CN102368906A - 用于灭活病毒的方法 - Google Patents
用于灭活病毒的方法 Download PDFInfo
- Publication number
- CN102368906A CN102368906A CN2010800144898A CN201080014489A CN102368906A CN 102368906 A CN102368906 A CN 102368906A CN 2010800144898 A CN2010800144898 A CN 2010800144898A CN 201080014489 A CN201080014489 A CN 201080014489A CN 102368906 A CN102368906 A CN 102368906A
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- Prior art keywords
- virus
- ammonium
- haloperoxidase
- bromide
- chloride
- Prior art date
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Abstract
本发明提供通过使病毒接触卤过氧化物酶、过氧化氢、氯离子、溴离子和铵离子而灭活所述病毒的方法。
Description
发明领域
本发明涉及用于灭活病毒和医疗装置或设备消毒或灭菌的方法。
发明背景
大多数真菌、病毒和病原细菌的营养细胞在70摄氏度时可在数分钟内杀死或灭活;然而,有些病原病毒(如脊髓灰质炎病毒)灭活困难得多。此外,因为敏感的医疗装置在接触所述高温时经常会缩短使用寿命,所以需要使用较低温度和温和条件同时保持病毒灭活能力的用于医疗装置消毒的方法。
特别值得注意的是小无包膜病毒,如脊髓灰质炎病毒(poliovirus)、细小病毒(parvovirus)和甲肝病毒(hepatitis A virus)。这些是小(直径23-27纳米)的无包膜人类病原,已经证明其可耐受去除和灭活过程,所述过程针对脂质包膜病毒如HIV、乙肝和丙肝病毒显示效用。
小无包膜病毒的高度稳定亚类是肠病毒(Enteroviruse)(例如脊髓灰质炎病毒和甲肝病毒),其耐受低于三的pH水平、洗涤剂、70%乙醇和其它脂质溶剂,如氯仿和醚。由于它们也耐受消毒剂如5%Lysol和1%季铵化合物,所以肠病毒特别难于灭活。
本发明提供用于灭活病毒的改进的酶促方法,其对于敏感性材料(如医疗装置)比传统方法更温和。
发明简述
本发明提供用于灭活病毒的方法,包括使所述病毒接触卤过氧化物酶、过氧化氢源、氯离子和/或溴离子,和铵离子。
在一个实施方案中,卤过氧化物酶是氯过氧化物酶或溴过氧化物酶。在另一个实施方案中,卤过氧化物酶是含有钒的卤过氧化物酶。
发明详述
卤过氧化物酶和展现卤过氧化物酶活性的化合物
适合于并入本发明方法的卤过氧化物酶是氯过氧化物酶、溴过氧化物酶和展现氯过氧化物酶或溴过氧化物酶活性的化合物。卤过氧化物酶形成一类酶,其能够在过氧化氢或过氧化氢生成系统存在下将卤化物(Cl-、Br-、I-)氧化为相应的次卤酸。
卤过氧化物酶依据它们对卤离子的特异性来分类。氯过氧化物酶(E.C.1.11.1.10)催化自氯离子形成次氯酸盐,自溴离子形成次溴酸盐和自碘离子形成次碘酸盐;而溴过氧化物酶催化自溴离子形成次溴酸盐和自碘离子形成次碘酸盐。然而,次碘酸盐经历自发歧化(spontaneous disproportionation)成碘,因此观察到的产物是碘。这些次卤酸盐化合物可随后与其它化合物起反应,形成卤代化合物。
在一个优选的实施方案中,本发明的卤过氧化物酶是氯过氧化物酶。
已经自多种生物体分离了卤过氧化物酶:哺乳动物、海洋动物、植物、藻类、地衣、真菌和细菌。普遍接受的是卤过氧化物酶是自然界中负责卤代化合物形成的酶,尽管可涉及其它酶。
已经自多种不同真菌分离了卤过氧化物酶,特别是真菌组暗色丝孢菌类(dematiaceous hyphomycetes),如卡尔黑霉属(Caldariomyces)(例如,烟色卡尔黑霉(C.fumago)),链格孢属(Alternaria),弯孢属(Curvularia)(例如疣状弯孢(C.verruculosa)和不等弯孢(C.inaequalis)),内脐蠕孢属(Drechslera),细基格孢属(Ulocladium)和葡萄孢属(Botrytis)。
已经自细菌分离了卤过氧化物酶,如假单胞菌属(Pseudomonas),例如吡咯菌素假单胞菌(P.pyrrocinia)和链霉菌属(Streptomyces),例如,金黄色链霉菌(S.aureofaciens)。
在一个优选的实施方案中,所述卤过氧化物酶是自弯孢属菌种,特别是疣状弯孢或不等弯孢,如WO 95/27046中所述的不等弯孢CBS 102.42可得到的钒卤过氧化物酶(即含有钒或钒酸盐的卤过氧化物酶),例如由WO 95/27046图2(通过提述全部并入)的DNA序列编码的钒卤过氧化物酶;或如WO 97/04102中所述的疣状弯孢CBS 147.63或疣状弯孢CBS 444.70可得到的钒卤过氧化物酶。优选地,所述卤过氧化物酶的氨基酸序列与自疣状弯孢(参见例如WO 97/04102中SEQ ID NO:2)或不等弯孢(例如由WO 95/27046图2中的DNA序列编码的成熟氨基酸序列)可得到的卤过氧化物酶的氨基酸序列具有至少90%同一性,优选95%同一性。
在另一个优选的实施方案中,所述卤过氧化物酶是含有钒的卤过氧化物酶;特别是钒氯过氧化物酶。所述钒氯过氧化物酶可以是自如WO 01/79459中所述的Drechslera hartlebii,如WO 01/79458中记载的Dendryphiella salina,如WO 01/79461中所述的Phaeotrichoconis crotalarie,或如WO 01/79460中所述的棉丝菌菌种(Geniculosporium)可得到的。所述钒卤过氧化物酶更优选是自Drechslera hartlebii(DSM 13444)、Dendryphiella salina(DSM 13443)、Phaeotrichoconis crotalarie(DSM 13441)或棉丝菌菌种(DSM 13442)可得到的。
卤过氧化物酶的浓度通常在0.01-100ppm酶蛋白质,优选0.05-50ppm酶蛋白质,更优选1-40ppm酶蛋白质,更优选0.1-20ppm酶蛋白质,和最优选0.5-10ppm酶蛋白质的范围内。
在一个实施方案中,卤过氧化物酶的浓度通常在5-50ppm酶蛋白质,优选5-40ppm酶蛋白质,更优选8-32ppm酶蛋白质的范围内。
卤过氧化物酶活性的测定
可通过如下进行测定卤过氧化物酶活性的测定法:混合100μl卤过氧化物酶样品(约0.2μg/mL)和100μl 0.3M磷酸钠pH 7缓冲液-0.5M溴化钾-0.008%酚红,将该溶液添加至10μl 0.3%H2O2,并测量595nm处的吸收作为时间的函数。
可通过测量290nm处吸收的降低(作为时间的函数)来进行使用单氯双甲酮(Sigma M4632,ε=20000M-1cm-1于290nm)作为底物的另一种测定法。该测定法是在0.1M磷酸钠或0.1M乙酸钠、50μM单氯双甲酮、10mMKBr/KCl、1mM H2O2和约1μμg/mL卤过氧化物酶的水溶液中进行的。一个卤过氧化物酶单位(HU)定义为于pH 5和30℃每分钟氯化或溴化1微摩尔单氯双甲酮。
过氧化氢
卤过氧化物酶所需要的过氧化氢可以作为用于原位生产过氧化氢的过氧化氢的水溶液或过氧化氢前体来提供。在溶解后释放卤过氧化物酶可利用的过氧化物的任何固体实体能充当过氧化氢的来源。在水或适当的水基介质中溶解后产生过氧化氢的化合物包括但不限于金属过氧化物、过碳酸盐、过硫酸盐、过磷酸盐、过氧酸、烃基过氧化物、酰基过氧化物、过氧化酯、脲过氧化物、过硼酸盐和过氧羧酸或其盐。
过氧化氢的另一种来源是过氧化氢生成酶系统,如氧化酶以及该氧化酶的底物。氧化酶和底物的组合的例子包括但不限于氨基酸氧化酶(参见例如US 6,248,575)和合适的氨基酸,葡萄糖氧化酶(参见例如WO 95/29996)和葡萄糖,乳酸氧化酶和乳酸盐,半乳糖氧化酶(参见例如WO 00/50606)和半乳糖,以及醛糖氧化酶(参见例如WO 99/31990)和合适的醛糖。
通过研究EC 1.1.3._、EC 1.2.3._、EC 1.4.3._和EC 1.5.3._或相似的类(根根据国际生物化学联合会),本领域技术人员可容易地识别这样的氧化酶和底物组合的其它例子。
可以在工艺开始或过程中添加过氧化氢或过氧化氢源,例如通常以对应于0.001mM至25mM的水平,优选对应于0.005mM至5mM的水平,和特别是对应于0.01-1mM过氧化氢的水平的量添加。也可以以对应于0.1mM至25mM的水平,优选对应于0.5mM至15mM的水平,更优选对应于1mM至10mM的水平,和最优选对应于2mM至8mM过氧化氢的水平的量使用过氧化氢。
氯和溴离子
根据本发明,与卤过氧化物酶反应所需要的氯和/或溴离子(Cl-和/或Br-)可以以许多不同方式,如通过添加氯化物和/或溴化物的盐来提供。在一个优选的实施方案中,所述氯化物和溴化物的盐是氯化钠(NaCl)、溴化钠(NaBr)、氯化钾(KCl)、溴化钾(KBr)、氯化铵(NH4Cl)或溴化铵(NH4Br);或其混合物。
在一个实施方案中,所述氯和/或溴离子只限于氯离子(Cl-)或溴离子(Br-)。在另一个实施方案中,氯和/或溴离子只限于氯离子(Cl-)和溴离子(Br-)。所述氯离子可以通过将氯化物的盐添加至水溶液中来提供。所述氯化物的盐可以是氯化钠、氯化钾或氯化铵;或其混合物。所述溴离子可以通过将溴化物的盐添加至水溶液中来提供。所述溴化物的盐可以是溴化钠、溴化钾或溴化铵;或其混合物。
每种氯化物和溴化物离子的浓度通常在0.01mM至1000mM的范围内,优选在0.05至500mM的范围内,更优选在0.1mM至100mM的范围内,最优选在0.1mM至50mM的范围内,和特别是在1mM至25mM的范围内。氯化物离子的浓度独立于溴化物离子的浓度;反之亦然。
在一个实施方案中,每种氯化物和溴化物离子的摩尔浓度是铵离子浓度的至少两倍,优选至少四倍,更优选至少六倍,最优选至少八倍,和特别是至少十倍。
铵离子
根据本发明方法灭活病毒所需要的铵离子(NH4 +)可以以许多不同方式,如通过添加铵盐来提供。在一个优选的实施方案中,所述铵盐是硫酸铵((NH4)2SO4)、碳酸铵((NH4)2CO3)、氯化铵(NH4Cl)、溴化铵(NH4Br),或碘化铵(NH4I);或其混合物。
铵离子的浓度通常在0.01mM至1000mM的范围内,优选在0.05mM至500mM的范围内,更优选在0.1mM至100mM的范围内,最优选在0.1mM至50mM的范围内,和特别是在1mM至25mM的范围内。
病毒
根据本发明用卤过氧化物酶、过氧化氢、氯离子和/或溴离子,和铵离子灭活的病毒包含所有种类的病毒。
在一个实施方案中,所述病毒选自下组:腺病毒(Adenoviruses)、沙粒病毒(Arenaviruses)、布尼亚病毒(Bunyaviruses)、杯状病毒(Caliciviruses)、冠状病毒(Coronaviruses)、Deltaviruse、丝状病毒(Filoviruses)、黄病毒(Flaviviruses)、肝DNA病毒(Hepadnaviruses)、疱疹病毒(Herpesviruses)、正粘病毒(Orthomyxoviruses)、乳多空病毒(Papovaviruses)、副粘病毒(Paramyxoviruses)、细小病毒(Parvoviruses)、微小RNA病毒(Picornaviruses)、痘病毒(Poxiviruses)、弹状病毒(Rhabdoviruses)、呼肠孤病毒(Reoviruses)、逆转录病毒(Retroviruses)和外衣病毒(Togaviruses)。
在另一个实施方案中,病毒选自下组:诺瓦克病毒(Norwalk virus)、脊髓灰质炎病毒(Poliovirus)、轮状病毒(Rotavirus)、呼吸道合胞病毒(RespiratorySyncytial Virus)、鼻病毒(Rhinovirus)、副流感病毒(Parainfluenza Virus)、冠状病毒、甲型和乙型流感病毒(Influenza A and B viruses)、人体免疫缺陷病毒(HIV)、甲肝病毒(Hepatitis A virus)、乙肝病毒(Hepatitis B virus)、丙肝病毒(Hepatitis Cvirus)、1型单纯疱疹病毒(Herpes simplex virus)和2型单纯疱疹病毒。
在另一个实施方案中,所述病毒是小无包膜病毒。小无包膜病毒的例子包括但不限于,微小RNA病毒,如人鼻病毒A、人鼻病毒B、口蹄疫病毒(Foot-and-mouth disease virus)、甲肝病毒和肠病毒(如脊髓灰质炎病毒)。
在一个优选的实施方案中,所述病毒是肠病毒。
表面活性剂
本发明的方法可包括施用表面活性剂(作为洗涤剂配制物的一部分或作为湿润剂)。适合于施用的表面活性剂可以是非离子的(包括半极性的)、阴离子的、阳离子和/或两性离子的;优选所述表面活性剂是阴离子的(如线性烷基苯磺酸酯(linear alkylbenzenesulfonate)、α-烯烃磺酸酯(alpha-olefinsulfonate)、烷基硫酸酯(脂肪醇硫酸酯)、醇乙氧基硫酸酯、仲烷磺酸酯、α-磺基脂肪酸甲酯、烷基或烯基琥珀酸或皂)或非离子的(如醇乙氧基化物、壬基酚乙氧基化物、烷基多聚糖苷、烃基二甲胺氧化物、乙氧基化脂肪酸单乙醇酰胺、脂肪酸单乙醇酰胺、多羟基烷基脂肪酸酰胺或葡糖胺的N-酰基N-烃基衍生物(“葡糖酰胺”)),或其混合物。
当包括在本发明方法中时,表面活性剂的浓度通常为按重量计约0.01%至约10%,优选约0.05%至约5%,和更优选约0.1%至约1%。
方法和用途
在第一个方面,本发明提供了一种用于灭活病毒的酶促方法,包括使所述病毒接触包含卤过氧化物酶、过氧化氢源、氯离子和/或溴离子,和铵离子的组合物。在一个优选的实施方案中,本发明提供了一种用于医疗装置或设备消毒或灭菌的方法,其包括使所述医疗装置或设备接触所述组合物。
所述组合物可配制为液体(例如含水的)或干产物配制物。所述干产物配制物可随后再水合以形成在本发明方法中有用的有活性的液体或半液体配制物。
当所述组合物配制成干配制物时,组分可以是混合的、安排在离散的层中的或分开包装的。
在第二个方面,本发明还涵盖一种组合物,其源自应用本发明的方法。在这种情况下,所述组合物包含卤过氧化物酶、过氧化氢、氯离子和/或溴离子、铵离子、活性或非活性病毒,和医疗装置或设备。
本发明的方法对于已经暴露于病毒(如生物战争剂)的场所的去污是有用的。
在本发明的语境中,术语“灭活病毒”意图指至少99%的病毒不能感染合适的细胞。优选99.9%,更优选99.99%,最优选99.999%,和特别是99.9999%的病毒不能感染合适的细胞。
在一个实施方案中,术语“消毒”指根据美国食品药品管理局2000年1月″Content and Format of Premarket Notification[510(k)]Submissions for LiquidChemical Sterilants/High Level Disinfectants″的高水平消毒。
根据本发明的方法可以在0-70摄氏度,优选5-60摄氏度,更优选10-60摄氏度,甚至更优选15-60摄氏度,甚至更优选20-60摄氏度,最优选20-50摄氏度,和特别是20-40摄氏度的温度进行。
本发明的方法可以采用10分钟至(至少)4小时,优选15分钟至(至少)3小时,更优选20分钟至(至少)2小时,最优选20分钟至(至少)1小时,和特别是30分钟至(至少)1小时的处理时间。
本发明的方法适合于灭活多种环境中的病毒。本发明的方法可理想地用于任何环境中以减少病毒感染,如医疗保健产业(例如动物医院、人类医院、动物诊所、人类诊所、疗养院、儿童或老年人的日间护理设施等)、食品工业(例如餐馆、食品加工厂、食品贮存场、杂货店等)、招待产业(例如旅馆、汽车旅馆、景点、游船等)、教育产业(例如学校和大学)等。
由于本发明方法利用相对较低的温度,它们对于医疗保健产业中所使用的设备如医疗装置(例如干的手术设备、麻醉设备、器皿等)的消毒或灭菌是非常有用的。消毒或灭菌后的设备会展现降低的变形和磨损,而且该设备便于在消毒或灭菌后基本上立即使用。当对复杂的或热敏感的医疗装置如包含不同材料的超声换能器(transducer)和内窥镜进行消毒或灭菌时,这是尤其有利的,因为这些装置的磨损显著降低,这导致这些常常是非常昂贵的装置有更长的使用寿命,这有效地降低了它们的操作成本。实际上,通过使用本发明,可以有效地对甚至其它非医疗类型的设备如重复使用的卫生用品消毒或灭菌。
在一个优选的实施方案中,医疗装置和/或非医疗类型的设备的消毒或灭菌在根据EN ISO 15883-1(或如美国食品药品管理局2002年2月″Class IISpecial Controls Guidance Document:Medical Washers and MedicalWasher-Disinfectors;Guidance for the Medical Device Industry and FDA ReviewStaff″中所述的)(医疗)清洗机-消毒机中使用本发明的方法而进行。
本发明的方法可理想地用于任何环境中以减少病毒感染,如一般房屋表面(例如地面、墙、天花板、家具外部等)、特殊设备表面(例如硬表面、制造设备、加工设备等)、纺织品(例如棉、毛、丝、合成纤维如聚酯、聚烯烃和丙烯酸酯类,纤维混纺物如棉聚酯等)、木材和基于纤维素的系统(例如纸)、土壤、动物躯体(例如皮、肉、毛发、羽等)、食物(例如果实、蔬菜、坚果、肉等)和水。
在一个实施方案中,本发明的方法针对纺织品的杀病毒处理。能用本发明组合物处理的纺织品的例子包括但不限于个人用品(例如衬衣、裤子、袜子、内衣等)、公共用品(例如毛巾、实验服、制服、围裙等)、招待用品(例如毛巾、餐巾、桌布等)。
用本发明组合物对纺织品的杀病毒处理可包括使纺织品接触本发明的组合物。此接触可以在洗涤纺织品之前发生。或者,此接触可以在洗涤纺织品期间发生以提供杀病毒活性和任选地提供清洁活性以自纺织品去除或减少尘土、污渍等。
本发明组合物所接触的病毒可以位于任何表面上,包括但不限于,例如乳制品、化学品或药品加工厂、医疗装置如内窥镜或其它医疗器具,实验室设备,洗涤机或水卫生系统中所使用的工艺设备的表面。本发明的组合物应当以有效灭活所述表面上的病毒的量使用。
通过将病毒浸没在组合物的含水配制物(例如洗涤过程)中,通过将组合物喷洒到病毒上,通过借助布将组合物施用至病毒,或通过技术人员认可的任何其它方法,可以使病毒接触本发明方法中所使用的组合物。任何将本发明的组合物施用于病毒,导致灭活所述病毒的方法是可接受的施用方法。
本发明的方法对于已经暴露于病毒(例如病原病毒)(如生物战争剂)的场所的去污也是有用的。此类场所包括但不限于衣物(如军服)、车辆(vehicle)内部和外部部分、建筑物内部和外部部分、任何种类的军事设施,和任何种类的上文所述环境。
通过下述实施例进一步描述本发明,它们不应解释为限制本发明的范围。
实施例
作为缓冲液和底物使用的化学品是至少试剂级的商业产品。
实施例1
脊髓灰质炎病毒的灭活
病毒和细胞
在VERO细胞中于34.5℃,5%CO2,使用Eagles MEM 2%FCS、100IU/mL青霉素、100mg/mL链霉素和20μg/mL庆大霉素繁殖从WHO获得的脊髓灰质炎病毒株Sabin(2H.010704型)。过滤培养物上清(0.45nm),分装并在-80℃保存至使用。
储液
制备下述储液。
溶液1:22.8mM NaCl和7.2mM NH4Cl,在20mM DMG(Sigma D4379)缓冲液pH 7.0中;
溶液2:10%H2O2,在MilliQ水中;
溶液3:8000ppm来自疣状弯孢的卤过氧化物酶(参见WO 97/04102中的SEQ ID NO:2),在20mM DMG缓冲液pH 7.0中;
磷酸盐缓冲盐水
消毒溶液
为了产生约50mL的活性消毒溶液,混合下述物质:
50mL溶液1;62μL溶液2;和100μL溶液3。
进一步用磷酸盐缓冲盐水(PBS)稀释未经稀释的消毒溶液,得到1∶10和1∶100的溶液。
细胞培养基
补充4%胎牛血清、100IU/mL青霉素、100mg/mL链霉素和20μg/mL庆大霉素的Eagles MEM培养基。
病毒的处理
在微孔板中将50μL测试溶液与50μL在PBS中稀释的脊髓灰质炎病毒储液混合并在34.5℃温育1小时。
为了比较酶消毒溶液和已知的消毒剂,使用次氯酸钠作为对照,浓度为0.5%、0.05%和0.005%。
对于所有处理,制备未加病毒的对照以鉴定测试溶液对于细胞系的任何细胞毒性作用。此外,包括病毒感染能力的阳性对照,其中未加入消毒溶液。
为了在处理后测试保持感染性的病毒,向新鲜的微孔板加入100μL测试溶液和包含4%FCS的100μL Eagles矿物培养基,其中已经建立了RD细胞系(人横纹肌肉瘤)的汇合生长。将平板在34.5℃和5%CO2温育5天,然后由专业评价人员使用显微镜为感染能力和细胞毒力评分。
表1.脊髓灰质炎病毒Sabin(2H.010704类型)抗病毒测试的结果
酶:卤过氧化物酶
次氯酸盐:次氯酸钠
I:感染
N:未感染
N*:未感染,有轻微毒性作用
T:毒性
结果显示本发明的方法抑制脊髓灰质炎病毒(2-4列)的程度与次氯酸钠(5-7列)相同。重复进行试验。即使稀释至1∶100,酶系统仍显示强的杀病毒活性。
实施例2
HIV-1病毒的灭活
病毒和细胞
在H9细胞(NIH AIDS Research and Reference Program)中于37℃,5%CO2,使用包含10%热灭活胎牛血清(FCS)、100IU/mL青霉素、100mg/mL链霉素、20μg/mL庆大霉素和10IU/mL制霉菌素的RPMI 1640繁殖HIV-1株HTLV-IIIB(NIH AIDS Research and Reference Program)。过滤培养物上清(0.45nm),分装并在-80℃保存至使用时。
储液
制备下述储液。
溶液1:22.8mM NaCl和7.2mM NH4Cl,在20mM DMG(Sigma D4379)缓冲液pH 7.0中;
溶液2:10%H2O2,在MilliQ水中;
溶液3:8000ppm来自疣状弯孢(参见WO 97/04102中的SEQ ID NO:2)的卤过氧化物酶,在20mM DMG缓冲液pH 7.0中;
磷酸盐缓冲盐水
消毒溶液
为了产生约50mL的活性消毒溶液,混合下述物质:
50mL溶液1;62μL溶液2;和100μL溶液3。
进一步用磷酸盐缓冲盐水(PBS)稀释未经稀释的消毒液,得到1∶10和1∶100的溶液。
细胞培养基
补充100IU/mL青霉素、100mg/mL链霉素、20μg/mL庆大霉素、10IU/mL制霉菌素和5%胎牛血清的RPMI 1640 Glutamax(Sigma R8758)。
MTT试剂
50mg MTT(Sigma M5655)
10mL PBS pH 7.4
MTT终止试剂
10mL Triton X-100(Sigma T8787)
4mL 1M HCl
异丙醇至100mL
病毒的处理
在微孔板中将50μL测试溶液与50μL在PBS中稀释的HIV病毒储液混合并在37℃温育5分钟。
为了比较酶消毒溶液和已知的消毒剂,使用次氯酸钠作为对照,浓度为0.5%、0.05%和0.005%。
对于所有处理,制备未加病毒的对照以鉴定测试溶液对于细胞系的任何细胞毒性作用。此外,包括病毒感染能力的阳性对照,其中未加入消毒溶液。
为了在处理后测试保持感染性的病毒,向新鲜的微孔板中加入100μL测试溶液和具有5%FCS包含0.3x103MT4细胞/mL的100μL RPMI1640细胞培养基。将平板在37℃和5%CO2温育6天,然后用MTT试验中测量活力。向每个孔加入30μL MTT试剂,并在37℃将平板温育1小时。去除150μL溶液并用130μL MTT终止溶液替代,将内容物混合。
在平板阅读器中测量540nm处的吸光度,并以690nm为参照。根据吸光度,将每孔评分为在包含病毒的孔中是感染的或非感染的;以及在细胞毒性的对照孔中是毒性的或未感染的。
表2.HIV-1病毒(HTLV-IIIB株)抗病毒测试的结果
酶:卤过氧化物酶
次氯酸盐:次氯酸钠
I:感染
N:未感染
N*:未感染,有轻微毒性作用
T:毒性
结果显示本发明的方法在1∶100稀释(第4列)中在5分钟温育期的过程中对于HIV病毒有抗病毒效果。
参考文献:(1983)Rapid colorimetric assay for cellular growth and survival:Application to profileration and cytotoxicity assays.J.Imm.Meth,65,55-63。
Claims (15)
1.一种用于灭活病毒的酶促方法,其包括使所述病毒接触卤过氧化物酶、过氧化氢、氯和/或溴离子,和铵离子。
2.权利要求1的方法,其中所述卤过氧化物酶是来自酶类EC 1.11.1.10的氯过氧化物酶。
3.权利要求1的方法,其中所述卤过氧化物酶是含有钒的卤过氧化物酶。
4.权利要求3的方法,其中所述卤过氧化物酶的氨基酸序列与自疣状弯孢(Curvularia verruculosa)或不等弯孢(Curvularia inequalis)可获得的卤过氧化物酶的氨基酸序列具有至少90%同一性,优选95%同一性。
5.权利要求1-4中任一项的方法,其中氯离子和/或溴离子是自氯化物和/或溴化物的盐得到的;优选所述氯化物和/或溴化物的盐包括氯化钠、溴化钠、氯化钾、溴化钾、氯化铵或溴化铵。
6.权利要求1-5中任一项的方法,其中所述铵离子是自铵盐得到的;优选所述铵盐是硫酸铵、碳酸铵、磷酸铵、氯化铵、溴化铵或碘化铵;或其混合物。
7.权利要求1-6中任一项的方法,其中所述氯离子浓度是铵离子浓度的至少两倍;优选至少四倍,更优选至少六倍,最优选至少八倍,和特别是铵离子浓度的至少十倍。
8.权利要求1-7中任一项的方法,其进一步包括使所述病毒接触表面活性剂。
9.权利要求1-8中任一项的方法,其中所述病毒是无包膜病毒,如小无包膜病毒。
10.权利要求1-9中任一项的方法,其中所述病毒是肠病毒。
11.权利要求1-10中任一项的方法,其中所述病毒是脊髓灰质炎病毒。
12.权利要求1-11中任一项的方法,其中所述病毒位于表面上,如医疗装置或设备的表面上。
13.权利要求1-12中任一项的方法,其为高水平消毒方法。
14.卤过氧化物酶、过氧化氢、氯化物盐和/或溴化物盐,和铵盐用于灭活病毒的用途。
15.权利要求14的用途,用于医疗装置或设备的高水平消毒。
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US (1) | US20120020946A1 (zh) |
EP (1) | EP2413700A2 (zh) |
JP (1) | JP2012522743A (zh) |
CN (1) | CN102368906A (zh) |
WO (1) | WO2010115735A2 (zh) |
Cited By (1)
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CN111363729A (zh) * | 2020-03-12 | 2020-07-03 | 苏州白垩纪生物科技有限公司 | 一种rna病毒灭活保存液及其应用 |
Families Citing this family (3)
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US9254922B2 (en) | 2012-03-05 | 2016-02-09 | Embry-Riddle Aeronautical University, Inc. | Hybrid clutch assembly for an aircraft |
US8791054B2 (en) | 2012-09-27 | 2014-07-29 | Halliburton Energy Services, Inc. | Methods of converting an inactive biocide into an active biocide using a chemical reaction |
CN110496525A (zh) * | 2019-08-30 | 2019-11-26 | 山东多芬农业有限公司 | 空气净化剂及其制备和使用方法 |
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CN1194003A (zh) | 1995-07-14 | 1998-09-23 | 诺沃挪第克公司 | 得自Curvularia verruculosa的卤过氧化物酶和编码该酶的核酸 |
ATE293359T1 (de) | 1997-12-22 | 2005-05-15 | Novozymes As | Kohlenhydratoxidase sowie verwendung derselben beim backen |
US6248575B1 (en) | 1998-05-18 | 2001-06-19 | Novozymes Biotech, Inc. | Nucleic acids encoding polypeptides having L-amino acid oxidase activity |
US6090604A (en) | 1999-02-24 | 2000-07-18 | Novo Nordisk Biotech, Inc. | Polypeptides having galactose oxidase activity and nucleic acids encoding same |
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WO2001079461A2 (en) | 2000-04-14 | 2001-10-25 | Novozymes A/S | Polypeptides having haloperoxidase activity |
AU2001246402A1 (en) | 2000-04-14 | 2001-10-30 | Novozymes A/S | Polypeptides having haloperoxidase activity |
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JP2002003317A (ja) * | 2000-06-21 | 2002-01-09 | Mitsubishi Gas Chem Co Inc | 殺菌剤組成物 |
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- 2010-03-26 WO PCT/EP2010/054029 patent/WO2010115735A2/en active Application Filing
- 2010-03-26 US US13/259,843 patent/US20120020946A1/en not_active Abandoned
- 2010-03-26 JP JP2012502604A patent/JP2012522743A/ja active Pending
- 2010-03-26 CN CN2010800144898A patent/CN102368906A/zh active Pending
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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CN111363729A (zh) * | 2020-03-12 | 2020-07-03 | 苏州白垩纪生物科技有限公司 | 一种rna病毒灭活保存液及其应用 |
CN111363729B (zh) * | 2020-03-12 | 2021-04-16 | 苏州白垩纪生物科技有限公司 | 一种rna病毒灭活保存液及其应用 |
Also Published As
Publication number | Publication date |
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US20120020946A1 (en) | 2012-01-26 |
WO2010115735A3 (en) | 2011-08-11 |
WO2010115735A2 (en) | 2010-10-14 |
EP2413700A2 (en) | 2012-02-08 |
JP2012522743A (ja) | 2012-09-27 |
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