CN113804637A - 一种海蛇毒的鉴定方法及其应用 - Google Patents
一种海蛇毒的鉴定方法及其应用 Download PDFInfo
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- CN113804637A CN113804637A CN202010546709.7A CN202010546709A CN113804637A CN 113804637 A CN113804637 A CN 113804637A CN 202010546709 A CN202010546709 A CN 202010546709A CN 113804637 A CN113804637 A CN 113804637A
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Abstract
本发明公开了一种海蛇蛇毒的鉴定方法,包括如下步骤:(1)选择产地为中国的海蛇毒素(含平颏海蛇毒和青环海蛇毒)冻干粉,溶解后配制成蛋白质溶液;(2)采用紫外光谱扫描进行海蛇毒的分析及初步鉴定;(3)采用SDS‑PAGE电泳分析及液质联用LC‑ESI‑MS/MS质谱法进行明确鉴定。此外,本发明还公开了该海蛇毒的鉴定方法在制备治疗海蛇毒中毒的抗海蛇毒血清中的应用。本发明可对海蛇毒进行实验室鉴定,更准确地对海蛇毒进行质控,进而生产出治疗海蛇毒中毒的特效药抗海蛇毒血清。本发明方法检测灵敏度高、正确检出率较高、不存在交叉反应,能有效缩短测试时间,并大大降低人工操作成本。
Description
技术领域
本发明属于生物制药领域,涉及一种海蛇毒的鉴定方法,特别涉及一种用蛇毒蛋白分析技术鉴定蛇毒的种类的方法;此外,本发明还涉及该鉴定方法的应用,鉴定的蛇毒可以用于抗蛇毒血清的生产。
背景技术
毒蛇咬伤常见于亚热带和热带地区。在亚洲、非洲、拉丁美洲和大洋洲的农村地区,毒蛇咬伤是特别突出的公共卫生问题,我国的蛇伤发病呈现出逐年增多的趋势。
毒液多为淡黄色或乳白色半透明黏稠状液体,成分达100多种。每种蛇毒含有多种不同的毒性成分,各种毒性组分在不同蛇毒中含量有较大差异,同种毒蛇的毒性组分可因地域分布、季节性、蛇龄等不同而异。蛇毒组分由酶、多肽、糖蛋白和金属离子等组成,其中毒性蛋白质达数十种,蛋白类占蛇毒总量的90%~95%以上。蛇毒可对机体神经系统、血液系统、肌肉组织、循环系统、泌尿系统、内分泌系统、消化系统等产生损害作用。
本研究的对象为海蛇毒,其成分复杂,含有多种蛋白,主要包括磷脂酶A2、三指毒素、金属蛋白酶、丝氨酸蛋白酶、血管内皮生长因子、蛇毒因子、蛋白酶抑制剂、磷脂酶B等。海蛇毒的组成成分与海蛇咬伤后的临床症状有直接关系,包括肌肉麻痹、呼吸麻痹、肌肉损伤等。
世界卫生组织(WHO)强调抗蛇毒血清是蛇伤的唯一有效的解毒药(WHO:Guidelines for the management of snakebites)。抗蛇毒血清是治疗毒蛇咬伤的高效、特效药物,一般认为在被毒蛇咬伤后应立即注射同种蛇毒抗血清以达到最佳中和治疗效果。在明确毒蛇种类后,采取相应的救治措施,迅速使用抗蛇毒血清中和体内蛇毒,防治可能发生的并发症。由于毒蛇咬伤如不及时有效救治常致人死亡,针对本文所述海蛇毒,应用抗海蛇毒血清进行治疗。
抗海蛇毒血清是利用脱毒的海蛇毒素免疫马匹,所得血浆经胃蛋白酶消化、硫酸铵沉淀、明矾沉淀、超滤等纯化步骤得到的马抗海蛇毒免疫球蛋白。为了生产出治疗海蛇毒中毒的特效药抗海蛇毒血清,需要对海蛇毒进行实验鉴定,以便更好的对海蛇毒进行质控。
目前研究蛇毒鉴定的技术包括凝集测试法、酶联免疫吸附法、适配子技术、聚合酶链反应等。凝集试验属于免疫学检测方法,需要将抗原与抗血清混合,使二者发生特异性结合,形成抗原-抗体复合物。但是,凝集测试法存在检测灵敏度不高、正确检出率偏低和交叉反应明显的问题。有学者揭示了酶联免疫法鉴定蛇毒蛋白时存在严重的交叉反应,对于蛇毒的鉴定非常不利。适配子技术存在操作过程十分复杂繁琐的缺点,如《一种利用适配子技术检测鉴定蛇毒种类的方法》(中国发明专利申请CN102323400A)。聚合酶链反应是从DNA的角度去鉴定,但是相关研究较少报道。
发明专利申请CN 110426522 A公开了一种圆斑蝰蛇蛇毒的鉴定方法及其应用,采用SDS-PAGE电泳结合质谱法的方法进行圆斑蝰蛇蛇毒的鉴定,该鉴定方法可明确鉴定蛋白的种类,但是质谱测试所用仪器设备昂贵,测试时间较长,人工操作工作量大。
因此,本领域亟需研发一种新的鉴定方法来克服上述传统方法的缺陷。
发明内容
本发明要解决的技术问题之一在于提供一种海蛇毒的鉴定方法,检测灵敏度高、正确检出率较高、不存在交叉反应,能有效缩短测试时间,并大大降低人工操作成本。
该方法我们摸索了海蛇毒蛋白稀释后的浓度及电泳最适蛋白胶浓度等,结合质谱进行海蛇毒的鉴定,同时,海蛇毒的鉴定方法增加紫外光谱扫描方法,该方法基于色氨酸和酪氨酸残基侧链基团对紫外光的吸收,其次是苯丙氨酸、组氨酸和半胱氨酸残基侧链基团以及肽键对紫外光的吸收,因不同的蛋白质氨基酸组成有所差别,其紫外光谱谱图各有不同。紫外光谱扫描方法能够灵敏和快速地鉴定海蛇毒,可以便捷地针对每个批次海蛇毒快速鉴定,初步鉴别海蛇毒素,之后使用SDS-PAGE电泳结合质谱法,3-5批海蛇毒样品归集后再进行测试,进一步明确鉴定蛋白的种类。
首先,我们用紫外光谱扫描分析方法,得到紫外光谱扫描曲线来鉴别和确定海蛇毒素。蛋白质产生紫外吸收主要是由于色氨酸和酪氨酸残基侧链基团对紫外光的吸收,其次是苯丙氨酸、组氨酸和半胱氨酸残基侧链基团以及肽键对紫外光的吸收。通过紫外光谱扫描分析可以对海蛇毒进行质量控制和对比分析。
其次,结合蛋白电泳和液质联用,即Liquid chromatography (LC)-electrosprayionization (ESI) tandem mass spectrometry (MS)(LC-ESI-MS/MS)LC-ESI-MS/MS的方法,从蛋白角度对海蛇毒进行鉴定,明确蛇毒的各种主要蛋白成分,针对性强,准确性好,较其他鉴定方法优势明显。
本发明要解决的技术问题之二在于提供该海蛇毒的鉴定方法在制备治疗海蛇毒中毒的抗海蛇毒血清中的应用。本发明可对海蛇毒进行实验室鉴定,更准确地对海蛇毒进行质控,进而生产出治疗海蛇毒中毒的特效药抗海蛇毒血清,对保证抗海蛇毒血清产品安全有效、质量可控,具有十分重要的意义。
为解决上述技术问题,本发明采用如下技术方案:
在本发明的一方面,提供一种海蛇毒的鉴定方法,包括如下步骤:
(1)选择产地为中国的海蛇毒素(含平颏海蛇毒和青环海蛇毒)冻干粉,溶解后配制成蛋白质溶液;海蛇毒素也称为海蛇蛇毒;
(2)采用紫外光谱扫描进行海蛇毒的分析及初步鉴定;
(3)采用SDS-PAGE电泳分析及液质联用LC-ESI-MS/MS质谱法进行明确鉴定。
作为本发明优选的技术方案,步骤(1)中,所述溶解后配制成蛋白质溶液具体为:用溶液A溶解后配制成蛋白浓度20-65mg/mL的蛋白质溶液(优选为蛋白浓度30mg/mL的蛋白质溶液);其中,溶液A的配方为:2.8-3.6g 氯化钠,4.2-4.5g硼酸,0.40-0.46g四硼酸钠,加注射用水定容至1L,pH值为 6.0-8.0。溶液A的配方优选为:3.5g 氯化钠,4.2g硼酸,0.45g四硼酸钠,加注射用水定容至1L,pH值为 6.8。
作为本发明优选的技术方案,步骤(2)中,所述进行紫外光谱扫描分析,紫外可见分光光度计的紫外波长设置为200-400nm,利用中速进行扫描。
作为本发明优选的技术方案,步骤(3)中,所述进行SDS-PAGE电泳分析,蛋白胶浓度为10-17.5%,蛋白胶浓度优选为17.5%。所述蛋白胶为分离胶,由如下表1组分组成:水、溶液B、溶液C、溶液D、溶液E、溶液F。
作为本发明优选的技术方案,步骤(3)中,所述进行SDS-PAGE电泳分析,蛋白胶浓度为4.5%,所述蛋白胶为浓缩胶,浓度为由如下表1组分组成:水、溶液B、溶液G、溶液D、溶液E、溶液F。
表1 蛋白胶浓度配方
表1中溶液的配制方法:
溶液B的配方:290g Acrylamide(丙烯酰胺),10g Bis-acrylamide(甲叉双丙烯酰胺),加注射用水定容至1L。
溶液C的配方:181.7g Tris(三羟甲基氨基甲烷),HCl(盐酸)调pH为7.5-9.5,加注射用水定容至1L。
溶液D的配方:100g SDS(十二烷基硫酸钠),加注射用水定容至1L。
溶液E的配方:100g APS(过硫酸铵),加注射用水定容至1L。
溶液F的配方:TEMED(四甲基乙二胺)溶液。
溶液G的配方:121g Tris(三羟甲基氨基甲烷),HCl(盐酸)调pH为6.0-8.0,加注射用水定容至1L。
作为本发明优选的技术方案,步骤(3)中,所述进行液质联用LC-ESI-MS/MS质谱鉴定,检测到的海蛇蛇毒蛋白主要有磷脂酶A2、三指毒素、金属蛋白酶、丝氨酸蛋白酶、血管内皮生长因子、蛇毒因子、蛋白酶抑制剂、磷脂酶B等。
此外,本发明还提供所述的一种海蛇毒的鉴定方法在制备治疗海蛇毒中毒的抗海蛇毒血清中的应用。
LC-ESI-MS/MS结合了液相色谱仪有效分离热不稳性及高沸点化合物的分离能力与质谱仪很强的组分鉴定能力,是一种分离分析复杂有机混合物的有效手段。串联质谱由两个质谱串联而成,其中第一个质量分析器将离子预分离或加能量修饰,由第二级质量分析器分析结果。分子离子经由第二级质谱通过与反应器的碰撞产生断裂,因此能提供更多的结构信息,所以二级质谱特别适合于复杂组分体系且干扰严重的样品中低含量组分分析测定。
液质联用鉴定蛋白质的方法虽然起步较晚,但是一次可以鉴定多个蛋白质,可以用蛋白质电泳分离和质谱技术相结合,对蛇毒做出鉴定。
紫外光谱扫描分析得到紫外光谱曲线,可以用来鉴别和确定海蛇毒素,并可对新购海蛇毒进行质量控制和对比分析。
与现有技术相比,本发明的有益效果在于:
本发明明确鉴定海蛇蛇毒,为海蛇蛇毒免疫马,并经生产得到的抗海蛇血清,提供重要的质量保证。本发明可对海蛇毒进行实验室鉴定,更准确地对海蛇毒进行质控,进而生产出治疗海蛇毒中毒的特效药抗海蛇毒血清,对保证抗海蛇毒血清产品安全有效、质量可控,具有十分重要的意义。本发明结合蛋白电泳和液质联用,即LC-ESI-MS/MS的方法,从蛋白角度对海蛇毒进行鉴定,明确蛇毒的各种主要蛋白成分,针对性强,准确性好,较其他鉴定方法优势明显。紫外光谱扫描分析可对新购海蛇毒进行质量控制和对比分析。通过检测生物样品中的蛇毒蛋白质鉴定蛇毒种类和质控,这是非常可靠的方法。
本发明相比于传统的凝集测试法、酶联免疫法等,创新点在于:
(1)首次系统地从蛋白角度鉴定了产地中国的海蛇蛇毒的组成成分。
(2)SDS-PAGE电泳实验中,蛇毒蛋白浓度和蛋白胶浓度十分关键,决定能否有效分离蛋白质,以对蛋白进行鉴定。本发明确定了可分离蛋白的最佳上样浓度和胶浓度条件:蛇毒蛋白上样浓度可为20-65mg/mL,30mg/ml最佳;SDS-PAGE电泳蛋白胶胶浓度可为10-17.5%,17.5%蛋白胶浓度最佳,可分辨出2条主蛋白条带。平颏海蛇毒和青环海蛇毒两种海蛇毒素组成类似,主要是:主蛋白带1为PLA2(15kDa);主蛋白带2为神经毒素(6-9kDa)。
(3)首次将基于液相色谱质谱联用技术的蛋白质组学研究方法运用于产地为中国的海蛇蛇毒的鉴定研究,为抗海蛇血清生产的原料海蛇蛇毒的鉴定和质控提供了新方法和新思路。
(4)质谱法解决了传统的凝集测试法存在检测灵敏度不高、正确检出率偏低和交叉反应明显的问题,克服了适配子技术存在操作过程十分复杂繁琐的缺点。本发明方法检测灵敏度高、正确检出率较高、不存在交叉反应,操作过程简单。
(5)通过紫外光谱扫描分析可以对海蛇毒进行质量控制和对比分析,是一个方便有效的检测方法。本发明首次将紫外光谱扫描技术运用于产地为中国的海蛇蛇毒的鉴定研究,可以便捷地针对每个批次海蛇毒快速鉴定,初步鉴别海蛇毒素,大大缩短了时间。按照发明专利申请CN 110426522 A公开的一种圆斑蝰蛇蛇毒的鉴定方法,采用SDS-PAGE电泳结合质谱法的方法进行圆斑蝰蛇蛇毒的鉴定,鉴定时间往往需要1-2周;而本发明采用紫外光谱扫描技术在1天内就可以初步鉴别海蛇毒素,大大缩短了测试时间,且将鉴定对象初步筛选至一定范围,再采用SDS-PAGE电泳分析及液质联用LC-ESI-MS/MS质谱法进行明确鉴定,大大降低了人工操作成本。
附图说明
图1为本发明实施例1中的紫外光谱扫描图。
图2为本发明实施例1中SDS-PAGE电泳图,其中,主带1、主带2分别代表2条主蛋白条
带。 图2中,M: 蛋白分子Marker;泳道1:平颏海蛇毒;泳道2:青环海蛇毒。
图3为本发明实施例2中的紫外光谱扫描图。
图4为本发明实施例2中SDS-PAGE电泳图,其中,主带1、主带2分别代表2条主蛋白条
带。 图4中,M: 蛋白分子Marker;泳道1:平颏海蛇毒;泳道2:青环海蛇毒。
具体实施方式
本发明通过下面的实施例可以对本发明作进一步的描述,然而,本发明的范围并不限于下述实施例。
实施例1 一种海蛇毒的鉴定方法
步骤如下:
(1)海蛇毒素(包括平颏海蛇毒和青环海蛇毒)用溶液A溶解后配制成30mg/mL;
(2)紫外光谱扫描分析
将海蛇蛇毒稀释至1mg/ml,光谱扫描波长设定为200-400nm,中速扫描,获得紫外光谱扫描的图谱(见图1),在210-220nm及280nm处有最高吸收峰。
(3)进行SDS-PAGE电泳,蛋白胶浓度17.5%,配方如下表2:
表2 蛋白胶浓度配方
(4)进行考马斯亮蓝染色;SDS-PAGE电泳图见图1,可分辨出2条主蛋白条带(分别15kD、6-9kD)(见图2中的主带1和主带2);
(5)进行LC-ESI-MS/MS质谱鉴定:样品在液相质谱中和流动相分离,被离子化后,经质谱的质量分析器将离子碎片按质量数分开,经检测器得到质谱图,通过搜索软件,在蛋白质数据库比对所鉴别的蛋白序列,以鉴定蛋白种类;
本发明方法检测到的海蛇蛇毒蛋白主要有PLA2、3-FTx、SVMP、SVSP、VEGF、VF、PI、PLB、ICI、CRVP、TCTP等蛋白,英文简写对应中文名如下表3。本实施例验证了本发明方法检测灵敏度高(蛋白电泳胶中10 ng蛋白即可通过质谱检出)、正确检出率较高(100%)、不存在交叉反应,操作过程简单。
表3 英文和中文名对照表
以上述及溶液的配制方法:
溶液A配方:3.5g 氯化钠,4.2g硼酸,四硼酸钠0.45g ,加注射用水定容至1L,pH 7.2。
溶液B的配方:290g Acrylamide(丙烯酰胺),10g Bis-acrylamide(甲叉双丙烯酰胺),加注射用水定容至1L。
溶液C的配方:181.7g Tris(三羟甲基氨基甲烷),HCl(盐酸)调pH至8.2,加注射用水定容至1L。
溶液D的配方:100g SDS(十二烷基硫酸钠),加注射用水定容至1L。
溶液E的配方:100g APS(过硫酸铵),加注射用水定容至1L。
溶液F的配方:TEMED(四甲基乙二胺)溶液
溶液G的配方:121g Tris(三羟甲基氨基甲烷),HCl(盐酸)调pH为6.0-8.0,加注射用水定容至1L。
实施例2 一种海蛇毒的鉴定方法在制备治疗海蛇毒中毒的抗海蛇毒血清中的应用
步骤如下:
(1)海蛇蛇毒(包括平颏海蛇毒和青环海蛇毒)冻干粉,溶液A(见实施例1)溶解后,进行紫外光谱扫描(见图3),与已有的光谱图(图1)进行比对;
(2)进行SDS-PAGE电泳和LC-ESI-MS/MS质谱鉴定,将SDS-PAGE电泳结果(图4)与已有的电泳图谱(图2)比对,确认蛋白为2个主要条带,即为对应海蛇蛇毒主蛋白;
(3)将已经鉴定的海蛇蛇毒甲醛脱毒后,免疫马匹;
(4)免疫海蛇蛇毒的马匹,经单采血浆机采血后,转移至生产部进行抗海蛇蛇毒血清的生产,得到抗海蛇蛇毒血清原液和产品;
以上抗海蛇蛇毒血清的生产流程,从源头上进行蛇毒种类的鉴别,控制海蛇蛇毒的质量和类型,为生产合格的抗海蛇蛇毒提供有力的保证。
Claims (11)
1.一种海蛇毒的鉴定方法,其特征在于,包括如下步骤:
(1)选择产地为中国的海蛇毒素冻干粉,溶解后配制成蛋白质溶液;所述产地为中国的海蛇毒素包括平颏海蛇毒和青环海蛇毒;
(2)采用紫外光谱扫描进行海蛇毒的分析及初步鉴定;
(3)采用SDS-PAGE电泳分析及液质联用LC-ESI-MS/MS质谱法进行明确鉴定。
2.根据权利要求1所述的一种海蛇毒的鉴定方法,其特征在于,步骤(1)中,所述溶解后配制成蛋白质溶液具体为:用溶液A溶解后配制成蛋白浓度20-65mg/mL的蛋白质溶液;其中,溶液A的配方为:2.8-3.6g 氯化钠,4.2-4.5g硼酸,0.40-0.46g四硼酸钠,加注射用水定容至1L,pH值为 6.0-8.0。
3.根据权利要求2所述的一种海蛇毒的鉴定方法,其特征在于,所述溶液A的配方为:3.5g 氯化钠,4.2g硼酸,0.45g四硼酸钠,加注射用水定容至1L,pH值为 7.2。
4.根据权利要求2所述的一种海蛇毒的鉴定方法,其特征在于,所述用溶液A溶解后配制成蛋白浓度为30mg/mL的蛋白质溶液。
5.根据权利要求1所述的一种海蛇毒的鉴定方法,其特征在于,步骤(2)中,所述紫外光谱扫描的波长扫描范围为200nm-400nm,所述紫外光谱扫描对海蛇毒进行质量控制和对比分析。
6.根据权利要求1所述的一种海蛇毒的鉴定方法,其特征在于,步骤(3)中,所述进行SDS-PAGE电泳分析,蛋白胶浓度为10-17.5%。
7.根据权利要求6所述的一种海蛇毒的鉴定方法,其特征在于,所述蛋白胶浓度为17.5%。
8.根据权利要求6或7所述的一种海蛇毒的鉴定方法,其特征在于,步骤(3)中,所述蛋白胶为分离胶,由如下组分组成:水、溶液B、溶液C、溶液D、溶液E、溶液F;所述溶液B的配方为:290g Acrylamide,10g Bis-acrylamide,加注射用水定容至1L;所述溶液C的配方为:181.7g Tris,HCl调pH值至7.5-9.5,加注射用水定容至1L;所述溶液D的配方为:100g SDS,加注射用水定容至1L;所述溶液E的配方为:100g APS,加注射用水定容至1L;所述溶液F的配方为:TEMED溶液。
9.根据权利要求1所述的一种海蛇毒的鉴定方法,其特征在于,步骤(3)中,所述进行SDS-PAGE电泳分析,蛋白胶浓度为4.5%,所述蛋白胶为浓缩胶,浓度为由如下组分组成:水、溶液B、溶液G、溶液D、溶液E、溶液F;所述溶液B的配方为:290g Acrylamide,10g Bis-acrylamide,加注射用水定容至1L;所述溶液G的配方为:121g Tris,HCl调pH值至6.0-8.0,加注射用水定容至1L;所述溶液D的配方为:100g SDS,加注射用水定容至1L;所述溶液E的配方为:100g APS,加注射用水定容至1L;所述溶液F的配方为:TEMED溶液。
10.根据权利要求1所述的一种海蛇毒的鉴定方法,其特征在于,步骤(3)中,所述进行液质联用LC-ESI-MS/MS质谱鉴定,检测到的海蛇蛇毒蛋白主要有磷脂酶A2、三指毒素、金属蛋白酶、丝氨酸蛋白酶、血管内皮生长因子、蛇毒因子、蛋白酶抑制剂、磷脂酶B、离子通道抑制剂、富含半胱氨酸的蛇毒蛋白、翻译控制肿瘤蛋白。
11.如权利要求1-10任一项所述的一种海蛇毒的鉴定方法在制备治疗海蛇毒中毒的抗海蛇毒血清中的应用。
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