WO2016004906A2 - 一种肿瘤血管阻断剂多肽、基因、表达载体及其应用 - Google Patents

一种肿瘤血管阻断剂多肽、基因、表达载体及其应用 Download PDF

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WO2016004906A2
WO2016004906A2 PCT/CN2015/089116 CN2015089116W WO2016004906A2 WO 2016004906 A2 WO2016004906 A2 WO 2016004906A2 CN 2015089116 W CN2015089116 W CN 2015089116W WO 2016004906 A2 WO2016004906 A2 WO 2016004906A2
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tumor
polypeptide
expression vector
gene
tumor vascular
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WO2016004906A3 (zh
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聂广军
李素萍
田艳华
赵颖
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北京华安科创生物技术有限公司
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Priority to US15/324,669 priority Critical patent/US9896498B2/en
Priority to JP2017521279A priority patent/JP6334061B2/ja
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

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  • the invention relates to the technical field of medicine for tumor treatment, in particular to a tumor vascular blocker 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 fusion protein can be localized to the surface of tumor vascular endothelial cells by pHLIP, and is expressed in tumor blood vessels. It is of great significance to treat tumors by swinging the coagulation function of tTF and generating thrombus, thereby blocking the blood supply to the tumor site and achieving the purpose of treating tumors.
  • the invention provides a tumor vascular blocker polypeptide, a gene, an expression vector and the use thereof in preparing a medicament for treating tumor.
  • the polypeptide is a fusion protein in which the above tTF is recombined with the tumor targeting molecule pHLIP, and the two are linked by 5 amino acids to ensure that the respective functions are not affected, and the fusion protein can be localized to the surface of the tumor vascular endothelial cells by pHLIP.
  • the tumor blood vessels to play the role of tTF coagulation, the production of blood clots, thereby blocking the blood supply to the tumor site, to achieve the purpose of treatment of tumors.
  • the invention includes the following:
  • the invention provides a tumor vascular blocker polypeptide having the amino acid sequence set forth in SEQ ID NO: 1.
  • amino acid sequence shown in SEQ ID NO: 1 is specifically as follows:
  • the tumor vasopressor polypeptide includes an active domain, a linking domain, and a targeting domain, each of which functions differently.
  • the active domain consists of 219 amino acids and the sequence is:
  • the sequence of the active domain is the same as the sequence of the amino acid residue of the tissue factor outside the cell membrane, and when it is in a free state, there is no clotting activity; when it is localized on the membrane of the tumor vascular endothelial cell through the targeting peptide, the tissue factor is exerted
  • the function activates the coagulation pathway and induces thrombosis.
  • the linkage domain consists of 5 amino acids and the sequence is: The sequence of the linker domain ensures that the function of the expressed fusion protein active domain and the targeting domain remains intact.
  • the targeting domain consists of 35 amino acids and the sequence is:
  • the sequence of the targeting domain is capable of producing a conformational change in the slightly acidic environment of the tumor site, forming an alpha-helical structure, and passing through the cell membrane to localize to the tumor vascular endothelial cell membrane.
  • the tumor vasopressor polypeptide can respond to the micro-acidity of the tumor to produce a conformational change, thereby localizing to the tumor vascular endothelial cells, and activating the coagulation activity of the active domain, thereby specifically forming a thrombus in the tumor blood vessel and inhibiting the tumor
  • the therapeutic purpose of growth in a normal physiological pH environment, the target domain cannot form an ⁇ -helical structure and does not have a transmembrane function, so the active domain cannot be localized on the cell, so that the active domain cannot exert clotting activity, thereby ensuring When the drug molecule circulates in the body, it does not form a blood vessel in the blood vessel at the normal site, thereby avoiding the occurrence of side effects.
  • the tumor vascular blocker polypeptide does not need to penetrate into the tumor tissue, and can directly play a role in the tumor blood vessel, so the amount of the drug used is reduced, and the drug resistance is not easily generated; the main part of the polypeptide is derived from the tissue factor cell of the body.
  • the outer zone is therefore less immunogenic and can reduce the immune clearance of the drug as it circulates in the body.
  • the tumor vasopressor polypeptide is closer to the native conformation of the tissue factor than the other tumor targeting peptide-mediated tissue factor that has been reported, and thus, its coagulation activity is better.
  • the invention provides a tumor vascular blocker gene having a nucleotide sequence encoding the tumor vasopressor polypeptide.
  • nucleotide sequence encoding the tumor vasopressor polypeptide of the present invention is not unique, and any of the tumor vasopressor polypeptides can be encoded and expressed. Nucleotide sequences are all to be understood as tumor vascular blocker genes of the invention.
  • the present invention particularly provides a tumor vascular blocker gene having the nucleotide sequence shown in SEQ ID NO: 5.
  • nucleotide sequence shown in SEQ ID NO: 5 is specifically as follows:
  • the invention provides a tumor vascular blocker expression vector comprising a nucleotide sequence encoding the tumor vasopressor polypeptide.
  • nucleotide sequence encoding the tumor vasopressor polypeptide of the present invention is not unique, and any of the tumor vasopressor polypeptides can be encoded and expressed.
  • Nucleotide sequences are to be understood as tumor vascular blocker genes of the invention, and thus any expression vector comprising a polypeptide encoding and expressing the tumor vasopressor polypeptide is to be understood as intended to be protected by the invention.
  • the present invention particularly provides an expression vector comprising the nucleotide sequence shown in SEQ ID NO: 5 above.
  • vector plasmid used in the expression vector of the present invention is not limited, as those skilled in the art can select the gene sequence of the present invention and can combine with the common knowledge of those skilled in the art.
  • a suitable vector plasmid is used for gene expression of the present invention.
  • the present invention specifically provides a vector plasmid which is a commonly used pET30a vector plasmid. Therefore, the expression vector of the present invention is preferably an expression vector constructed using the pET30a vector plasmid.
  • the present invention provides the use of the tumor vasopressor polypeptide in the preparation of a medicament for treating a tumor.
  • the tumor vascular blocker polypeptide can specifically construct a fusion protein expression plasmid by designing a corresponding gene sequence, and transfer it into, for example, BL21 E. coli, IPTG induces expression and purification, and obtains tumor targeting property. And clotting activity of tumor vascular blockers.
  • the main part of the tumor vascular blocker is an extracellular region of tissue factor derived from itself, and therefore, the immunogenicity is small, and the immune system can be well evaded;
  • the tumor vascular blocker skillfully utilizes an acid-responsive tumor targeting peptide to localize tissue factor to tumor vascular endothelial cells, closer to tissue than other ligand-receptor localization factors. Factor natural structure, better clotting activity;
  • the present invention can be applied to other hemorrhagic diseases by changing the targeting molecule, and has broad application prospects.
  • FIG. 1 is a schematic diagram of a composition domain of a tumor vascular blocker polypeptide (fusion protein) of the present invention (A) and a SDS-PAGE electrophoresis result of the fusion protein (B); wherein the tumor vascular blocker polypeptide includes an active domain,
  • the linking domain and the targeting domain are three parts; M represents the standard molecular weight of the protein, 1 represents the lane of the fusion protein, and the arrow indicates the fusion protein.
  • FIG. 2 is a therapeutic effect of the tumor vascular blocker provided by the present invention after injecting into a nude mouse breast cancer tumor model through the tail vein for 12 hours; wherein, (A) is the appearance of the tumor-bearing mouse after injection of physiological saline (control), (B) is a control tumor, (C) is a control tumor pathological section (arrows represent tumor blood vessels, no thrombosis); (D) is the appearance of tumor-bearing mice after injection of tumor vascular blockers, (E) is injection The tumor after the tumor vascular blocker, (F) is the tumor pathological section after the injection of the tumor vascular blocker (the arrow represents the tumor blood vessel, there is obvious thrombosis).
  • the gene sequence of the extracellular 219 amino acid sequence of the tissue factor (as shown in SEQ ID NO: 2) was found from the NCBI website, and secondly, the tumor targeting peptide (as shown in SEQ ID NO: 3) and the linking portion were The amino acid sequence is translated into its gene sequence to obtain a fusion protein gene sequence as shown in SEQ ID NO: 5.
  • the fusion protein gene was synthesized by whole-genome synthesis, and 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-described restriction enzyme site to obtain a fusion protein expression vector.
  • the bacterial solution induced by the above IPTG was centrifuged (6000 ⁇ rpm, 5 min), and the supernatant was discarded and collected.
  • the fusion protein was identified by SDS-PAGE electrophoresis. As a result, as shown in Fig. 1(B), a clear and pure band was formed, and the size was above 30 KDa, which was in agreement with the expectation.
  • a nude mouse breast cancer tumor model was injected through the tail vein according to the amount of 833 ug/kg body weight or 20 ug/mouse, and the therapeutic effect observed after 12 hours was injected. Saline was used as a control group. The result is shown in Figure 2.
  • the tumor vascular blocker of the present invention can cause macroscopic red color (D) at the tumor site; after dissection, the control tumor (B) is normal meat. Red, while tumors injected with tumor vascular blocker (E) showed obvious dark red color caused by thrombus; tumor pathological sections showed that compared with the control group (C), injected tumor vascular blocker group (F) Significant thrombosis is formed in the tumor blood vessels (shown by the scissors).

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Abstract

本发明涉及一种肿瘤血管阻断剂多肽、基因、表达载体及其应用。所述肿瘤血管阻断剂多肽具有SEQ ID NO：1所示的氨基酸序列。所述多肽包括截断的组织因子tTF和肿瘤靶向分子pHLIP，二者之间由5个氨基酸连接，保证各自功能不受影响，所述融合蛋白能够通过pHLIP定位到肿瘤血管内皮细胞表面，并在肿瘤血管中发挥tTF的凝血功能，产生血栓，从而阻断肿瘤部位血液供应，达到治疗肿瘤的目的。本发明的多肽对于肿瘤的治疗具有重要意义，可用于制备治疗肿瘤的药物中。

Description

一种肿瘤血管阻断剂多肽、基因、表达载体及其应用 技术领域
本发明涉及肿瘤治疗的药物技术领域，尤其涉及一种肿瘤血管阻断剂多肽、基因、表达载体及其应用。
背景技术
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。
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的作用。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；但当它被锚定在细胞膜上，并暴漏于血液中时，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)。鉴于此特点，如果用具有肿瘤靶向功能的分子将tTF特异的锚定于肿瘤组织中，则能够特异性的在肿瘤血管中引发血栓形成事件，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。
肿瘤靶向分子pHLIP是一种由35个氨基酸构成的多肽。当其在体内(pH＝7.35-7.45)循环时，处于自由伸展状态，并不能锚定于特定组织；但当其到达肿瘤(pH＜7)部位时，则会产生酸响应的构象变化，形成α-螺旋结构，并与细胞膜发生相互作用，插入细胞膜中从而锚定于内皮细胞表面。
如果将上述截断的组织因子tTF与肿瘤靶向分子pHLIP重组成融合蛋白，以保证各自功能不受影响，该融合蛋白能够通过pHLIP定位到肿瘤血管内皮细胞表面，并在肿瘤血管中发 挥tTF的凝血功能，产生血栓，从而阻断肿瘤部位血液供应，达到治疗肿瘤的目的，则具有肿瘤治疗的重要意义。
发明内容
本发明提供一种肿瘤血管阻断剂多肽、基因、表达载体及其在制备治疗肿瘤的药物中的应用。所述多肽是将上述tTF与肿瘤靶向分子pHLIP重组的融合蛋白，二者之间由5个氨基酸连接，保证各自功能不受影响，所述融合蛋白能够通过pHLIP定位到肿瘤血管内皮细胞表面，并在肿瘤血管中发挥tTF的凝血功能，产生血栓，从而阻断肿瘤部位血液供应，达到治疗肿瘤的目的。
本发明包括以下内容：
在第一方面，本发明提供一种肿瘤血管阻断剂多肽，所述多肽具有SEQ ID NO：1所示的氨基酸序列。
其中，SEQ ID NO：1所示的氨基酸序列具体如下：
Figure PCTCN2015089116-appb-000001
所述肿瘤血管阻断剂多肽包括活性域、连接域和靶向域，它们各自发挥不同功能。
所述活性域由219个氨基酸构成，序列为：
Figure PCTCN2015089116-appb-000002
Figure PCTCN2015089116-appb-000003
所述活性域的序列与组织因子位于细胞膜外的氨基酸残基序列相同，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。
所述连接域由5个氨基酸构成，序列为：
Figure PCTCN2015089116-appb-000004
所述连接域的序列能够保证表达得到的融合蛋白活性域和靶向域的功能保持完整。
所述靶向域由35个氨基酸构成，序列为：
Figure PCTCN2015089116-appb-000005
所述靶向域的序列在肿瘤部位的微酸性环境中能够产生构象变化，形成α-螺旋结构，并穿过细胞膜，从而定位于肿瘤血管内皮细胞膜。
所述肿瘤血管阻断剂多肽，能够响应肿瘤微酸性的特点，产生构象变化，从而定位于肿瘤血管内皮细胞，并激活活性域的凝血活性，从而能够特异地在肿瘤血管形成血栓，达到抑制肿瘤生长的治疗目的；而在正常生理pH环境中，其靶向域不能形成α-螺旋结构，不具有穿膜功能，因此不能将活性域定位于细胞上，使得活性域不能发挥凝血活性，从而保证该药物分子在体内循环时，不会在正常部位血管形成血栓，避免副作用的产生。此外，所述肿瘤血管阻断剂多肽无需渗透到肿瘤组织中，在肿瘤血管处即可直接发挥作用，因此所用药量减少，且不易产生耐药性；多肽主体部分来源于自身的组织因子胞外区，因此免疫原性小，能够减少药物在体内循环时被免疫清除。
本发明中，所述肿瘤血管阻断剂多肽与已经报道的其他肿瘤靶向肽介导组织因子相比，更接近组织因子天然的构象，因此，其凝血活性更好。
在第二方面，本发明提供一种肿瘤血管阻断剂基因，所述基因具有编码所述肿瘤血管阻断剂多肽的核苷酸序列。
本领域的技术人员应理解，由于密码子的简并性，本发明编码所述肿瘤血管阻断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阻断剂多肽的核苷酸序列都应当理解为本发明的肿瘤血管阻断剂基因。
虽然如此，本发明特别提供一种肿瘤血管阻断剂基因，其具有SEQ ID NO：5所示的核苷酸序列。
其中，SEQ ID NO：5所示的核苷酸序列具体如下：
Figure PCTCN2015089116-appb-000006
Figure PCTCN2015089116-appb-000007
在第三方面，本发明提供一种肿瘤血管阻断剂表达载体，所述表达载体包含编码所述肿瘤血管阻断剂多肽的核苷酸序列。
本领域的技术人员应理解，由于密码子的简并性，本发明编码所述肿瘤血管阻断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阻断剂多肽的核苷酸序列都应当理解为本发明的肿瘤血管阻断剂基因，因此任何包含编码并表达所述肿瘤血管阻断剂多肽的表达载体都应当理解为本发明意欲保护的范围。
虽然如此，本发明特别提供一种表达载体，其包含上述SEQ ID NO：5所示的核苷酸序列。
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限制，因为本领域的技术人员在得知本发明的基因序列的基础上，结合本领域技术人员的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。
虽然如此，本发明特别提供一种载体质粒，其为常用的pET30a载体质粒。因此，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。
在第四方面，本发明提供一种所述肿瘤血管阻断剂多肽在制备治疗肿瘤的药物中的应用。
本发明中，所述肿瘤血管阻断剂多肽具体可以通过设计相应的基因序列，构建融合蛋白表达质粒，并将其转入如BL21大肠杆菌中，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阻断剂。
本发明的有益效果表现在：
(1)所述肿瘤血管阻断剂的主体部分为自身来源的组织因子胞外区域，因此，免疫原性小，能够很好的躲避免疫系统的清除；
(2)所述肿瘤血管阻断剂巧妙的利用了一种酸响应的肿瘤靶向肽将组织因子定位于肿瘤血管内皮细胞，比用其他配体-受体定位组织因子的方法，更接近组织因子天然的结构，凝血活性更好；
(3)本发明可以通过改变靶向分子，运用于其他的出血性疾病，具有广泛的应用前景。
附图说明
图1是本发明的肿瘤血管阻断剂多肽(融合蛋白)组成结构域示意图(A)和融合蛋白SDS-PAGE电泳鉴定结果(B)；其中，所述肿瘤血管阻断剂多肽包括活性域、连接域和靶向域三部分；M表示蛋白质标准分子量，1表示融合蛋白的泳道，箭头指示处即为融合蛋白。
图2是本发明提供的肿瘤血管阻断剂通过尾静脉注入裸鼠乳腺癌肿瘤模型12小时后的治疗效果；其中，(A)为注射生理盐水(对照)后荷瘤小鼠体外形貌，(B)为对照肿瘤、(C)为对照肿瘤病理切片(箭头代表肿瘤血管，没有血栓形成)；(D)为注射肿瘤血管阻断剂后荷瘤小鼠体外形貌，(E)为注射肿瘤血管阻断剂后肿瘤、(F)为注射肿瘤血管阻断剂后肿瘤病理切片(箭头代表肿瘤血管，有明显血栓形成)。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述。本领域技术人员将会理解，以下实施例仅为本发明的优选实施例，以便于更好地理解本发明，因而不应视为限定本发明的范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法；所用的实验材料，如无特殊说明，均为自常规生化试剂厂商购买得到的。
实施例1：融合蛋白质粒的构建
首先，从NCBI网站查得组织因子胞外219个氨基酸序列(如SEQ ID NO：2所示)的基因序列，其次，将肿瘤靶向肽(如SEQ ID NO：3所示)及连接部分的氨基酸序列翻译出其基因序列，得到如SEQ ID NO：5所示的融合蛋白基因序列。用全基因合成的方式，合成融合蛋白基因，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，从而得到融合蛋白表达载体。
实施例2：融合蛋白的表达和纯化
(1)融合蛋白的表达
将上述融合蛋白表达载体转化入BL21大肠杆菌中，先接5μL菌液到5mL LB液体培养基中，37℃，200×rpm，摇床培养16h。将培养的菌液转接到500mL LB液体培养基中，37℃，200×rpm，培养至OD＝0.6～0.8，用IPTG(0.5mM)诱导表达4h。
(2)融合蛋白的纯化
将上述IPTG诱导表达的菌液离心(6000×rpm，5min)，弃上清，收菌。沉淀用25mL 10mM Tris-HCl(pH＝8.0)溶液吹散，超声破菌，12000×rpm，离心10min，上清去除干净，用25mL10mM Tris-HCl(pH＝8.0)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(pH＝8.0)溶液重悬沉淀，再加8mL含8M尿素的10mM Tris-HCl(pH＝8.0)溶液溶解蛋白，12000×rpm，离心10min，收集上清。
通过SDS-PAGE电泳鉴定融合蛋白，结果如图1(B)所示，可见形成清晰、纯净的条带，大小在30KDa以上，与预期相符。
实施例3：肿瘤血管阻断剂效果实验
采用本发明实施例2所制得的肿瘤血管阻断剂，按照833ug/kg体重或20ug/只小鼠的量通过尾静脉注入裸鼠乳腺癌肿瘤模型，12小时后观察的治疗效果，以注射生理盐水作为对照组。结果如图2所示。
与注射生理盐水荷瘤小鼠(A)相比，本发明的肿瘤血管阻断剂能够引起肿瘤部位出现肉眼可见的深红色(D)；解剖后，可见对照组肿瘤(B)为正常的肉红色，而注射肿瘤血管阻断剂(E)的肿瘤则出现明显的血栓引起的暗红色；肿瘤病理切片则可以看出与对照组(C)相比，注射肿瘤血管阻断剂组(F)在肿瘤血管中形成了明显的血栓(剪头所示)。
申请人声明，本发明通过上述实施例来说明本发明的详细特征以及详细方法，但本发明并不局限于上述详细特征以及详细方法，即不意味着本发明必须依赖上述详细特征以及详细方法才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明选用组分的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。

Claims (8)

  1. 一种肿瘤血管阻断剂多肽，其特征在于，所述多肽具有SEQ ID NO：1所示的氨基酸序列。
  2. 一种肿瘤血管阻断剂基因，其特征在于，所述基因具有编码权利要求1所述多肽的核苷酸序列。
  3. 根据权利要求2所述的基因，其特征在于，所述基因具有SEQ ID NO：5所示的核苷酸序列。
  4. 一种肿瘤血管阻断剂表达载体，其特征在于，所述表达载体包含编码权利要求1所述多肽的核苷酸序列。
  5. 根据权利要求4所述的表达载体，其特征在于，所述表达载体包含SEQ ID NO：5所示的核苷酸序列。
  6. 根据权利要求4或5所述的表达载体，其特征在于，所述表达载体采用pET30a载体质粒构建。
  7. 如权利要求1所述的多肽在制备治疗肿瘤的药物中的应用。
  8. 权利要求1所述的多肽用于治疗肿瘤的用途。
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112300264A (zh) * 2020-11-09 2021-02-02 青岛大学附属医院 一种新型放射性标记抗癌药的合成标记方法

Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104045717B (zh) * 2014-07-08 2016-07-06 北京华安科创生物技术有限公司 一种肿瘤血管阻断剂多肽、基因、表达载体及其应用
CN104387474B (zh) * 2014-11-17 2018-01-23 北京华安科创生物技术有限公司 一种肿瘤血管梗塞剂多肽、基因、表达载体及其应用
CN105218647A (zh) * 2015-10-28 2016-01-06 苏州普罗达生物科技有限公司 Vegfr2阻断剂多肽及其应用
CN105770901B (zh) * 2016-03-01 2017-05-31 中国人民解放军南京军区南京总医院 交联pH响应跨膜小肽的金纳米星材料及其应用
CN107400169B (zh) * 2017-08-01 2021-05-14 北京华安科创生物技术有限公司 一种肿瘤血管阻断剂融合蛋白的纯化方法
CN107686523B (zh) * 2017-09-15 2021-05-14 山西大学 一种肿瘤酸度响应自噬诱导多肽及其制备方法和应用
CN109467607B (zh) * 2017-12-28 2020-10-30 北京泽勤生物医药有限公司 一种靶向肿瘤的酸性敏感融合肽及其应用
CN109293780B (zh) * 2018-09-04 2021-05-07 厦门宏谱福生物科技有限公司 一种人组织因子凝血复合物及其制备方法
CN109517073A (zh) * 2018-11-30 2019-03-26 北京泽勤生物医药有限公司 一种靶向治疗肿瘤的融合肽及其应用
CN109467593A (zh) * 2018-11-30 2019-03-15 北京泽勤生物医药有限公司 低pH插入肽的胞外段作为抗原的应用
CN110790841A (zh) * 2019-11-11 2020-02-14 国家纳米科学中心 一种蛋白核酸复合物及其制备方法和应用
CN112426438B (zh) * 2019-11-14 2023-12-05 上海鑫湾生物科技有限公司 用于调控酸性环境免疫应答的组合物、其制备方法和用途

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996001653A1 (en) * 1994-07-11 1996-01-25 Board Of Regents, The University Of Texas System Methods and compositions for the specific coagulation of vasculature
WO2001003735A1 (en) * 1999-07-12 2001-01-18 Maine Medical Center Research Institute Cancer treatment using angiopoietins targeted to aminophospholipids
US20100184695A1 (en) * 2007-01-09 2010-07-22 Yuichi Koyamatsu Vascular endothelial cell-binding peptide
EP4098272A3 (en) * 2010-07-13 2023-03-08 University of Rhode Island Board of Trustees Compositions comprising a ph-sensitive membrane insertion polipeptide
CN102153653B (zh) * 2010-12-30 2012-08-15 厦门大学 肿瘤血管靶向多肽与组织因子的融合蛋白及其制备方法
CN103705465B (zh) * 2012-10-09 2016-01-13 复旦大学 一种微酸环境靶向多肽修饰的肿瘤靶向纳米给药系统及其制备方法
CN104045717B (zh) * 2014-07-08 2016-07-06 北京华安科创生物技术有限公司 一种肿瘤血管阻断剂多肽、基因、表达载体及其应用

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112300264A (zh) * 2020-11-09 2021-02-02 青岛大学附属医院 一种新型放射性标记抗癌药的合成标记方法

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