WO2020107565A1 - 一种用于清除氧自由基的小分子蛋白质及其应用 - Google Patents

一种用于清除氧自由基的小分子蛋白质及其应用 Download PDF

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WO2020107565A1
WO2020107565A1 PCT/CN2018/122066 CN2018122066W WO2020107565A1 WO 2020107565 A1 WO2020107565 A1 WO 2020107565A1 CN 2018122066 W CN2018122066 W CN 2018122066W WO 2020107565 A1 WO2020107565 A1 WO 2020107565A1
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small molecule
molecule protein
free radicals
oxygen free
chemotherapy
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李翔
杨海洋
钱政江
陈瑜
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深圳先进技术研究院
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    • C12N9/0004—Oxidoreductases (1.)
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  • the present application relates to the field of biotechnology, in particular to a small molecule protein for scavenging oxygen free radicals and its application.
  • Radiotherapy and chemotherapy refer to the use of radiation or chemical drugs to treat cancer.
  • the method of using drugs to kill tumor cells is chemotherapy. It is an important tumor treatment method today. Its principle is to use different cells for different sensitivity to drugs. That is, the cells that proliferate vigorously are more sensitive to chemotherapy than cells that proliferate slowly, and cancer cells belong to the most proliferating cells.
  • Radiation therapy (radiotherapy) of tumors is a local treatment method that uses radiation to treat tumors, such as the use of ⁇ , ⁇ , and ⁇ rays produced by radioisotopes. About 70% of cancer patients require radiation therapy during the treatment of cancer, about 40 % Of cancers can be cured with radiation therapy. Radiation therapy has become increasingly prominent in the role and status of cancer therapy, and has become one of the main means of treating malignant tumors.
  • CN102847142A Provides a new oral biologic medicine for treating cancer patients with radiotherapy and chemotherapy syndrome.
  • the bionew medicine ingredients include recombinant human superoxide dismutase, antioxidant vitamin C and fat-soluble vitamin E, and antioxidant metallothionein ) MT, mannitol, trehalose, and small molecules of water.
  • CN1554443 discloses an oral liquid for improving blood image and enhancing immunosuppressive tumors in cancer radiotherapy and chemotherapy.
  • Each kilogram of oral liquid contains the following components: Ganoderma lucidum deep fermentation liquid 50-100 grams, SOD (superoxide dismutase) 800-16000
  • the active unit is 100-200 grams of lentinan extract, 75-125 grams of mixed Chinese herbal extract, and also contains a certain amount of Cordyceps mycelium, so that the adenosine content reaches 36-108 mg, and the mannitol content reaches 4-6 grams.
  • the balance is water.
  • superoxide dismutase can scavenge oxygen free radicals, so that the body maintains a low concentration of dynamic free radical oxygen radicals.
  • this application provides a small molecule protein for scavenging oxygen free radicals and its application.
  • the small molecule protein is named GEM, which can combine with divalent manganese ions and can remove the body Oxygen free radicals, which maintain the dynamic balance of low concentration of free radicals.
  • the mechanism of action is similar to superoxide gasification enzyme 2 (Mn SOD). It can be used to prevent and treat cancer and the many side effects of postoperative radiotherapy and chemotherapy to suppress cancer.
  • Mn SOD superoxide gasification enzyme 2
  • the present application provides a small molecule protein for scavenging oxygen free radicals.
  • the amino acid sequence of the small molecule protein is SEQ ID NO.1.
  • the SEQ ID NO.1 is as follows:
  • the affinity metal ions of the small molecule protein include divalent manganese ions.
  • nucleotide sequence of the small molecule protein is shown in SEQ ID NO. 2:
  • the SEQ ID NO. 2 is as follows:
  • GEM novel small molecule protein with a molecular weight of about 10KD
  • the small molecule protein specifically binds to divalent manganese ions and can be developed based on the binding of divalent manganese ions
  • the application can eliminate oxygen free radicals in the body and maintain the dynamic balance of low concentration of free radicals.
  • the mechanism of action is similar to superoxide gasification enzyme 2 (Mn SOD). It can be used to prevent and treat cancer and postoperative radiotherapy and chemotherapy. Numerous side effects, inhibit the re-formation or spread of cancer cells, as a raw material for the preparation of cancer recovery and chemotherapy-assisted recovery drugs, has high economic benefits and social value.
  • the present application provides a plasmid that includes a nucleotide sequence encoding the small molecule protein of the first aspect.
  • the present application provides a lentivirus, which is obtained by co-transfection and packaging of the plasmid and the auxiliary plasmid of the second aspect.
  • the present application provides a host cell, the host cell comprising the plasmid according to the second aspect.
  • the present application provides a small molecule protein according to the first aspect, a plasmid according to the second aspect, a lentivirus according to the third aspect, or a host cell according to the fourth aspect for scavenging oxygen free The purpose of the base.
  • the oxygen radical scavenging agent may be a drug and/or an agent.
  • the present application provides a medicament comprising the small molecule protein of the first aspect, the plasmid of the second aspect, the lentivirus of the third aspect, or the host cell of the fourth aspect.
  • the present application provides a use of the medicine according to the sixth aspect for preparing an adjuvant drug for radiotherapy and chemotherapy.
  • the small-molecule protein can scavenge oxygen free radicals in the body and maintain the dynamic balance of low concentration of free radicals.
  • the mechanism of action is similar to superoxide gasification enzyme 2 (Mn SOD), which can be used to prevent and treat cancer and postoperative Many side effects brought by radiotherapy and chemotherapy inhibit the formation or spread of cancer cells.
  • Mn SOD superoxide gasification enzyme 2
  • this application has high economic benefits and social value.
  • the GEM protein described in this application as an adjuvant drug for radiochemotherapy includes four processes: construction of GEM plasmids, batch expression in microorganisms, purification of GEM proteins, optimized addition of GEM proteins; modification of synthesized proteins has the effect of scavenging oxygen free radicals, It can assist radiotherapy and chemotherapy, and the small molecular weight has high medicinal value.
  • the small molecule protein provided by this application is a modified small molecule protein, with clear composition, safe and no side effects, and a molecular weight smaller than the currently used SOD molecular weight, so it is easier for the body to absorb, and the efficiency of scavenging oxygen free radicals is higher, and its role
  • the mechanism is similar to the naturally occurring SOD, so it can be used as an alternative or mixed use of SOD; because the DNA sequence is clear and short, it is more conducive to later transformation and optimization.
  • Figure 1 is a graph of ion mass spectrometry detection results of this application.
  • FIG. 2 is a graph of the MRI (nuclear magnetic resonance imaging) detection result of the application
  • FIG. 3 is an immunofluorescence detection chart of the present application, in which FIG. 3(A) is a fluorescence chart in a normal state, and FIG. 3(B) is a fluorescence chart after treatment.
  • the DNA sequence SEQ ID NO. 2 of the protein (GEM) described in this application is as follows:
  • This sequence is the core sequence of this small molecule protein.
  • the protein encoded by it can be specifically bound to the divalent manganese ion; in order to facilitate the subsequent experimental detection process (immunohistochemistry and western blot), we have 5'in this sequence Add a 3xFlag Tag segment to the segment; at the same time, in order to facilitate gene cloning, we introduced BamHI (GGATCC) and EcoRI (GAATTC) two more commonly used restriction sites at both ends. The choice of introduction of the restriction site does not affect The specific role of this protein; that is, its structure is BamHI-3xFlag-GEM-EcoRI, and its specific DNA sequence SEQ ID NO. 3 see below:
  • this vector is also a commonly used vector, and the choice of vector will not affect the purpose of this application.
  • the purified plasmid was transfected into HeLa cells by lipofection (or lentivirus infection). After 48 hours, the cells were collected and the changes of metal ions in cells were detected by ICP-MS metal ion mass spectrometry. The results are shown in Figure 1; As can be seen from Figure 1, the results show that GEM can specifically bind manganese ions, and other trace metal ions in the cell are not affected.
  • GEM and GFP were transferred into HeLa cells respectively. After 48 hours of expression, the cells were collected and T1-weighted images were detected by MRI.
  • the MRI parameters were (3.0T; scan time 2-6min per sequence; pixel resolution, 250 ⁇ 250 ⁇ 1500 ⁇ m), the specific results are shown in Figure 2;
  • the present application provides an engineered small molecule protein that can bind manganese ions in the body, the mechanism of action is similar to that of superoxide gasification enzymes in the body, and has antioxidant and scavenging oxygen free radicals. Therefore, it can be used as an adjuvant drug/technology for radiotherapy and chemotherapy, with broad application prospects and huge market value.

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Abstract

提供了一种用于清除氧自由基的小分子蛋白质及其应用，所述小分子蛋白质的氨基酸序列为SEQ ID NO.1；所述小分子通过结合2价锰离子，能够清除体内氧自由基，维持自由基低浓度的动态平衡，作用机制类似于超氧化物歧化酶2(Mn SOD)，能够用于预防和治疗癌症及术后放疗、化疗带来的众多副作用，抑制癌细胞的再形成或扩散，作为一种制备癌症放、化疗的辅助药物的原料，具有很高的经济效益和社会价值。

Description

一种用于清除氧自由基的小分子蛋白质及其应用 技术领域
本申请涉及生物技术领域，尤其涉及一种用于清除氧自由基的小分子蛋白质及其应用。
背景技术
放、化疗是指利用放射线或者化学药物治疗癌症的的手段，其中利用药物杀死肿瘤细胞的方法即为化疗，是当今重要的肿瘤治疗手段，其原理是利用不同细胞对于药物敏感性不同进行的，即增殖旺盛的细胞对于化疗的敏感性高于增殖缓慢的细胞，癌细胞则属于增殖最为旺盛的细胞。肿瘤的放射治疗(放疗)是利用放射线治疗肿瘤一种局部治疗方法，例如利用放射性同位素产生的α、β、γ射线等，大约70％的癌症患者在治疗癌症过程中需要放射治疗，约有40％的癌症可以用放疗根治。放射治疗在肿瘤治疗中的作用和地位日益突出，已成为治疗恶性肿瘤的主要手段之一。
但是目前限制放疗化疗使用并对患者造成巨大痛苦的原因就是放、化疗过程中、后对患者造成的许多对机体有害的副作用，例如恶心呕吐，毛发脱落，免疫力降低等，导致患者的生存质量普遍下降，甚至不能耐受而终止治疗。众多放、化疗副作用中，其中一个重要的原因就是氧自由基造成的。在放疗或化疗过程中，药物和放射线会作用于机体组织内的水分子发生电离，导致体内产生大量的氧自由基，这些自由基和生物大分子相互作用，会引起骨髓损伤，白血球减少，脱发等严重的不良反应，甚至会造成肿瘤的再次发生以及转移。
CN102847142A提供一种治疗癌症病人放疗、化疗综合症的一种口服生物新药，该生物新药成份包括重组人源超氧化物歧化酶、抗氧化剂维生素C和脂溶 性维生素E，抗氧化金属硫蛋白(metallothionein)MT，甘露醇、海藻糖，以及小分子团水。CN1554443公开了一种在癌症放化疗中提高血象增强免疫抑制肿瘤的口服液，每千克口服液中含有以下组分：灵芝深层发酵液50-100克，SOD(超氧化物歧化酶)8000-16000活性单位，香菇多糖浸液100-200克，混合中药提取液75-125克，还含有一定量的虫草菌丝体，使得腺苷含量达到36-108毫克，甘露醇含量达到4-6克，余量为水。在正常机体内，超氧化物歧化酶能清除氧自由基，使机体维持低浓度动态平衡的氧自由基。但是放疗或化疗的使用会降低内源性超氧化物气化酶的活性，并且会产生更多的氧自由基，打破氧自由基低浓度的动态平衡。目前常用辅助化疗药物包括添加外源性超氧化物歧化酶，或者利用提取的中草药复合物缓解疼痛等。外源的超氧化物歧化酶分子量较大，机体吸收效率较低，因此其总的清除氧自由基的效率会较低。新兴的利用中草药提取的复合物作为放化疗的辅助药物，首先其临床效果没有明确的科学机制支撑，其次这种复合物的成分并不明确，存在很大的安全隐患。
因此，研发出一种效果较好的放疗辅助药物对于放疗化疗技术的使用具有十分重要的意义。
发明内容
针对现有技术的不足及实际的需求，本申请提供一种用于清除氧自由基的小分子蛋白质及其应用，所述小分子蛋白质命名为GEM，其可以结合2价锰离子，能够清除体内氧自由基，维持自由基低浓度的动态平衡，作用机制类似于超氧化物气化酶2(Mn SOD)，能够用于预防和治疗癌症及术后放疗、化疗带来的众多副作用，抑制癌细胞的再形成或扩散，作为一种制备癌症放、化疗的辅助恢复药物的原料，具有很高的经济效益和社会价值。
为达此目的，本申请采用以下技术方案：
第一方面，本申请提供一种用于清除氧自由基的小分子蛋白质，所述小分子蛋白质的氨基酸序列为SEQ ID NO.1。
所述SEQ ID NO.1如下所示：
Figure PCTCN2018122066-appb-000001
优选地，所述小分子蛋白质的亲和金属离子包括2价锰离子。
优选地，所述小分子蛋白质的核苷酸序列如SEQ ID NO.2所示：
所述SEQ ID NO.2如下所示：
Figure PCTCN2018122066-appb-000002
本申请中，申请人发现一种新型小分子蛋白质，所述小分子蛋白质的分子量大约为10KD，命名为GEM，所述小分子蛋白质特异性结合2价锰离子，能够开发基于结合2价锰离子的应用，能够清除体内氧自由基，维持自由基低浓度的动态平衡，作用机制类似于超氧化物气化酶2(Mn SOD)，能够用于预防和治疗癌症及术后放疗、化疗带来的众多副作用，抑制癌细胞的再形成或扩散，作为一种制备癌症放、化疗的辅助恢复药物的原料，具有很高的经济效益和社 会价值。
本申请保护上述基因序列，但不局限于此，在此基础上所做的基因突变和改造都属于本申请的保护点。
本申请并不局限于某一形式的药物或者技术，凡是在此蛋白功能基础上开发的放化疗辅助药物都属于本申请专利的保护范围。
第二方面，本申请提供一种质粒，所述质粒包括编码第一方面所述的小分子蛋白质的核苷酸序列。
第三方面，本申请提供一种慢病毒，所述慢病毒通过第二方面所述的质粒和辅助质粒共转染包装得到。
第四方面，本申请提供一种宿主细胞，所述宿主细胞包括第二方面所述的质粒。
第五方面，本申请提供一种如第一方面所述的小分子蛋白质、第二方面所述的质粒、第三方面所述的慢病毒或第四方面所述的宿主细胞用于清除氧自由基的用途。
本申请中，所述清除氧自由基的可以是药物和/或试剂。
第六方面，本申请提供一种药物，所述药物包含第一方面所述的小分子蛋白质、第二方面的质粒、第三方面所述的慢病毒或第四方面所述的宿主细胞。
第七方面，本申请提供一种如第六方面所述的药物用于制备放化疗辅助药物的用途。
本申请中，所述小分子蛋白质能够清除体内氧自由基，维持自由基低浓度的动态平衡，作用机制类似于超氧化物气化酶2(Mn SOD)，可用于预防和治疗癌症及术后放疗、化疗带来的众多副作用，抑制癌细胞的再形成或扩散，作为一种制备癌症放、化疗的辅助恢复药物的原料，本申请具有很高的经济效益和 社会价值。
本申请所述GEM蛋白作为放化疗辅助药物包含四个过程：GEM质粒的构建，微生物内批量的表达，GEM蛋白质的纯化，GEM蛋白质的优化添加；改造合成的蛋白质具有清除氧自由基的作用，能够辅助放化疗，并且分子量小具有很高的药用价值，
由于本申请在于GEM蛋白作为放化疗辅助药物，清除氧自由基的作用，并且所用技术(包括质粒构建、批量表达、蛋白纯化加工，辅助药物的合成)等工艺皆为目前通用技术，因此此部分不在本专利的讨论范围，并且本专利的并不局限于某一种形式的辅助药物。
目前现有的产品技术路线有两种，第一种是外源超氧化物气化酶(SOD)；另一种是中草药复合物的组合；这两种技术产品有一下几点缺点：1.分子量大，难以吸收，作用效率低；2.成分不明确，存在安全隐患。
与现有技术相比，本申请具有如下有益效果：
本申请提供的小分子蛋白质为改造过的小分子蛋白质，成分明确，安全无副作用，分子量比目前常用的SOD分子量小，因此更易于机体的吸收，清除氧自由基的效率更高，而其作用机制与天然存在的SOD类似，因此可作为SOD的一种替代选择或者混合使用；由于DNA序列明确，并且较短，因此更利于后期的改造和优化。
附图说明
图1为本申请的离子质谱检测结果图；
图2为本申请的MRI(核磁共振成像)检测结果图；
图3为本申请的免疫荧光检测图，其中图3(A)为正常状态的荧光图，图3(B)为处理后的荧光图。
具体实施方式
为更进一步阐述本申请所采取的技术手段及其效果，以下结合附图并通过具体实施方式来进一步说明本申请的技术方案，但本申请并非局限在实施例范围内。
实施例1 质粒构建
本申请所述蛋白质(GEM)的DNA序列SEQ ID NO.2如下：
Figure PCTCN2018122066-appb-000003
此段序列为此小分子蛋白质的核心序列，其编码的蛋白质经过检测可以特异性的结合2价锰离子；为了便于后续实验检测过程(免疫组化及western blot)，我们在此段序列5’段加入一段3xFlag Tag片段；同时为了便于基因克隆，我们分别在两端引入了BamHI(GGATCC)和EcoRI(GAATTC)两个较为常用的酶切位点，酶切位点的引入的选择并不影响此蛋白的具体作用；即其结构为BamHI-3xFlag-GEM-EcoRI，其具体DNA序列SEQ ID NO.3见下：
Figure PCTCN2018122066-appb-000004
Figure PCTCN2018122066-appb-000005
通过人工合成的方法得到此段DNA片段后，通过基因克隆将其插入到pUltra-smurf载体中(此载体亦为常用载体，载体的选择并不会影响本申请的目的)，具体方法见下：
(1)将人工合成的DNA片段通过BamHI/EcoRI限制性内切酶切，同时用相同的酶切载体，利用胶回收的方法分别得到粘性末端的片段和载体；
(2)将回收的产物利用T4连接酶连接3小时，转入大肠杆菌感受态细胞并涂板，后挑取单克隆，摇菌培养；
(3)提取质粒，并通过酶切的方法检测挑取阳性克隆；阳性的质粒经过BamHI/EcoRI酶切后，会出现500bp大小左右的条带，得到阳性质粒后扩大培养即可。
实施例2 实验检测
一.GEM结合锰离子能力的检测
离子质谱检测
将提纯得到的质粒利用脂质体转染(或慢病毒感染)转入HeLa细胞中，48小时后收集细胞利用ICP-MS金属离子质谱检测细胞内金属离子的变化，结果如图1所示；由图1可知，结果证明GEM可以特异性的结合锰离子，并且细 胞内其他微量金属离子并不受影响。
MRI检测
由于2价锰离子的特殊性质，其在核磁共振成像T1加权像下会呈现高信号，因此可以利用此方法检测GEM结合锰离子的能力。
分别把GEM和GFP(对照)转入HeLa细胞中，表达48小时后收集细胞，利用MRI检测T1加权像，MRI参数为(3.0T；扫描时间2-6min per sequence；像素分辨率，250×250×1500μm)，具体结果如图2所示；
由图2可知，MRI(核磁共振成像)检测结果表明GEM在细胞中能结合锰离子的能力远远大于对照组。
二.胞内模拟化疗(氧化应激危害)
为了在细胞内模拟放化疗对机体氧化应激的危害，采用高浓度过氧化氢处理细胞；分别将GEM、GFP转入HeLa细胞，稳定表达后利用过氧化氢处理细胞模拟化疗的危害，免疫荧光检测处理前(图3(A))和处理两小时后(图3(B))的荧光信号，结果如图3(A)和图3(B)所示；
由图3(A)和图3(B)所示，GEM蛋白在过氧化氢处理后，很大一部分由线粒体转移至细胞核，这一转移过程是一种经典的蛋白入核修复氧化应激对DNA的损伤作用，证明了GEM蛋白作为化放疗辅助药物的清除氧自由基的功能。
综上所述，本申请提供一种改造的小分子蛋白质，所述小分子蛋白质可以在体内结合锰离子，作用机制类似于生物体内的超氧化物气化酶，具有抗氧化，清除氧自由基的功能，因此可作为放化疗辅助药物/技术，具有广阔的应用前景和巨大的市场价值。
申请人声明，本申请通过上述实施例来说明本申请的详细方法，但本申请 并不局限于上述详细方法，即不意味着本申请必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本申请的任何改进，对本申请产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本申请的保护范围和公开范围之内。

Claims (9)

  1. 一种用于清除氧自由基的小分子蛋白质，其氨基酸序列为SEQ ID NO.1。
  2. 根据权利要求1所述的小分子蛋白质，其中，所述小分子蛋白质的亲和金属离子包括2价锰离子。
  3. 根据权利要求2所述的小分子蛋白质，其中，所述小分子蛋白质的核苷酸序列为SEQ ID NO.2。
  4. 一种质粒，其包括编码权利要求1-3中任一项所述的小分子蛋白质的核苷酸序列。
  5. 一种慢病毒，其通过权利要求4所述的质粒和辅助质粒共转染包装得到。
  6. 一种宿主细胞，其包括权利要求4所述的质粒。
  7. 如权利要求所述1-3中任一项所述的小分子蛋白质、权利要求4所述的质粒、权利要求5所述的慢病毒或权利要求6所述的宿主细胞用于清除氧自由基的用途。
  8. 一种药物，其包含权利要求1-3中任一项所述的小分子蛋白质、权利要求4所述的质粒、权利要求5所述的慢病毒或权利要求6所述的宿主细胞中的任意一种或至少两种的组合。
  9. 如权利要求8所述的药物用于制备放化疗辅助药物的用途。
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