood supply to tumors.
  • the invention provides a tumor vascular infarction polypeptide, a gene, an expression vector and application thereof.
  • the tumor vascular infarct polypeptide comprises two parts: a polypeptide having a blood coagulation function and a targeting peptide having a tumor targeting function, the targeting peptide being linked to the C-terminus of the polypeptide having a blood coagulation function.
  • the tumor vascular infarction polypeptide provided by the invention has the characteristics of high targeting, safety and low immunogenicity, and induces ischemic death of tumor cells by blocking nutrient supply of tumor sites and excretion of metabolites, and is a novel type.
  • a tumor treatment targeting agent based on blocking blood supply to the tumor.

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Abstract

The present invention provides a tumor vascular infarction agent polypeptide, gene and expression vector and application thereof. The tumor vascular infarction agent polypeptide is composed of two sections: a polypeptide with blood coagulation function and a targeting peptide with tumor targeting function, wherein the targeting peptide is connected to C-terminal end of the polypeptide with blood coagulation function.

Description

一种肿瘤血管梗塞剂多肽、基因、表达载体及其应用Tumor vascular infarction polypeptide, gene, expression vector and application thereof 技术领域Technical field
本发明属于肿瘤血管靶向药物技术领域，具体地说，涉及一种肿瘤血管梗塞剂多肽、基因、表达载体及其应用。The invention belongs to the technical field of tumor vascular targeted drugs, in particular to a tumor vascular infarction polypeptide, a gene, an expression vector and an application thereof.
背景技术Background technique
肿瘤血管是肿瘤细胞获取营养物质及排除代谢产物的通道，也是肿瘤细胞逃逸、转移的重要途径之一，其形态学和功能都有别于机体的正常血管系统，因而是肿瘤靶向治疗的关键靶点之一。肿瘤血管靶向治疗主要包括两种模式：抑制新生血管生成和阻断已有肿瘤血管。其中阻断肿瘤已有血管疗法主要是通过选择性的破坏肿瘤已有的血管，切断肿瘤血供，诱发肿瘤细胞发生缺血性坏死，从而达到治疗肿瘤的目的。因此，如何实现特异的阻断肿瘤部位的血管，而对机体正常组织血管没有影响，成为研究的热点。Tumor vasculature is a channel for tumor cells to acquire nutrients and exclude metabolites. It is also one of the important ways for tumor cells to escape and metastasize. Its morphology and function are different from the normal vascular system of the body, so it is the key to tumor targeted therapy. One of the targets. Tumor vascular targeted therapy mainly includes two modes: inhibiting neovascularization and blocking existing tumor blood vessels. Among them, vascular therapy for blocking tumors mainly aims to treat tumors by selectively destroying the existing blood vessels of the tumor, cutting off the blood supply of the tumor, and inducing ischemic necrosis of the tumor cells. Therefore, how to achieve specific blocking of blood vessels in the tumor site, but has no effect on the normal tissue of the body, has become a research hotspot.
组织因子(TF)是一个分子量约为47kDa的跨膜糖蛋白，在血栓形成过程中起重要作用。正常情况下，组织因子位于血管壁外膜细胞，不存在于循环中或不与循环血液接触。当血管壁的完整性遭到破坏时，组织因子就会暴露于循环血液，通过激活凝固级联反应发挥止血作用。组织因子由263个氨基酸残基组成，其中氨基端的219个氨基酸残基位于细胞膜外，是组织因子的活性部位。研究表明，当该区域处于游离状态时，并没有凝血活性；但当它被锚定在磷脂膜表面，并暴露于血液中时，则会产生类似于全长因子的凝血活性，因此该段序列被称之为截断的组织因子(tTF)。鉴于此，如果用具有肿瘤血管靶向功能的分子将tTF特异的定位到肿瘤血管壁表面，则能够特异性的在肿瘤血管中诱发血栓形成事件，从而切断肿瘤部位的血供和代谢产物排除途径，达到治疗肿瘤的目的。Tissue factor (TF) is a transmembrane glycoprotein with a molecular weight of approximately 47 kDa and plays an important role in the thrombosis process. Normally, the tissue factor is located in the adventitial cells of the vessel wall and is not present in the circulation or in contact with circulating blood. When the integrity of the vessel wall is disrupted, the tissue factor is exposed to circulating blood, which exerts a hemostatic effect by activating the coagulation cascade. The tissue factor consists of 263 amino acid residues, of which the 219 amino acid residues at the amino terminus are located outside the cell membrane and are the active sites of tissue factor. Studies have shown that when the region is in a free state, there is no clotting activity; but when it is anchored on the surface of the phospholipid membrane and exposed to the blood, it produces a clotting activity similar to the full-length factor, so the sequence It is called truncated tissue factor (tTF). In view of this, if a molecule having a tumor vascular targeting function specifically targets tTF to the surface of a tumor blood vessel wall, it is possible to specifically induce a thrombotic event in the tumor blood vessel, thereby cutting off blood supply and metabolite elimination pathway at the tumor site. To achieve the purpose of treating tumors.
发明内容 Summary of the invention
本发明的目的是提供一种肿瘤血管梗塞剂多肽、基因、表达载体及其在制备用于治疗肿瘤的药物中的应用。It is an object of the present invention to provide a tumor vascular infarction polypeptide, gene, expression vector and use thereof in the preparation of a medicament for treating a tumor.
为了实现本发明目的，本发明利用一种特异的肿瘤血管靶向肽，将具有促凝活性的截断的组织因子tTF特异性地定位于肿瘤血管内皮表面，从而诱发血栓形成，阻断肿瘤血供。本发明提供的一种肿瘤血管梗塞剂多肽，由具有凝血功能的多肽与具有肿瘤靶向功能的靶向肽两部分组成，所述靶向肽连接于具有凝血功能的多肽的C末端。In order to achieve the object of the present invention, the present invention utilizes a specific tumor vascular targeting peptide to specifically localize a truncated tissue factor tTF having procoagulant activity to the surface of a tumor vascular endothelium, thereby inducing thrombosis and blocking tumor blood supply. . The invention provides a tumor vascular infarction polypeptide consisting of a polypeptide having a coagulation function and a targeting peptide having a tumor targeting function, the targeting peptide being linked to the C-terminus of the polypeptide having coagulation function.
优选地，所述具有凝血功能的多肽为截断的组织因子tTF，其氨基酸序列如SEQ ID NO:2所示。该序列即为促凝因子-组织因子位于细胞膜外的氨基酸残基序列，当其处于游离状态时，没有凝血活性；当其通过靶向肽定位于肿瘤血管内皮细胞膜上时，则会发挥组织因子的功能，激活凝血途径，诱发血栓形成。Preferably, the polypeptide having coagulation function is a truncated tissue factor tTF having an amino acid sequence as shown in SEQ ID NO: 2. The sequence is the amino acid residue sequence of the procoagulant-tissue factor located outside the cell membrane. When it is in the free state, it has no clotting activity; when it is localized on the tumor vascular endothelial cell membrane through the targeting peptide, it will play the tissue factor. The function activates the coagulation pathway and induces thrombosis.
优选地，所述靶向肽的氨基酸序列如SEQ ID NO:3所示。本发明采用的肿瘤血管靶向分子是由5个氨基酸构成的一种多肽(CREKA)。该多肽通过噬菌体抗体库筛选技术而获得，能够特异性识别肿瘤血管壁表面的纤维蛋白-纤连蛋白复合物(fibrin-fibronectin complex)，从而定位到肿瘤血管内皮表面。Preferably, the amino acid sequence of the targeting peptide is set forth in SEQ ID NO: 3. The tumor vascular targeting molecule used in the present invention is a polypeptide consisting of 5 amino acids (CREKA). The polypeptide is obtained by phage antibody library screening technology, and can specifically recognize the fibrin-fibronectin complex on the surface of the tumor blood vessel wall, thereby localizing the surface of the tumor vascular endothelium.
更优选地，本发明的肿瘤血管梗塞剂多肽的氨基酸序列如SEQ ID NO:1所示。其是将上述tTF与肿瘤血管靶向肽CREKA重组的融合蛋白，该融合蛋白能够通过CREKA定位到肿瘤血管内皮细胞表面，从而在肿瘤血管中启动tTF的凝血功能，诱发血栓形成，阻断肿瘤部位血液供应，以“饿死”肿瘤的方式达到治疗肿瘤的目的。More preferably, the amino acid sequence of the tumor vascular infarct polypeptide of the present invention is shown in SEQ ID NO: 1. It is a fusion protein that recombines the above tTF with the tumor vascular targeting peptide CREKA, which can localize to the surface of tumor vascular endothelial cells through CREKA, thereby initiating the coagulation function of tTF in tumor blood vessels, inducing thrombosis, and blocking tumor sites. Blood supply, to achieve the purpose of treating tumors by "starving" tumors.
本发明还提供编码上述肿瘤血管梗塞剂多肽的基因。The invention also provides a gene encoding the above-described tumor vascular infarct polypeptide.
本领域的技术人员应理解，由于密码子具有简并性，本发明编码所述肿瘤血管梗塞剂多肽的核苷酸序列并不唯一，任何能够编码并表达所述肿瘤血管梗塞剂多肽的核苷酸序列都属于本发明要求保护的基因。 It will be understood by those skilled in the art that due to the degeneracy of the codon, the nucleotide sequence encoding the tumor vascular infarct polypeptide of the present invention is not unique, and any nucleoside capable of encoding and expressing the tumor vasorant polypeptide can be encoded. The acid sequences are all genes claimed in the present invention.
本发明还提供含有编码上述肿瘤血管梗塞剂多肽的基因的载体。所述载体包括克隆载体和表达载体。The present invention also provides a vector comprising a gene encoding the above-described tumor vascular infarct polypeptide. The vector includes a cloning vector and an expression vector.
本领域的技术人员应理解，本发明对表达载体所采用的载体质粒并没有特别限制，本领域技术人员在本发明的基因序列的基础上，结合本领域公知常识能够选择合适的载体质粒用于本发明的基因表达。It will be understood by those skilled in the art that the vector plasmid used in the expression vector of the present invention is not particularly limited, and those skilled in the art can select a suitable vector plasmid for use on the basis of the gene sequence of the present invention in combination with common knowledge in the art. Gene expression of the invention.
本发明的表达载体优选为采用pET30a载体质粒构建的表达载体。The expression vector of the present invention is preferably an expression vector constructed using the pET30a vector plasmid.
本发明还提供含有编码上述肿瘤血管梗塞剂多肽的基因的转基因细胞系及工程菌。The present invention also provides a transgenic cell line and an engineered bacterium comprising a gene encoding the above-described tumor vascular infarction polypeptide.
本发明还提供含有上述载体的转基因细胞系及工程菌。The present invention also provides a transgenic cell line and an engineered bacteria comprising the above vector.
本发明还提供一种肿瘤血管梗塞剂组合物，其包含所述的肿瘤血管梗塞剂多肽。The invention also provides a tumor vascular infarct composition comprising the tumor vascular infarct polypeptide.
本发明还提供一种用于治疗肿瘤的药物，其有效成分含有所述的肿瘤血管梗塞剂多肽。The present invention also provides a medicament for treating a tumor, the active ingredient of which comprises the tumor vascular infarct polypeptide.
本发明还提供所述肿瘤血管梗塞剂多肽、其编码基因、含有其编码基因的载体或工程菌在制备用于治疗肿瘤的药物中的应用。The present invention also provides the use of the tumor vascular infarct polypeptide, a gene encoding the same, a vector containing the gene encoding the same, or an engineered bacterium for preparing a medicament for treating a tumor.
本发明进一步提供所述肿瘤血管梗塞剂多肽编码基因或含有其编码基因的载体在肿瘤基因治疗中的应用。The invention further provides the use of the tumor vascular infarct polypeptide encoding gene or the vector containing the gene encoding the same in tumor gene therapy.
本发明中述及的肿瘤包括但不限于黑色素瘤、乳腺癌。Tumors described in the present invention include, but are not limited to, melanoma, breast cancer.
本发明所述肿瘤血管梗塞剂多肽具体可以通过设计合成相应的基因序列，构建融合蛋白表达质粒，并将其转入如BL21大肠杆菌中，通过IPTG诱导表达并纯化，得到具有肿瘤靶向性及凝血活性的肿瘤血管梗塞剂多肽。The tumor vascular infarction polypeptide of the present invention can specifically synthesize the corresponding gene sequence, construct a fusion protein expression plasmid, and transfer it into, for example, BL21 E. coli, induce expression by IPTG and purify, and obtain tumor targeting property and Coagulation-active tumor vascular infarct polypeptide.
在本发明的一个具体实施方案中，首先通过全基因合成融合蛋白的基因序列，两端分别设计Nde I和Xho I酶切位点。其次，将该基因序列连接到pET30a表达质粒中，将此表达质粒转化入BL21感受态大肠杆菌中。最后，用IPTG诱导表达、纯化得到目的蛋白。将目的蛋白后用C57BL/6小鼠黑色素瘤模型检测其对肿瘤的治疗作用，尾静脉 将该融合蛋白注入荷瘤小鼠体内，然后利用游标卡尺测量肿瘤的长度和宽度，记录肿瘤体积。In a specific embodiment of the present invention, the gene sequence of the fusion protein is first synthesized by whole genes, and Nde I and Xho I restriction sites are designed at both ends. Next, the gene sequence was ligated into the pET30a expression plasmid, and the expression plasmid was transformed into BL21 competent E. coli. Finally, expression and purification were induced by IPTG to obtain the desired protein. The target protein was tested for its therapeutic effect on tumors by C57BL/6 mouse melanoma model, tail vein The fusion protein was injected into tumor-bearing mice, and then the length and width of the tumor were measured using a vernier caliper, and the tumor volume was recorded.
本发明具有以下优点：The invention has the following advantages:
(一)本发明提供的肿瘤血管梗塞剂多肽具有高靶向性、安全性，免疫原性低的特点，其主体部分为自身来源的TF胞外区域，因此，能够很好的躲避免疫系统的识别和清除。(1) The tumor vascular infarction polypeptide provided by the invention has the characteristics of high targeting, safety and low immunogenicity, and the main part thereof is a TF extracellular region derived from itself, and therefore can well evade the immune system. Identify and clear.
(二)本发明的肿瘤血管梗塞剂多肽利用一种特异的肿瘤靶向肽将tTF定位于肿瘤血管内皮表面，与其他配体-受体定位tTF的方法相比，特异性更强。(b) The tumor vascular infarction polypeptide of the present invention utilizes a specific tumor targeting peptide to localize tTF to the surface of the tumor vascular endothelium, and is more specific than other ligand-receptor localization methods of tTF.
(三)本发明可以通过改变靶向分子，将促凝因子-组织因子的胞外功能区(tTF)应用于其他出血性疾病治疗，应用范围广泛。(C) The present invention can be applied to the treatment of other hemorrhagic diseases by changing the targeting molecule, and the extracellular functional region (tTF) of the procoagulant factor-tissue factor can be applied in a wide range of applications.
附图说明DRAWINGS
图1为本发明实施例2中融合蛋白CREKA-tTF的SDS-PAGE电泳检测结果。1 is a result of SDS-PAGE electrophoresis detection of the fusion protein CREKA-tTF in Example 2 of the present invention.
图2为本发明实施例3中肿瘤血管梗塞剂多肽对黑色素瘤肿瘤生长抑制作用评价结果。2 is a result of evaluating the growth inhibitory effect of a tumor vascular infarction polypeptide on melanoma tumors in Example 3 of the present invention.
图3为本发明实施例4中肿瘤血管梗塞剂多肽对乳腺癌肿瘤生长抑制作用评价结果。Fig. 3 is a graph showing the results of evaluating the growth inhibition effect of tumor vascular infarction polypeptide on breast cancer in Example 4 of the present invention.
图2和图3中，箭头代表给药时间点，Saline为生理盐水组，Free CREKA为单靶向肽组，CREKA/tTF为单靶向肽和tTF蛋白的混合物给药组，CREKA-tTF为融合蛋白药物组。In Fig. 2 and Fig. 3, the arrows indicate the time of administration, Saline is the saline group, Free CREKA is the single targeting peptide group, CREKA/tTF is the mixture of the single targeting peptide and the tTF protein, and CREKA-tTF is Fusion protein drug group.
具体实施方式detailed description
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例均按照常规实验条件，如Sambrook等分子克隆实验手册(Sambrook J&Russell DW,Molecular cloning:a laboratory manual,2001)，或按照制造厂商说明书建议的条件。The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are based on routine experimental conditions, such as the Sambrook J&Russell DW, Molecular cloning: a laboratory manual, 2001, or as suggested by the manufacturer's instructions.
实施例1融合蛋白CREKA-tTF表达质粒的构建 Example 1 Construction of fusion protein CREKA-tTF expression plasmid
根据NCBI公布的促凝因子-组织因子(TF)的胞外功能区(tTF)219个氨基酸序列(SEQ ID NO:2)及其基因序列，并通过噬菌体抗体库筛选技术获得能够特异性识别肿瘤血管壁表面的纤维蛋白-纤连蛋白复合物(fibrin-fibronectin complex)，从而定位到肿瘤血管内皮表面的靶向肽，该靶向肽是具有肿瘤内皮结合功能的五氨基酸多肽CREKA(SEQ ID NO:3)。将靶向肽CREKA连接于具有凝血功能的多肽tTF的C末端，获得融合蛋白CREKA-tTF(即肿瘤血管梗塞剂多肽，SEQ ID NO:1)，然后采用全基因合成的方式，合成该融合蛋白的表达序列，并在两端分别设计Nde I和Xho I酶切位点；最后，将融合蛋白基因通过上述酶切位点，连接入pET30a载体中，从而构建得到融合蛋白表达载体。The 219 amino acid sequence (SEQ ID NO: 2) and its gene sequence of the extracellular domain (tTF) of the procoagulant factor-tissue factor (TF) published by NCBI, and the ability to specifically recognize tumors by phage antibody library screening technology A fibrin-fibronectin complex on the surface of the vessel wall to localize to a targeting peptide on the surface of the vascular endothelium of the tumor. The targeting peptide is a five amino acid polypeptide CREKA (SEQ ID NO) with tumor endothelial binding function. :3). The targeting peptide CREKA is ligated to the C-terminus of the polypeptide tTF having coagulation function to obtain the fusion protein CREKA-tTF (ie, tumor vaso-infarct polypeptide, SEQ ID NO: 1), and then the fusion protein is synthesized by whole-gene synthesis. The expression sequence of Nde I and Xho I restriction sites were designed at both ends; finally, the fusion protein gene was ligated into the pET30a vector through the above-mentioned restriction site, thereby constructing a fusion protein expression vector.
实施例2融合蛋白CREKA-tTF的表达、纯化Example 2 Expression and purification of fusion protein CREKA-tTF
1、融合蛋白CREKA-tTF的表达1. Expression of fusion protein CREKA-tTF
将实施例1中构建的融合蛋白表达载体转化入BL21大肠杆菌中，将5μL菌液接种到5mL LB液体培养基中，37℃200rpm，摇床培养16h；将培养的菌液转接到500mL LB液体培养基中，37℃200rpm，培养至OD600＝0.6-0.8，用IPTG(终浓度0.5mM)诱导表达4h。The fusion protein expression vector constructed in Example 1 was transformed into BL21 E. coli, and 5 μL of the bacterial solution was inoculated into 5 mL of LB liquid medium, and cultured at 37 ° C, 200 rpm for 16 hours on a shaker; and the cultured bacterial solution was transferred to 500 mL of LB. In liquid medium, culture was carried out at 37 ° C at 200 rpm until OD 600 = 0.6-0.8, and expression was induced with IPTG (final concentration 0.5 mM) for 4 h.
2、融合蛋白CREKA-tTF的纯化2. Purification of fusion protein CREKA-tTF
将上述IPTG诱导表达的菌液离心(6000rpm，5min)，弃上清，菌体沉淀用25mL 10mM Tris-HCl(pH8.0)溶液吹散，超声破菌，12000rpm，离心10min，上清去除干净，用25mL 10mM Tris-HCl(pH8.0)溶液重悬超声离心得到的沉淀，静置10min。重复上述操作一次，得到沉淀。加入少量的10mM Tris-HCl(pH8.0)溶液重悬沉淀，再加入8mL含8M尿素的10mM Tris-HCl(pH8.0)溶液溶解蛋白，12000rpm，离心10min，收集的上清液中含有所述融合蛋白CREKA-tTF。对融合蛋白进行SDS-PAGE电泳检测，结果见图1。The above-mentioned IPTG induced expression of the bacterial liquid was centrifuged (6000 rpm, 5 min), the supernatant was discarded, and the bacterial cell pellet was blown off with 25 mL of 10 mM Tris-HCl (pH 8.0) solution, ultrasonically disrupted, centrifuged at 12,000 rpm for 10 min, and the supernatant was removed. The precipitate obtained by ultrasonic centrifugation was resuspended in 25 mL of 10 mM Tris-HCl (pH 8.0) solution and allowed to stand for 10 min. The above operation was repeated once to obtain a precipitate. The pellet was resuspended by adding a small amount of 10 mM Tris-HCl (pH 8.0) solution, and 8 mL of 10 mM Tris-HCl (pH 8.0) solution containing 8 M urea was added to dissolve the protein, and centrifuged at 12,000 rpm for 10 min. The collected supernatant contained Said fusion protein CREKA-tTF. The fusion protein was subjected to SDS-PAGE electrophoresis, and the results are shown in Fig. 1.
实施例3肿瘤血管梗塞剂多肽在黑色素瘤模型中的抗肿瘤效果评价 Example 3 Evaluation of anti-tumor effect of tumor vascular infarction polypeptide in melanoma model
构建C57BL/6小鼠黑色素瘤模型，当肿瘤体积达到约0.15cm3时，实验组以750μg/kg体重融合蛋白CREKA-tTF给药量尾静脉注射小鼠，对照组Free CREKA(单靶向肽组)、CREKA/tTF组(单靶向肽和tTF蛋白的混合物给药组)分别以与实验组相同当量的给药量尾静脉注射小鼠，生理盐水组则以与实验组相同体积的量尾静脉注射小鼠。每隔2-3天注射一次并测量肿瘤体积大小，注射10天后，统计分析肿瘤体积变化情况。肿瘤体积按照以下公式计算：The C57BL/6 mouse melanoma model was constructed. When the tumor volume reached about 0.15 cm 3 , the experimental group was injected with 750 μg/kg body weight of fusion protein CREKA-tTF in the tail vein, and the control group was free CREKA (single targeting peptide). Group), CREKA/tTF group (mixed group of single-target peptide and tTF protein) were injected into the tail vein in the same amount as the experimental group, and the normal saline group was in the same volume as the experimental group. The mice were injected into the tail vein. The tumor volume was measured every 2-3 days and the tumor volume was measured after 10 days of injection. The tumor volume is calculated according to the following formula:
肿瘤体积＝(d2×D)/2Tumor volume = (d 2 × D) / 2
其中，d为肿瘤的最小直径，D为肿瘤的最大直径。注射生理盐水组为阴性对照组。Where d is the smallest diameter of the tumor and D is the largest diameter of the tumor. The saline injection group was a negative control group.
疗效分析结果显示，给荷瘤小鼠注射CREKA-tTF融合蛋白4次后，能够有效地抑制肿瘤的生长，并使50％以上肿瘤发生消退，与对照组相比具有显著的统计学意义。(图2)The results of curative effect analysis showed that the injection of CREKA-tTF fusion protein into tumor-bearing mice could effectively inhibit tumor growth and cause more than 50% of tumors to subside, which was statistically significant compared with the control group. (figure 2)
实施例4肿瘤血管梗塞剂多肽在乳腺癌模型中的抗肿瘤效果评价Example 4 Evaluation of anti-tumor effect of tumor vascular infarction polypeptide in breast cancer model
构建MDA-MB-231小鼠乳腺癌肿瘤模型，当肿瘤体积达到约0.15cm3时，实验组以750μg/kg体重融合蛋白CREKA-tTF的给药量尾静脉注射小鼠，对照组Free CREKA(单靶向肽组)、CREKA/tTF组(单靶向肽和tTF蛋白的混合物给药组)分别以与实验组相同当量给药量尾静脉注射小鼠，生理盐水组则以与实验组相同体积的量尾静脉注射小鼠。每隔3天注射一次并测量肿瘤体积大小，注射12天后，统计分析肿瘤体积变化情况。肿瘤体积按照以下公式计算：The MDA-MB-231 mouse breast cancer tumor model was constructed. When the tumor volume reached about 0.15 cm 3 , the experimental group injected the mice with the dose of 750 μg/kg body weight fusion protein CREKA-tTF, and the control group was free CREKA ( The single-target peptide group), the CREKA/tTF group (mixed group of single-target peptide and tTF protein) were injected into the tail vein in the same amount as the experimental group, and the saline group was the same as the experimental group. Volume of the tail was injected intravenously into mice. The tumor volume was measured every 3 days and the tumor volume was measured statistically after 12 days of injection. The tumor volume is calculated according to the following formula:
肿瘤体积＝(d2×D)/2Tumor volume = (d 2 × D) / 2
其中，d为肿瘤的最小直径，D为肿瘤的最大直径。注射生理盐水组为阴性对照组。Where d is the smallest diameter of the tumor and D is the largest diameter of the tumor. The saline injection group was a negative control group.
疗效分析结果显示，给荷瘤小鼠注射CREKA-tTF融合蛋白3次后，能够有效地抑制肿瘤的生长，并使50％以上肿瘤发生消退，与对照组相比具有显著的统计学意义。(图3) The results of curative effect analysis showed that the injection of CREKA-tTF fusion protein into tumor-bearing mice could effectively inhibit tumor growth and cause more than 50% of tumors to subside, which was statistically significant compared with the control group. (image 3)
本发明以CREKA多肽作为靶向因子，tTF作为效应因子，构建了一种可选择性诱导肿瘤血管栓塞的促凝剂(梗塞剂)，通过阻断肿瘤部位的营养供给和代谢产物排泄，诱导肿瘤细胞缺血性死亡，是一种新型的基于阻断肿瘤血供的肿瘤治疗靶向剂。The invention uses CREKA polypeptide as a targeting factor and tTF as an effector to construct a coagulant (infarcting agent) which can selectively induce tumor vascular embolization, and induces tumor by blocking nutrient supply and excretion of metabolites at the tumor site. Ischemic death of cells is a novel tumor therapy targeting agent based on blocking blood supply to tumors.
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。Although the present invention has been described in detail with reference to the preferred embodiments of the present invention, it will be apparent to those skilled in the art Therefore, such modifications or improvements made without departing from the spirit of the invention are intended to be within the scope of the invention.
工业实用性Industrial applicability
本发明提供一种肿瘤血管梗塞剂多肽、基因、表达载体及其应用。所述肿瘤血管梗塞剂多肽由两部分组成：具有凝血功能的多肽和具有肿瘤靶向功能的靶向肽，所述靶向肽连接于具有凝血功能的多肽的C末端。本发明提供的肿瘤血管梗塞剂多肽具有高靶向性、安全性，免疫原性低的特点，通过阻断肿瘤部位的营养供给和代谢产物排泄，诱导肿瘤细胞缺血性死亡，是一种新型的基于阻断肿瘤血供的肿瘤治疗靶向剂。 The invention provides a tumor vascular infarction polypeptide, a gene, an expression vector and application thereof. The tumor vascular infarct polypeptide comprises two parts: a polypeptide having a blood coagulation function and a targeting peptide having a tumor targeting function, the targeting peptide being linked to the C-terminus of the polypeptide having a blood coagulation function. The tumor vascular infarction polypeptide provided by the invention has the characteristics of high targeting, safety and low immunogenicity, and induces ischemic death of tumor cells by blocking nutrient supply of tumor sites and excretion of metabolites, and is a novel type. A tumor treatment targeting agent based on blocking blood supply to the tumor.
Figure PCTCN2015094576-appb-000001
Figure PCTCN2015094576-appb-000001
Figure PCTCN2015094576-appb-000002
Figure PCTCN2015094576-appb-000002

Claims (10)

  1. 一种肿瘤血管梗塞剂多肽，其特征在于，所述的肿瘤血管梗塞剂多肽由具有凝血功能的多肽与具有肿瘤靶向功能的靶向肽两部分组成，所述靶向肽连接于具有凝血功能的多肽的C末端。A tumor vascular infarction polypeptide, characterized in that the tumor vascular infarction polypeptide consists of a polypeptide having a coagulation function and a targeting peptide having a tumor targeting function, the targeting peptide being linked to a coagulation function The C-terminus of the polypeptide.
  2. 根据权利要求1所述的肿瘤血管梗塞剂多肽，其特征在于，所述具有凝血功能的多肽，其氨基酸序列如SEQ ID NO:2所示。The tumor vascular infarction polypeptide according to claim 1, wherein the polypeptide having a blood coagulation function has an amino acid sequence as shown in SEQ ID NO: 2.
  3. 根据权利要求1或2所述的肿瘤血管梗塞剂多肽，其特征在于，所述靶向肽的氨基酸序列如SEQ ID NO:3所示。The tumor vascular infarction polypeptide according to claim 1 or 2, wherein the amino acid sequence of the targeting peptide is shown in SEQ ID NO: 3.
  4. 根据权利要求1-3任一项所述的肿瘤血管梗塞剂多肽，其特征在于，所述肿瘤血管梗塞剂多肽的氨基酸序列如SEQ ID NO:1所示。The tumor vascular infarction polypeptide according to any one of claims 1 to 3, wherein the amino acid sequence of the tumor vascular infarct polypeptide is as shown in SEQ ID NO: 1.
  5. 编码权利要求1-4任一项所述肿瘤血管梗塞剂多肽的基因。A gene encoding a tumor vascular infarction polypeptide according to any one of claims 1 to 4.
  6. 含有权利要求5所述基因的载体。A vector comprising the gene of claim 5.
  7. 含有权利要求6所述载体的工程菌。An engineered fungus comprising the vector of claim 6.
  8. 一种肿瘤血管梗塞剂组合物，其包含如权利要求1-4任一项所述的肿瘤血管梗塞剂多肽。A tumor vascular infarction composition comprising the tumor vascular infarct polypeptide of any one of claims 1-4.
  9. 权利要求1-4任一项所述肿瘤血管梗塞剂多肽、权利要求5所述基因、权利要求6所述载体或权利要求7所述工程菌在制备用于治疗肿瘤的药物中的应用。Use of the tumor vascular infarction polypeptide according to any one of claims 1 to 4, the gene of claim 5, the vector of claim 6, or the engineered bacteria of claim 7 for the preparation of a medicament for treating a tumor.
  10. 权利要求5所述基因或权利要求6所述载体在肿瘤基因治疗中的应用。 Use of the gene of claim 5 or the vector of claim 6 in tumor gene therapy.
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