CN111617108A - 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 PDFInfo
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- CN111617108A CN111617108A CN202010623330.1A CN202010623330A CN111617108A CN 111617108 A CN111617108 A CN 111617108A CN 202010623330 A CN202010623330 A CN 202010623330A CN 111617108 A CN111617108 A CN 111617108A
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Abstract
本发明涉及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。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，病毒性流感和肺炎是日常生活中比较常见的病例；除此以外，乙肝病毒、艾滋病毒也会通过多种途径感染人类致病。病毒性引起的呼吸道疾病目前的治疗手段无论是疫苗还是药物效果都不太理想，病毒性肝炎和艾滋病的治疗效果也有待进一步提高，故开发一种对病毒具有广谱抑制能力而副作用小的产品已成为一种临床急需。眼镜蛇科蛇突触后神经毒素、心脏毒素、细胞毒素、磷脂酶A2及粗毒都显示出其在抗病毒感染上的有效性，有可能成为新的抗病毒产品的候选者。
Description
技术领域：
本发明涉及眼镜蛇科蛇主要毒素成分及粗毒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致病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，病毒性流感和肺炎是日常生活中比较常见的病例；除此以外，乙肝病毒、艾滋病毒也会通过多种途径感染人类致病。
病毒性流感和肺炎是一种比较难以预防和治疗的疾病，首先由于其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出能够抵抗新病毒的特异性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可能由于病毒的变异而无法真正的抑制病毒；对乙肝病人来说，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；对艾滋病的治疗也面临同样的境地。故开发一种对病毒具有广谱抑制能力的的产品已成为一种急需，这也是医药领域科学工作者的一个努力方向。
根据以往的研究发现，蛇毒具有广谱的抗菌性，比如对金黄色葡萄球菌、克雷伯杆菌、绿脓杆菌、大肠杆菌、痢疾杆菌等多种细菌，蛇毒都显示出其抑菌作用。蛇毒抗菌的机理一般被认为是蛇毒中的膜毒素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；1990年，张宏和李云龙也观察到了多种蛇毒对不同细菌的抑制作用。
另一方面，国外一些研究也发现了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、圆环病毒等的复制，起到了有效的杀灭病毒的作用，有助于猪场疫病净化。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。
关于蛇毒抗菌的详细机理，目前的研究认为是蛇毒中有一类被称作为膜活性多肽的成分，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，其中包括突触后神经毒素、心脏毒素、细胞毒素。这些毒素是蛇毒的主要毒性组份，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还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；[6-11]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，以及细胞毒素轻易进入活的肿瘤细胞内并浓集于溶酶体，[12]最终导致细胞破裂死亡。
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同样是蛇毒抗病毒的机制。
眼镜蛇科蛇突触后神经毒素另一个共同特点是能可逆性的和烟碱型乙酰胆碱受体结合，[13-15]而烟碱型乙酰胆碱受体是影响细胞免疫反应的一个靶点，[16]同时也不排除这种受体是病毒进入细胞的另一个通道，所以有流行病学报告显示吸烟人群因烟碱和烟碱型乙酰胆碱受体结合而减低了呼吸道病毒感染的机会。
而眼镜蛇科磷脂酶A2的抗病毒共性可能是它们能溶解蛋白，[16]从而破坏病毒的蛋白外壳，起到抑制病毒的作用。
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。但眼镜蛇毒素粗毒中各种毒素种类繁多，已知成分有突触前神经毒素、突触后神经毒素、细胞毒素、心脏毒素、神经生长因子、溶血素(DLP)、CVA蛋白、其他膜活性多肽、眼镜蛇蛇毒因子等；另外，它的成分还包括碱性磷酸单酯酶、磷酸二酯酶、磷脂酶A2、乙酰胆碱酯酶、L-氨基酸氧化酶、核糖核酸酶、蛋白水解酶等。
通过我们的实验可以发现眼镜蛇科毒素中一系列不同成分的单种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、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抗病毒作用，本发明公开了这些毒素的成熟蛋白或多肽的氨基酸序列，具体如下：(SEQ ID No.1-SEQ ID No.60)
银环蛇突触后神经毒素
SEQ ID No.1
SEQ ID No.2
SEQ ID No.3
SEQ ID No.4
SEQ ID No.5
黑曼巴眼镜蛇突触后神经毒素
SEQ ID No.6
SEQ ID No.7
SEQ ID No.8
眼镜王蛇突触后神经毒素
SEQ ID No.9
SEQ ID No.10
SEQ ID No.11
SEQ ID No.12
SEQ ID No.13
SEQ ID No.14
SEQ ID No.15
SEQ ID No.16
SEQ ID No.17
SEQ ID No.18
SEQ ID No.19
SEQ ID No.20
SEQ ID No.21
SEQ ID No.22
SEQ ID No.23
SEQ ID No.24
SEQ ID No.25
金环蛇突触后神经毒素
SEQ ID No.26
SEQ ID No.27
中华眼镜蛇突触后神经毒素
SEQ ID No.28
SEQ ID No.29
SEQ ID No.30
孟加拉眼镜蛇突触后神经毒素
SEQ ID No.31
SEQ ID No.32
SEQ ID No.33
SEQ ID No.34
中华眼镜蛇磷脂酶A2
Sequence ID No.35
Sequence ID No.36
银环蛇磷脂酶A2
Sequence ID No.37
Sequence ID No.38
Sequence ID No.39
眼镜王蛇磷脂酶A2
Sequence ID No.40
金环蛇磷脂酶A2
Sequence ID No.41
Sequence ID No.42
Sequence ID No.43
孟加拉眼镜蛇磷脂酶A2
Sequence ID No.44
Sequence ID No.45
Sequence ID No.46
Sequence ID No.47
中华眼镜蛇心脏毒素
Sequence ID No.48
Sequence ID No.49
Sequence ID No.50
Sequence ID No.51
Sequence ID No.52
Sequence ID No.53
Sequence ID No.54
孟加拉眼镜蛇心脏毒素
Sequence ID No.55
中华眼镜蛇细胞毒素
Sequence ID No.56
Sequence ID No.57
Sequence ID No.58
孟加拉眼镜蛇细胞毒素
Sequence ID No.59
Sequence ID No.60
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、细胞毒素、心脏毒素和磷脂酶A2
将中华眼镜蛇粗毒经过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；
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)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，分离出突触后神经毒素、细胞毒素、心脏毒素及磷脂酶A2等。
实施例2.将分离出的中华眼镜蛇突触后神经毒素、细胞毒素、心脏毒素、磷脂酶A2，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、心脏毒素、细胞毒素、磷脂酶A2，及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、心脏毒素、磷脂酶A2、细胞毒素，及中华眼镜蛇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、心脏毒素、磷脂酶A2、细胞毒素和蛇毒粗毒进行药物最大细胞无毒浓度测试
I.将测试的中华眼镜蛇突触后神经毒素、心脏毒素、细胞毒素、磷脂酶A2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、心脏毒素、磷脂酶A2、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、0.39、0.23、0.32、0.89(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细胞上；然后按上述同样方法进行I-VI步骤，以下为各种药物对以上所述这些病毒的空斑减数率％。
空斑减数率％
以上各种药物组与病毒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、心脏毒素、细胞毒素、磷脂酶A2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蛇包括、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)和它们的突触后神经毒素、心脏毒素、磷脂酶A2、细胞毒素(参见氨基酸序列表)都显示出和以上实验类似的空斑减数率％，证明它们有同样的抗病毒感染能力。
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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、艾滋病病毒、新型冠状病毒(2019-nCoV)、呼吸道合胞病毒、乙肝病毒。
5.权利要求(3)中所述的疾病，其特征在于它们是甲型流感、乙型流感、艾滋病、新型冠状病毒(2019-nCoV)引起的肺炎、呼吸道合胞病毒引起的肺炎、乙型肝炎。
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.60中的蛋白或多肽的序列，或分别与SEQ ID No.1至SEQ ID No.60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.60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)以上所述重组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，根据重组生产方案所用的宿主，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。
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| WO2022001075A1 (zh) * | 2020-06-29 | 2022-01-06 | 沈喆景 | 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 |
| CN114716567A (zh) * | 2022-03-01 | 2022-07-08 | 上海赛伦生物技术股份有限公司 | 一种嵌合体抗原和抗金环蛇蛇毒制剂的制备方法及其应用 |
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