WO2022001075A1 - 眼镜蛇科蛇突触后神经毒素、心脏毒素、细胞毒素、磷脂酶a2及粗毒在抗病毒感染上的应用 - Google Patents

眼镜蛇科蛇突触后神经毒素、心脏毒素、细胞毒素、磷脂酶a2及粗毒在抗病毒感染上的应用 Download PDF

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WO2022001075A1
WO2022001075A1 PCT/CN2021/000131 CN2021000131W WO2022001075A1 WO 2022001075 A1 WO2022001075 A1 WO 2022001075A1 CN 2021000131 W CN2021000131 W CN 2021000131W WO 2022001075 A1 WO2022001075 A1 WO 2022001075A1
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cobra
virus
venom
polypeptide
protein
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沈喆景
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沈喆景
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    • A61K35/56—Materials from animals other than mammals
    • A61K35/58—Reptiles
    • A61K35/583—Snakes; Lizards, e.g. chameleons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • A61K38/46—Hydrolases (3)
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • C—CHEMISTRY; METALLURGY
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • C12Y301/01004—Phospholipase A2 (3.1.1.4)

Definitions

  • the invention relates to the crude venom of Cobra snake and its main toxin, namely the application of a membrane toxin (MT) called membrane active polypeptide (membrane active polypeptide) in the treatment of diseases caused by virus infection, belonging to Biochemical and biopharmaceutical fields.
  • MT membrane toxin
  • membrane active polypeptide membrane active polypeptide
  • the earth is a space where multiple organisms coexist.
  • other organisms that originally coexist peacefully with us may also undergo corresponding changes. People and these organisms The relationship between the two will also change accordingly, and the infection of humans after the virus mutates is a relatively common example in daily life.
  • the respiratory tract is vulnerable to virus invasion and infection due to its open anatomical structure to the outside world.
  • Viral respiratory tract infections such as rhinitis, pharyngitis, bronchitis, and pneumonia are common diseases in daily life.
  • viral infections of the respiratory tract such as hepatitis B virus, HIV also infects humans through various routes.
  • Inflammation caused by viral respiratory infection is a relatively difficult disease to prevent and treat.
  • vaccines developed based on previously circulating viruses cannot be produced when a new mutated virus strikes.
  • the specific antibodies of the virus cannot deal with the invasion of the new virus; at the same time, the limited drugs for the treatment of the virus cannot really suppress the virus due to the mutation of the virus; in the end, many patients are infected by the virus because they missed the opportunity of immunization, and the current treatment does not matter.
  • more ideal drugs need to appear. Therefore, the development of a product with broad-spectrum inhibitory ability to the virus has become an urgent need, and it is also an effort of scientists in this field. .
  • Snake venom is a liquid secreted by snakes from venom glands.
  • the main components are toxic proteins, enzymes and toxins of dozens of species.
  • Enzyme A2 is the main component of the snake venom (crude venom) they secrete.
  • snake venom has broad-spectrum antibacterial properties.
  • snake venoms have been found to have bacteriostatic effects, such as Staphylococcus aureus, Klebsiella, Pseudomonas aeruginosa, Escherichia coli, and Shigella.
  • the antibacterial mechanism of snake venom is generally considered to be that the cytotoxins (membrane toxins, direct hemolytic factors, etc.) in snake venom have strong and broad-spectrum antibacterial and bactericidal effects.
  • Du Yucang et al. observed that five membrane toxins of Chinese cobra have the ability to inhibit the growth of E. coli.
  • Zhang Hong and Li Yunlong also observed the inhibitory effect of various snake venoms on various bacteria in 1990; YFV) antiviral effect. [1-5]
  • snake venom In animal husbandry, in order to prevent livestock animals from being infected by viruses, snake venom is widely added to feed for antiviral effects.
  • the snake venom of the Cobra family has been proved to have a relatively strong killing effect on circovirus, PRRS virus, viral diarrhea virus, swine fever, small and pseudorabies virus; The replication of ring virus, etc., helps to purify the pig farm disease, and indeed plays an effective role in killing the virus.
  • the above provides some reference for snake venom as a potential candidate source of new antiviral drugs.
  • MT membrane toxin
  • MT membrane active polypeptide
  • the membrane toxins of cobra venom include postsynaptic neurotoxin, cardiotoxin, cytotoxin and phospholipase A2. Among them, post-synaptic neurotoxin, cardiotoxin and cytotoxin have a common functional structure, that is, a three-finger structure.
  • Cobra membrane toxins may be similar to its antibacterial mechanism. Although the virus has no other functional structures in the cell except for the genetic material, its protein coat is the same as the main component of the cell membrane. Moreover, membrane toxins can easily pass through the membrane structure and then destroy internal structures such as mitochondria. The characteristics of lysosomes may also be the common mechanism of snake venom membrane toxins against viruses.
  • the common membrane toxicity and antiviral mechanism of Cobra phospholipase A2 may be related to their common properties of solubilizing proteins [17] and necrosis of proteins [18], resulting in the dissolution or necrosis of the protein coat of the virus, thereby destroying the
  • the protein coat of the virus plays a role in inhibiting the virus; at the same time, phospholipase A2 can enzymatically decompose the nucleic acid in the virus.
  • Cobra venom is a liquid substance secreted from venom glands by various snakes of the cobra family.
  • Postsynaptic neurotoxin, cytotoxin, cardiotoxin, and phosphatase A2 are several main components of cobra toxin.
  • they are all membrane active polypeptides, and their activity is achieved by destroying the cell membrane.
  • Such membrane active polypeptides Also known as membrane toxins.
  • the membrane toxins in cobra toxins are very sensitive to influenza A virus, influenza B virus, new coronavirus (2019-nCoV) ), respiratory syncytial virus, HIV, hepatitis B virus, etc. have broad-spectrum anti-virus effects, and the broad-spectrum inhibitory effect on these viruses has been confirmed for the first time.
  • Toxin has better broad-spectrum antiviral effect than any single toxin.
  • the present invention also discloses a group of post-synaptic neurotoxins, cytotoxins, cardiotoxins and phospholipase A2 isolated from Cobra venom.
  • Their mature proteins or amino acid sequences starting from the N-terminus are as follows: (SEQ ID No. 1-SEQ ID No. 28):
  • Bungaro phospholipase A2 (SEQ ID No. 10)
  • Example 1 Separation and purification of Chinese cobra crude venom to prepare postsynaptic neurotoxin, phospholipase A2, cardiotoxin and cytotoxin
  • the crude venom of Chinese cobra is subjected to cation exchange through TSK CM-650 (M) column, and the method for separating various toxins comprises the following steps:
  • Example 2 The isolated postsynaptic neurotoxin (SEQ ID NO.1), phospholipase A2 (SEQ ID NO.2), cardiotoxin I (SEQ ID NO.3), cytotoxin (SEQ ID NO.3) 7) and Chinese cobra crude venom used in the antiviral activity experiment, that is, the virus plaque reduction rate % experiment
  • the released virus can only expand from the initially infected cell to the periphery, that is to say, the propagated virus particles can only spread to adjacent cells.
  • a localized area of diseased cells forms, eventually forming a plaque-like plaque.
  • a plaque is formed by the replication and spread of a virus particle that initially infected the cell and is therefore an accurate measure of the ability of the virus to infect.
  • plaque reduction rate % is an indicator of the ability to inhibit virus infection.
  • Cobra snake postsynaptic neurotoxin (SEQ ID NO.1), phospholipase A2 (SEQ ID NO.2), cardiotoxin I (SEQ ID NO.3), cytotoxin (SEQ ID NO.3) tested in the present invention .7) and the inhibition rate of crude cobra venom to the virus is expressed by plaque reduction rate %.
  • the implementation method of the inhibition test (plaque reduction rate % test) of influenza A virus infectivity comprises the following steps:
  • the tested Chinese cobra postsynaptic neurotoxin, phospholipase A2, cardiotoxin, cytotoxin and Chinese cobra toxin crude venom were prepared into appropriate concentrations with maintenance solution, and then diluted by doubling method and added to well-growing cells In the culture plate, there are 6 wells for each concentration of each drug, and a control group (no drug group) is set up;
  • Influenza A virus PR8 strain the MDCK cells of routine preparation are grown into monolayer with 48-well culture plate and inoculate the virus liquid of 100TCID50 (tissue half infection dose), add 10% fetal bovine serum and 100U after adsorption for 2 hours /ML penicillin and 100U/ML streptomycin in DMEM medium;
  • influenza B virus strain was inoculated on MDCK cells, and the new coronavirus (2019-nCoV) was inoculated on Vero-E6 cells; hepatitis B virus was inoculated on primary human hepatocytes (PHH); AIDS Virus (HIV-1) was inoculated on MT4 cells; Respiratory syncytial virus (RSV) was inoculated on HEp-2 cells; then I-VI steps were carried out in the same manner as above, and the following are various drugs (SEQ ID No1-4) and The plaque reduction rate % of crude cobra venom against these viruses mentioned above.
  • plaque reduction rate % (the number of plaques in the virus control group - the number of plaques in the drug treatment group)/the number of plaques in the virus control group X 100%, after converting the plaque numbers of each group into plaque reduction rate %, the plaques of influenza A virus % reduction rate, % plaque reduction rate for influenza B virus, % plaque reduction rate for novel coronavirus (2019-nCoV), % plaque reduction rate for HIV, and % plaque reduction rate for respiratory syncytial virus From the perspective of the number rate % and the plaque reduction rate % of hepatitis B virus, cobra postsynaptic neurotoxin, phospholipase A2, cardiotoxin, cytotoxin and crude cobra toxin all have the ability to inhibit the above viruses from infecting cells,
  • cobras described in the present invention include the original venom (crude venom) secreted from the venom glands of cobra, king cobra, Bengal cobra, golden ring snake, silver ring snake and black mamba cobra and other amino acids listed in the present invention
  • the sequences of postsynaptic neurotoxin, cardiotoxin, phospholipase A2, and cytotoxin all showed similar % plaque reduction rates to the experiments above.

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Abstract

病毒感染是引起人们呼吸道感染的主要原因，同时也是引起肝炎、艾滋病的原因。引起呼吸道感染的病毒有多种，而引起肝炎、艾滋病的这些病毒会经常变异，所以用一种能够光谱抑制病毒的药品已成为临床上的一种急需。眼镜蛇科蛇的粗毒及其主要毒素突触后神经毒素、心脏毒素、细胞毒素、磷脂酶A2，是一种膜活性多肽(membrane active polypeptide)的膜毒素(membrane toxin，MT)，它们能通过对病毒蛋白外壳的破坏起着广谱抑制病毒的作用，所以在治疗由病毒感染引起的疾病上有着特殊的意义和应用。

Description

眼镜蛇科蛇突触后神经毒素、心脏毒素、细胞毒素、磷脂酶A2及粗毒在抗病毒感染上的应用 技术领域：
本发明涉及眼镜蛇科蛇的粗毒及其主要毒素，即一种被称之为膜活性多肽(membrane active polypeptide)的膜毒素(membrane toxin，MT)在治疗病毒感染引起的疾病上的应用，属于生化和生物制药领域。
背景技术：
地球是一个多生物体共存的空间，当无论由于自然原因或人为原因造成了这个空间环境的巨大变化时，本来与我们和平共处的其他生物体也可能会发生相应的变化，人和这些生物体的关系也会随之发生变化，病毒突变后感染人类就是日常生活中比较常见的一个例子。
呼吸道由于它对外界开放式的解剖结构而容易受到病毒的侵袭和感染，病毒性呼吸道感染如鼻炎、咽喉炎、气管炎、肺炎是日常生活中比较常见的疾病。另外，除了呼吸道的病毒感染外，如乙肝病毒，艾滋病病毒也会通过多种途径感染人类。
病毒性呼吸道感染产生的炎症是一种比较难以预防和治疗的疾病，首先由于病毒易于变异而导致根据以前流行的病毒来开发的疫苗在新的变异后的病毒来袭时身体无法产生能够抵抗新病毒的特异抗体而不能应对新病毒的侵袭；同时有限的治疗病毒的药物也由于病毒的变异而无法真正抑制病毒；最后有很多患者由于错失了免疫接种机会而被病毒感染，而目前的治疗无论从疗效还是病人所要面对的毒副作用上来说都需要有更理想的药物能够出现，故开发一种对病毒具有广谱抑制能力的产品成为一种急需，也是这个领域科学工作者的一个努力方向。
蛇毒是蛇从毒腺中分泌出来的一种液体，主要成份是毒性蛋白质，酶类和毒素约几十种，而对眼镜蛇科的蛇来说，突触后神经毒素、心脏毒素、细胞毒素、磷脂酶A2是它们分泌的蛇毒(粗毒)中的主要成分。
根据以往的研究发现，蛇毒具有广谱抗菌性，比如现已发现多种蛇毒具有抑菌作用，比如金黄色葡萄球菌、克雷伯杆菌、绿脓杆菌，大肠杆菌，痢疾杆菌。蛇毒抗菌的机理一般被认为是蛇毒中的细胞毒素(膜毒素、直接溶血因子等)有较强而广谱的抑菌和杀菌作用。1985年，杜雨苍等观察到中华眼镜蛇5个膜毒素对大 肠杆菌有抑制生长的能力。张宏和李云龙也在1990年观察了多种蛇毒对多种细菌的抑制作用；另一方面，一些研究也发现蛇毒及其成分对麻疹病毒、仙台病毒、登革热病毒(DENV)、黄热病病毒(YFV)的抗病毒作用。[1-5]
在畜牧业上，为了预防家畜动物被病毒感染而致病，蛇毒会被添加在饲料中广泛的用于抗病毒作用。在实践中眼镜蛇科的蛇毒被证明对圆环病毒、蓝耳病毒、病毒性腹泻病毒、猪瘟、细小、伪狂犬病毒均有比较强的杀灭效果；并且能有效切断蓝耳病病毒、圆环病毒等复制，有助于猪场疫病净化，的确起到了有效的杀灭病毒的作用。以上这些为蛇毒可能成为新的抗病毒药物的潜在候选来源提供了一定的参考依据。
关于蛇毒抗菌的机制，目前的研究认为因为蛇毒中有一类被称作为膜活性多肽(membrane active polypeptide)的膜毒素(membrane toxin，MT)，[6-10]医学上这种膜毒素被定义为是“一种可以结合于细胞表面，破坏蛋白质膜从而引起细胞裂解的蛋白质”，眼镜蛇科蛇毒的膜毒素包括突触后神经毒素、心脏毒素、细胞毒素以及磷脂酶A2。其中突触后神经毒素、心脏毒素、细胞毒素它们具有共同的功能结构即三指结构。在结构组成上，它们含大量疏水性残基的强碱性多肽，含有较多的赖氨酸和亮氨酸，8-10个半胱氨酸交叉联接成4-5对二硫键。它们都属于三指毒素家族(three finger toxins)，其4-5对高度保守的二硫键使得它们的空间结构呈“三指”状，有3个指环状(Loop)结构从球形区突出。三指指端为疏水性氨基酸带负电荷，指侧则带有正电荷的精氨酸、赖氨酸，使它们呈具有明显极性的两性分子，这种两性指状结构有利于它们与细胞膜结合，发生寡聚；眼镜蛇科的膜毒素对细胞膜的破坏作用被认为是由于他们共同的三指结构，使得他们依赖其特征性的空间结构-“三指”插入细胞膜中形成孔道；[11-15]在其他一些实验中，研究学者在激光共聚焦显微镜下分别观察到细胞毒素在数分钟内进入线粒体并破坏线粒体结构，以及眼镜蛇毒细胞毒素轻易进入活的肿瘤细胞内并浓集于溶酶体。[16]
关于眼镜蛇科的膜毒素的共同抗病毒机制可能和其抗菌机制类同，虽然病毒除了遗传物质外没有细胞内的其他功能结构体，但它的蛋白质外壳和细胞膜的主要构成成分一样都是蛋白质，而且膜毒素能轻易通过膜结构然后破坏内部构造如线粒体，溶酶体的特质也可能是蛇毒膜毒素抗病毒的共同机制。
而眼镜蛇科磷脂酶A2的共同膜毒性抗病毒机制可能是和它们能溶解蛋白[17]和使蛋白质坏死[18]这一共同特性相关，从而导致病毒的蛋白外壳被溶解或坏死，由此破坏病毒的蛋白外壳，起到抑制病毒的作用；同时磷脂酶A2能酶解病毒中的核酸。
发明内容：
眼镜蛇毒素是由眼镜蛇科的各种蛇从毒腺中分泌出的一种液态状物质，进过干燥后呈固体状，我们也把它称之为眼镜蛇毒素原毒或粗毒。突触后神经毒素、细胞毒素、心脏毒素、磷酯酶A2、是眼镜蛇毒素中的几种主要成分，同时它们都是膜活性多肽，其活性是通过破坏细胞膜来实现的，这类膜活性多肽也被称为膜毒素。
通过我们的实验可以发现眼镜蛇科毒素中的这种膜毒素如突触后神经毒素、细胞毒素、心脏毒素及磷脂酶A2对甲型流感病毒的、乙型流感病毒、新型冠状病毒(2019-nCoV)、呼吸道合胞病毒、艾滋病病毒、乙肝病毒等具有广谱的抗多种病毒的作用，而对这些病毒的广谱抑制作用还是首次被证实，同时我们的研究还发现眼镜蛇科蛇毒的原始粗毒比任何一种单种毒素具有更好的广谱抗病毒作用。
本发明同时公开了一组从眼镜蛇科蛇毒中分离出来的突触后神经毒素，细胞毒素，心脏毒素及磷脂酶A2，它们的成熟蛋白或N端起始的氨基酸序列如下：(SEQ ID No.1-SEQ ID No.28)：
中华眼镜蛇突触后神经毒素(SEQ ID No.1)
Figure PCTCN2021000131-appb-000001
中华眼镜蛇磷脂酶A2(SEQ ID No.2)
Figure PCTCN2021000131-appb-000002
中华眼镜蛇心脏毒素I(SEQ ID No.3)
Figure PCTCN2021000131-appb-000003
中华眼镜蛇心脏毒素(SEQ ID No.4)
Figure PCTCN2021000131-appb-000004
中华眼镜蛇心脏毒素(SEQ ID No.5)
Figure PCTCN2021000131-appb-000005
中华眼镜蛇心脏毒素(SEQ ID No.6)
Figure PCTCN2021000131-appb-000006
Figure PCTCN2021000131-appb-000007
中华眼镜蛇细胞毒素(SEQ ID No.7)
Figure PCTCN2021000131-appb-000008
银环蛇突触后神经毒素(SEQ ID No.8)
Figure PCTCN2021000131-appb-000009
银环蛇突触后神经毒素(SEQ ID No.9)
Figure PCTCN2021000131-appb-000010
银环蛇磷脂酶A2(SEQ ID No.10)
Figure PCTCN2021000131-appb-000011
孟加拉眼镜蛇突触后神经毒素(SEQ ID No.11)
Figure PCTCN2021000131-appb-000012
孟加拉眼镜蛇突触后神经毒素(SEQ ID No.12)
Figure PCTCN2021000131-appb-000013
孟加拉眼镜蛇磷脂酶A2(SEQ ID No.13)
Figure PCTCN2021000131-appb-000014
孟加拉眼镜蛇心脏毒素(SEQ ID No.14)
Figure PCTCN2021000131-appb-000015
孟加拉眼镜蛇细胞毒素(SEQ ID No.15)
Figure PCTCN2021000131-appb-000016
孟加拉眼镜蛇细胞毒素(SEQ ID No.16)
Figure PCTCN2021000131-appb-000017
眼镜王蛇突触后神经毒素(SEQ ID No.17)
Figure PCTCN2021000131-appb-000018
眼镜王蛇突触后神经毒素(SEQ ID No.18)
Figure PCTCN2021000131-appb-000019
眼镜王蛇突触后神经毒素(SEQ ID No.19)
Figure PCTCN2021000131-appb-000020
眼镜王蛇突触后神经毒素(SEQ ID No.20)
Figure PCTCN2021000131-appb-000021
眼镜王蛇突触后神经毒素(SEQ ID No.21)
Figure PCTCN2021000131-appb-000022
眼镜王蛇磷脂酶A2(SEQ ID No.22)
Figure PCTCN2021000131-appb-000023
金环蛇突触后神经毒素(SEQ ID No.23)
Figure PCTCN2021000131-appb-000024
金环蛇突触后神经毒素(SEQ ID No.24)
Figure PCTCN2021000131-appb-000025
金环蛇磷脂酶A2(SEQ ID No.25)
Figure PCTCN2021000131-appb-000026
黑曼巴眼镜蛇突触后神经毒素(SEQ ID No.26)
Figure PCTCN2021000131-appb-000027
黑曼巴眼镜蛇突触后神经毒素(SEQ ID No.27)
Figure PCTCN2021000131-appb-000028
黑曼巴眼镜蛇突触后神经毒素(SEQ ID No.28)
Figure PCTCN2021000131-appb-000029
实施案例：
下面结合具体实施案例对本发明做进一步说明。
实施例1.对中华眼镜蛇粗毒进行分离纯化来制备突触后神经毒素、磷脂酶A2、心脏毒素和细胞毒素
将中华眼镜蛇粗毒经通过TSK CM-650(M)柱进行阳离子交换，分离各种毒素的方法包括下述步骤：
I.样品准备-将1g中华眼镜蛇粗毒溶解在25ml 0.025摩尔PH6.0的醋酸铵缓冲液中，低温离心，取上清液；
II.平衡-用0.025摩尔PH6.0的醋酸铵溶液平衡TSK CM-650(M)柱；
III.洗脱-上样后用0.1～0.5摩尔和0.7～1.0摩尔，pH5.9醋酸铵缓冲液进行2厢阶梯梯度洗脱，紫外检测参数：280nm；洗脱流速：48ml/h；
IV.按记录谱图收集各种毒素组分，收集液中洗脱出12个蛋白峰；(参见申请公布号：CN110090296A)
V.对12个蛋白峰进一步进行阳离子交换，对分离出来的每一个峰用反相高效液相色谱法(RP-HPLC)柱(4.6×250mm，VYDAC RP-C8，5μm)对其蛋白进行纯化和脱盐；
VI.最后对纯化后的蛋白质的一级结构用Edman降解法和蛋白覆盖率进行氨基酸测序以分离出突触后神经毒素、磷脂酶A2、心脏毒素及细胞毒素等。
实施例2.将分离出的突触后神经毒素(SEQ ID NO.1)、磷脂酶A2(SEQ ID NO.2)、心脏毒素I(SEQ ID NO.3)、细胞毒素(SEQ ID NO.7)及中华眼镜蛇粗毒用于抗病毒活性实验，即病毒空斑减数率％实验
病毒空斑原理
在覆盖一薄层琼脂的一片单层细胞上，病毒感染某一细胞后，由于固体介质的限 制，释放的病毒只能由最初感染的细胞向周边扩展，也就是说增殖后的病毒颗粒只能扩散至临近的细胞。经过几个增殖周期，便形成一个局限性病变细胞区域，最终形成一个与噬菌斑类似的空斑。一个空斑由最初感染细胞的一个病毒颗粒复制扩散形成，所以是病毒感染能力的精确计量方法。
空斑减数率％是一种抑制病毒感染能力的指标，具体算法如下：空斑减数率％＝病毒对照组空斑数-药物处理组空斑数/病毒对照组空斑数X100％
本发明中所测试的眼镜蛇科蛇突触后神经毒素(SEQ ID NO.1)、磷脂酶A2(SEQ ID NO.2)、心脏毒素I(SEQ ID NO.3)、细胞毒素(SEQ ID NO.7)和眼镜蛇粗毒对病毒的抑制率用空斑减数率％来表达。
中华眼镜蛇突触后神经毒素(SEQ ID NO.1)、磷脂酶A2(SEQ ID NO.2)、心脏毒素I(SEQ ID NO.3)、细胞毒素(SEQ ID NO.7)、及粗毒对甲型流感病毒感染能力的抑制试验(空斑减数率％试验)实施方法包括下述步骤：
A.将从中华眼镜蛇蛇毒中分离纯化出来的突触后神经毒素、磷脂酶A2、心脏毒素、细胞毒素和眼镜蛇毒素粗毒进行药物毒性试验
I.将测试的中华眼镜蛇突触后神经毒素、磷脂酶A2、心脏毒素、细胞毒素和中华眼镜蛇毒素粗毒分别用维持液配制成适当的浓度，然后按倍比法稀释后加入生长良好的细胞培养板中，每药每种浓度6孔，并设对照组(无药物组)；
II.每3天换同浓度药物一次，到第8天观察细胞病变(CPE)情况，100％CPE为4；75％CPE为3；55％CPE为2；25％CPE为1；无细胞病变(CPE)为0。根据Reed-Muench法计算最大对细胞的无毒浓度CT0；
III.突触后神经毒素、磷脂酶A2、心脏毒素、细胞毒素和眼镜蛇毒素粗毒的最大无毒浓度CT0分别为1.03、0.23、0.39、0.32、1.09。(单位：μg/ml)
B.空斑减数率％试验
I.甲型流感病毒PR8株，用48孔培养板将常规制备的MDCK细胞长成单层后接种100TCID50(组织半数感染量)的病毒液，吸附2小时后加入含10％胎牛血清及100U/ML青霉素和100U/ML链霉素的DMEM培养剂；
II.放入35度5％CO2培养箱继续培养2h，使病毒充分吸附；
III.用维持液将上述5种药物配制成最大无毒浓度的溶液，加入48孔培养板中，每药6孔，同时留下6孔为无药对照组；
IV.加入2％甲基纤维素孔覆盖培养剂，放入35度5％CO2培养箱继续培养，每24h观察细胞病变(CPE)情况；
V.当无药对照组的细胞已发生75％～100％病变(CPE)时，吸弃培养剂；
VI.加入5％甲醛进行孔固定5分钟，弃甲醛，加入结晶紫进行孔染色20分钟，自来水缓缓冲洗染液，计算出药物组和对照组的平均孔斑数。
VII.按上述方法，将乙型流感病毒株接种在MDCK细胞上，将新型冠状病毒(2019-nCoV)接种在Vero-E6细胞上；乙肝病毒接种在人类原代肝细胞(PHH)上；艾滋病病毒(HIV-1)接种在MT4细胞上；呼吸道合胞病毒(RSV)接种在HEp-2细胞；然后按上述同样方法进行I-VI步骤，以下为各种药物(SEQ ID No1-4)及中华眼镜蛇粗毒对以上所述这些病毒的空斑减数率％。
空斑减数率％
Figure PCTCN2021000131-appb-000030
以上各种药物组与对照组的平均空斑数之间有显著性差异；眼镜蛇毒素粗毒组与各单种毒素组之间空斑数也有显著性差异，根据算法：空斑减数率％＝(病毒对照组空斑数-药物处理组空斑数)/病毒对照组空斑数X100％，把各个组的空斑数转换成空斑减数率％后从甲型流感病毒的空斑减数率％、乙型流感病毒的空斑减数率％、新型冠状病毒(2019-nCoV)的空斑减数率％、艾滋病病毒的空斑减数率、呼吸道合胞病毒的空斑减数率％和乙肝病毒的空斑减数率％来看，眼镜蛇突触后神经毒素、磷脂酶A2、心脏毒素、细胞毒素和眼镜蛇毒素粗毒都具有抑制以上病毒感染细胞的能力，而眼镜蛇毒素粗毒的抗病毒活性要高于单种类的毒素。
本发明所述的其他眼镜蛇科包括、眼镜王蛇、孟加拉眼镜蛇、金环蛇、银环蛇及黑曼巴眼镜蛇毒腺中分泌出的原毒(粗毒)和本发明所列出的其他具有氨基酸序列的突触后神经毒素，心脏毒素，磷脂酶A2，细胞毒素都显示出和以上实验类似的空斑减数率％。
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Claims (10)

  1. 一种抗病毒的蛇毒，其特征为眼镜蛇科包括中华眼镜蛇、眼镜王蛇、孟加拉眼镜蛇、金环蛇、银环蛇及黑曼巴眼镜蛇毒腺中分泌出的原毒(粗毒)。
  2. 一种抗病毒的蛇毒毒素，其特征为权利(1)中所述的眼镜蛇科蛇的突触后神经毒素、心脏毒素、细胞毒素及磷脂酶A2。
  3. 一组治疗由病毒感染引起的疾病的药物组合物，其特征在于，它们是权利要求(1)中眼镜蛇科蛇毒腺中分泌出的原毒(粗毒)，或权利要求(2)中所述的抗病毒的眼镜蛇科蛇毒毒素中任何一种单体或任意的混合物，及药物可接受的载体。
  4. 权利要求(1)和(2)中所述的病毒，其特征在于它们是甲型流感病毒、乙型流感病毒、新型冠状病毒(2019-nCoV)、呼吸道合胞病毒、艾滋病病毒、乙肝病毒。
  5. 权利要求(3)中所述的疾病，其特征在于它们是艾滋病、乙型肝炎、及甲型流感病毒、乙型流感病毒、新型冠状病毒(2019-nCoV)、呼吸道合胞病毒引起的呼吸道感染，包括鼻炎、咽喉炎、气管炎及肺炎；同时也包括由甲型流感病毒、乙型流感病毒、新型冠状病毒(2019-nCoV)、呼吸道合胞病毒合并细菌感染引起的呼吸道疾病，包括鼻炎、咽喉炎、气管炎及肺炎。
  6. 根据权利要求(2)所述的眼镜蛇科蛇的突触后神经毒素、心脏毒素、细胞毒素、磷脂酶A2，其特征在于它们的成熟蛋白或多肽的氨基酸序列为SEQ ID No.1至SEQ ID No.28中的蛋白或多肽的序列，或分别与SEQ ID No.1至SEQ ID No.28中的蛋白或多肽具有70％或以上同源性的成熟蛋白或多肽，该成熟蛋白或多肽的功能与SEQ ID No.1至SEQ ID No.28所示的氨基酸序列的成熟蛋白或多肽具有相同或相似的抗病毒功能。
  7. 权利要求(2)所述的眼镜蛇科蛇突触后神经毒素、心脏毒素、细胞毒素和 磷脂酶A2，其特征还在于，它们可来自于从天然蛇毒中分离提取、或化学多肽合成、或是使用重组技术从原核或真核宿主(例如，细菌、酵母、高等植物、昆虫和哺乳动物细胞)中产生。
  8. 根据权利要求(7)以上所述生产的眼镜蛇科的蛇突触后神经毒素、心脏毒素、细胞毒素和磷脂酶A2，本发明的蛋白或多肽可以是糖基化的，或可以是非糖基化的；可以是包含二硫键的，或可以是不包含二硫键的。本发明中所述的蛋白或多肽还可包括或不包括起始的甲硫氨酸残基。
  9. 权利要求(1，2，3，6，7，8)以上所述眼镜蛇科蛇粗毒、突触后神经毒素、心脏毒素、细胞毒素、磷脂酶A2的蛋白或多肽，其特征还在于本发明中所述的蛋白或多肽可包括上述各种眼镜蛇科蛇毒素分子蛋白或多肽经过水解或酶解后的片段、用物理和化学方法处理后的衍生物和类似物，他们是基本保持着与上述眼镜蛇科蛇毒素分子蛋白或多肽相同的生物学功能或活性的多肽。本发明中所述的片段、衍生物或类似物可以是一个或多个氨基酸残基被取代的多肽或蛋白；或在一个或多个氨基酸残基中具有取代基团的多肽或蛋白；或与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇、脂肪链融合)所形成的多肽或蛋白，或附加的氨基酸序列融合到此多肽或蛋白序列而形成的多肽或蛋白。根据本文的描述，这些片段、衍生物和类似物都属于本领域熟练技术人员公知的范围。
  10. 权利要求(3)所述的药物组合物，其使用方法包括雾化吸入、静脉注射、肌肉注射、皮下注射、口服、舌下、鼻腔、直肠、真皮内、腹膜内或鞘內给药或经皮给药；剂量包括从1μg/Kg到350μg/kg每次，给药频率从每天一次到每天多次；或一年多次。
PCT/CN2021/000131 2020-06-29 2021-06-24 眼镜蛇科蛇突触后神经毒素、心脏毒素、细胞毒素、磷脂酶a2及粗毒在抗病毒感染上的应用 WO2022001075A1 (zh)

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