CN116676273A - 一种蛋白水解靶向流感病毒及其制备方法和应用 - Google Patents
一种蛋白水解靶向流感病毒及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供了一种蛋白水解靶向流感病毒及其制备方法和应用。所述蛋白水解靶向流感病毒含有水解靶向的M1蛋白,所述水解靶向的M1蛋白的C端顺次插入了TEVp识别位点和被泛素‑蛋白酶体系统识别的蛋白水解靶向分子,所述泛素‑蛋白酶体系统识别的蛋白水解靶向分子包括SEQ ID No.1~52所示的氨基酸序列。所述水解靶向的M1蛋白能被泛素‑蛋白酶体系统识别而水解,病毒的复制能力减弱,具有较高的安全性。本发明提供的蛋白水解靶向流感病毒的亚型种类丰富,对于流感病毒疫苗的研发具有重要作用。
Description
技术领域
本发明属于生物技术领域,具体涉及一种蛋白水解靶向流感病毒及其制备方法和应用。
背景技术
流感病毒分为甲(A)、乙(B)、丙(C)三种类型,其中甲型流感病毒的暴发最为频繁且影响广泛。甲型流感病毒的基因组由8个独立的单链RNA片段组成,编码10种蛋白,包括血凝素蛋白(HA)、基质蛋白(M)、神经氨酸酶(NA)、核壳蛋白(NP)、非结构蛋白(NS)以及PB1、PB2和PA三种聚合酶。其中基质蛋白包括M1和M2,非结构蛋白包括NS1和NEP,其中,基质蛋白M1和M2在维持病毒颗粒形态与病毒的致病性中发挥重要作用。
甲型流感病毒可季节性地感染人类和禽类,大规模流行可引起极高的发病率和死亡率,严重威胁人类的健康。目前,接种疫苗是最主要的预防流感、控制流感传播的手段。
CN102899294A公开了一种H1N1型猪流感病毒疫苗株及其应用,所述H1N1型猪流感病毒疫苗株为预防猪流感的灭活疫苗,对同源强毒攻毒的猪可以提供很好的保护,具有良好的免疫原性,不论单苗或联苗,均可有效预防H1N1型猪流感。
CN106075424A公开了一种禽流感病毒疫苗,所述禽流感病毒疫苗的抗原为灭活的H9亚型禽流感病毒,所述禽流感病毒疫苗所使用的H9亚型禽流感病毒FJ11株新毒株的HA及EID50效价高、免疫原性好并能抵御各地方流行分离的H9亚型禽流感病毒的攻击。所述疫苗的安全性好,保存期试验中经过性状、安全性试验和效力试验数据的分析,分析结果与同类产品相比较,各项指标均稳定有效,且所述H9亚型禽流感灭活疫苗产生抗体快。
但是灭活疫苗在灭活过程中,有可能破坏或改变有效抗原成分,影响免疫效果,灭活疫苗的免疫效果维持时间较短,需要多次接种加强,一般成本较高;同时,上述流感病毒疫苗仅能抵御少量几种亚型的病毒。因此,开发一种能够安全可控、靶向降解和亚型种类丰富的流感病毒库,对于流感病毒疫苗的研发具有重要作用。
发明内容
针对现有技术存在的不足,本发明的目的在于提供一种蛋白水解靶向流感病毒及其制备方法和应用。所述蛋白水解靶向流感病毒含有水解靶向的M1蛋白,所述水解靶向的M1蛋白能被泛素-蛋白酶体系统识别,同时能被烟草蚀斑病毒蛋白酶(Tobacco etch virusprotease,TEVp)切割,在过表达TEVp的细胞系中能被大量制备,而在正常的细胞系中,泛素-蛋白酶体系统会识别与病毒蛋白融合的蛋白水解靶向分子,从而降解病毒蛋白,病毒的复制能力被减弱,甚至完全失去复制能力,所得的蛋白水解靶向流感病毒具有较高的安全性。所述蛋白水解靶向流感病毒还可以作为活疫苗、减毒疫苗用于流感的预防;所述蛋白水解靶向流感病毒还可以应用于肿瘤的治疗中,作为溶瘤病毒使用。本发明提供的蛋白水解靶向流感病毒的亚型种类丰富,对于流感病毒疫苗的研发具有重要作用。
为达到此发明目的,本发明采用以下技术方案:
第一方面,本发明提供一种蛋白水解靶向流感病毒,所述蛋白水解靶向流感病毒含有水解靶向的M1蛋白;
所述水解靶向的M1蛋白的C端顺次插入了TEVp识别位点和被泛素-蛋白酶体系统识别的蛋白水解靶向分子;
所述泛素-蛋白酶体系统识别的蛋白水解靶向分子包括SEQ ID No.1~52所示的氨基酸序列。
本发明中,所述蛋白水解靶向病毒的设计原理如下所示:
(1)引入到病毒蛋白特定位点的蛋白水解靶向分子可以被正常宿主细胞中的泛素-蛋白酶体系统识别,从而将相应的病毒蛋白降解,使病毒失活;
(2)引入到病毒蛋白特定位点的蛋白水解靶向分子可以在特定的病毒生产系统中被抑制,或者通过连接链被选择性地切割,而与病毒蛋白分离,从而避免或减少病毒蛋白被泛素-蛋白酶体系统降解;
(3)引入到病毒蛋白特定位点的蛋白水解靶向分子在正常宿主细胞中不能被抑制,或者连接蛋白水解靶向分子与病毒蛋白的连接链在正常宿主细胞中不能被切割。因此制备的病毒在动物和人体等的宿主细胞中可以被泛素-蛋白酶体系统识别、降解,从而使复制能力降低,甚至完全失去复制、繁殖能力,增加了病毒的安全性。
本发明中,所述水解靶向的M1蛋白的C端分别引入了可被条件性切割的52种蛋白水解靶向分子,由于人体和动物的正常细胞中存在泛素-蛋白酶体系统,可以识别与病毒蛋白融合表达的蛋白水解靶向分子,能将病毒的M1蛋白通过泛素-蛋白酶体系统降解,从而将病毒蛋白降解。含有所述水解靶向的M1蛋白的水解靶向流感病毒在动物和人体中复制减弱甚至不能进行复制繁殖,增加了病毒的安全性,获得的水解靶向流感病毒可以制备成流感病毒活疫苗。
所述蛋白水解靶向分子的氨基酸序列如下所示:
SEQ ID No.1:LDPETGEYL;
SEQ ID No.2:LDPETGEFL;
SEQ ID No.3:LDEETGEFL;
SEQ ID No.4:AFFAQLQLDEETGEFL;
SEQ ID No.5:PVPDYTSIHIV;
SEQ ID No.6:NIDFYAQVSDI;
SEQ ID No.7:ASFEYTILDPS;
SEQ ID No.8:MPPPGAPSFPSPPTEPSSEVPEQPSAQPLPGSPPRR;
SEQ ID No.9:NGNNYVYIDPT;
SEQ ID No.10:PGAPPGRDLA;
SEQ ID No.11:YYCFG;
SEQ ID No.12:YKKVGTMAAG;
SEQ ID No.13:RWGRRG;
SEQ ID No.14:RPCQRG;
SEQ ID No.15:RAPRQRSRDG;
SEQ ID No.16:SWRLTGFSGMKG;
SEQ ID No.17:RGPSSGG;
SEQ ID No.18:PPPMAGG;
SEQ ID No.19:RGPSSGG;
SEQ ID No.20:EAIGLLGG;
SEQ ID No.21:HLRGSPPPMAGG;
SEQ ID No.22:RGSPPPMAGG;
SEQ ID No.23:SPPPMAGG;
SEQ ID No.24:PPMAGG;
SEQ ID No.25:PMAGG;
SEQ ID No.26:RRGPSSGG;
SEQ ID No.27:VLIRVTYCGL;
SEQ ID No.28:KADTTTPTT;
SEQ ID No.29:PEQDCAVTSGE;
SEQ ID No.30:DEVTSTTSSS;
SEQ ID No.31:KAASADSTTEGTPAD;
SEQ ID No.32:EPEEPEADQHQ;
SEQ ID No.33:GPPSVFPPEPEEPEADQHQ;
SEQ ID No.34:ECEETEVDQHV;
SEQ ID No.35:LPDLV;
SEQ ID No.36:LPDMV;
SEQ ID No.37:LGLPDLVAKYN;
SEQ ID No.38:LGLPDMVAKHN;
SEQ ID No.39:ELNNNL;
SEQ ID No.40:DINNNN;
SEQ ID No.41:PTDVRDVDI;
SEQ ID No.42:PTDVRDIDL;
SEQ ID No.43:PTDVTAIHL;
SEQ ID No.44:KKERLLDDRHDSGLDS;
SEQ ID No.45:KAWQQQSYLDSGIHS;
SEQ ID No.46:LDSGIHS;
SEQ ID No.47:SPLPSGLLTPPQSG;
SEQ ID No.48:CSLIPTPDKE;
SEQ ID No.49:FELLPTPPLS;
SEQ ID No.50:PPTPPGSH;
SEQ ID No.51:TPEAPPCYMDVI;
SEQ ID No.52:KFMPPPTYTEVD。
优选地,所述TEVp识别位点包括SEQ ID No.53所示的氨基酸序列。
SEQ ID No.53:ENLYFQG。
优选地,所述M1蛋白的C端和TEVp识别位点之间通过柔性接头1连接。
优选地,所述柔性接头1包括SEQ ID No.54所示的氨基酸序列。
SEQ ID No.54:GSGG。
优选地,所述TEVp识别位点和蛋白水解靶向分子之间通过柔性接头2连接。
优选地,所述柔性接头2包括SEQ ID No.55所示的氨基酸序列。
SEQ ID No.55:GSG。
优选地,所述蛋白水解靶向流感病毒为A型病毒。
优选地,所述流感病毒的亚型包括H1N1、H1N2、H1N3、H1N8、H1N9、H2N2、H2N3、H2N8、H3N1、H3N2、H3N8、H4N2、H4N4、H4N6、H4N8、H5N1、H5N2、H5N3、H5N6、H5N8、H5N9、H6N1、H6N2、H6N4、H6N5、H6N6、H6N8、H7N1、H7N2、H7N3、H7N7、H7N8、H7N9、H8N4、H9N1、H9N2、H9N5、H9N8、H10N3、H10N4、H10N7、H10N8、H10N9、H11N2、H11N6、H11N9、H12N1、H12N3、H12N5、H13N6、H13N8、H14N5、H15N2、H15N8、H16N3、H17N10或H18N11中任意一种或至少两种的组合。
在本发明中,发明人发现通过把TEVp识别位点和蛋白水解靶向的分子的核苷酸序列引入到流感病毒的基因组中,在TEVp过表达的细胞系中,含有蛋白水解靶向分子的流感病毒基因组可以随着病毒基因组的复制而复制,并且可以随着病毒蛋白的翻译而融合表达于病毒蛋白中,从而得到被蛋白水解靶向分子定点修饰的病毒,即蛋白水解靶向病毒(Proteolysis-Targeting chimeric virus,PROTAC病毒)。
在正常的细胞中,泛素-蛋白酶体系统会识别与病毒M1蛋白融合的蛋白水解靶向分子,从而降解病毒蛋白,病毒的复制能力被减弱甚至完全失去复制能力,因此,PROTAC病毒具有很高的安全性。同时,由于所述PROTAC病毒中包含泛素-蛋白酶体系统的特异识别序列和位点,所述PROTAC病毒在肿瘤的治疗中,可以使冷肿瘤转变成热肿瘤,起到溶瘤的作用。
本发明中,所述PROTAC病毒可以在特定的人工改造的细胞系中高效复制、大量生产制备。此外,PROTAC病毒还可以进一步被修饰,例如引入免疫增强剂到病毒蛋白的特定区域或者特定氨基酸上,从而得到性能改善的病毒,从而得到免疫原性增强的PROTAC病毒。
在本发明中,所使用的柔性接头和TEVp识别位点(连接链)及柔性接头和蛋白水解靶向分子的氨基酸序列结构,还可以应用于B型流感,或者应用于流感病毒的其他亚型,还可以用于其他种类的病毒的改造,例如艾滋病毒、新冠病毒、手足口病毒、丁型肝炎病毒或戊型肝炎病毒中任意一种或至少两种的组合。
第二方面,本发明提供一种核酸分子,所述核酸分子编码第一方面所述的水解靶向的M1蛋白。
第三方面,本发明提供一种重组载体,所述重组载体含有至少一个拷贝的第二方面所述的核酸分子。
第四方面,本发明提供一种第一方面所述的蛋白水解靶向流感病毒的制备方法,所述制备方法包括如下步骤:
(1)构建用于制备蛋白水解靶向流感病毒的表达载体;
(2)用步骤(1)所得的表达载体替换流感病毒拯救系统中表达流感病毒M基因的质粒,于细胞系中共转染,得到所述蛋白水解靶向流感病毒。
本发明提供了52种蛋白水解靶向病毒,所述蛋白水解靶向病毒的M1蛋白的C端包含一个被泛素-蛋白酶体系统识别的蛋白水解靶向分子,M1蛋白和蛋白水解靶向分子之间通过TEVp识别位点进行连接,TEVp识别位点能够被选择性切割,且在M1蛋白和TEVp识别位点之间及TEVp识别位点与蛋白水解靶向分子之间通过两个柔性分子进行连接,通过所述方法改造病毒操作简单,得到的蛋白水解靶向流感病毒安全可靠、免疫性高。
优选地,步骤(1)中,所述表达载体中包括水解靶向的M1蛋白的编码序列。
优选地,步骤(2)中,所述细胞系为过表达TEVp的人工细胞系。
优选地,步骤(2)中,所述流感病毒拯救系统包括WSN流感病毒的12质粒拯救系统。
优选地,所述制备方法还包括将所述蛋白水解靶向流感病毒在过表达TEVp的人工改造的细胞系中进行复制,大规模生产的步骤。
在本发明中,所述PROTAC病毒只有在过表达TEVp的细胞系中才可以复制,利用所述PROTAC病毒对所述过表达TEVp的细胞系的依赖性,可以过表达在TEVp的细胞系中进行流感病毒的大量制备。
作为本发明的优选技术方案,所述制备方法包括如下步骤:
(1)构建用于制备蛋白水解靶向流感病毒的表达载体,使用基因工程的方法对WSN流感病毒的12质粒拯救系统中的表达流感病毒M基因的质粒进行改造,在M1蛋白的基因序列的C端和终止密码子之前引入一段插入序列,得到表达载体,所述表达载体包括所述水解靶向的M1蛋白的编码序列;
(2)用步骤(1)所得的表达载体替换流感病毒拯救系统中表达流感病毒M基因的质粒,将替换后的流感病毒拯救系统与过表达TEVp的人工细胞系共转染,得到所述蛋白水解靶向流感病毒;
将所述蛋白水解靶向病毒在过表达TEVp的人工改造的细胞系中进行复制,以大规模生产所述蛋白水解靶向病毒。
第五方面,本发明提供一种流感病毒疫苗,所述流感病毒疫苗包括第一方面所述的蛋白水解靶向流感病毒。
优选地,所述流感病毒疫苗为减毒疫苗、复制缺陷活病毒疫苗或复制可控活病毒疫苗中的任意一种。
第六方面,本发明提供第一方面所述的蛋白水解靶向流感病毒、第二方面所述的核酸分子、第三方面所述的重组载体、第四方面所述的蛋白水解靶向流感病毒的制备方法或第五方面所述的流感病毒疫苗中任意一种或至少两种的组合在制备治疗流感的药物和/或溶瘤药物中的应用。
本发明所述的数值范围不仅包括上述列举的点值,还包括没有列举出的上述数值范围之间的任意的点值,限于篇幅及出于简明的考虑,本发明不再穷尽列举所述范围包括的具体点值。
相对于现有技术,本发明具有以下有益效果:
(1)本发明利用泛素-蛋白酶体系统的水解作用和TEVp的切割作用,在过表达TEVp的细胞系中大量制备蛋白水解靶向流感病毒,而在正常的细胞系中,泛素-蛋白酶体系统会识别与病毒蛋白融合的蛋白水解靶向分子,从而降解病毒蛋白,病毒的复制能力被减弱,甚至完全失去复制能力。因此,所述蛋白水解靶向流感病毒具有很高的安全性,所述蛋白水解靶向流感病毒是一种减毒、复制缺陷和复制可控的活病毒。
(2)所述蛋白水解靶向流感病毒的制备方法操作简单、安全可靠;本发明中的蛋白水解靶向流感病毒通过哺乳动物稳定细胞系生产,克服了传统使用鸡胚繁殖病毒的缺点(使用鸡胚繁殖病毒易引起人体过敏等不良反应),所得的蛋白水解靶向流感病毒免疫原性高,可以用于疫苗的制备,和用于治疗病毒感染相关的药物的开发,具有很强的研究和应用价值。
(3)所述蛋白水解靶向流感病毒还可以进一步被修饰,例如,引入免疫增强剂到病毒蛋白的特定区域或者特定氨基酸上,得到性能改善的病毒,从而得到免疫原性增强的蛋白水解靶向流感病毒。
(4)所述蛋白水解靶向流感病毒可以激活肿瘤微环境,将冷肿瘤变为热肿瘤,增加肿瘤治疗效果。
附图说明
图1为实施例1中蛋白水解靶向流感病毒的制备过程示意图。
具体实施方式
下面通过具体实施方式来进一步说明本发明的技术方案。本领域技术人员应该明了,所述实施例仅仅是帮助理解本发明,不应视为对本发明的具体限制。
实施例中未注明具体技术或条件者,按照本领域内的文献所描述的技术或条件,或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可通过正规渠道商购获得的常规产品。
实施例1
本实施例提供一系列蛋白水解靶向流感病毒,所述蛋白水解靶向流感病毒中含有水解靶向的M1蛋白,所述水解靶向的M1蛋白的C端顺次插入了TEVp识别位点和被泛素-蛋白酶体系统识别的蛋白水解靶向分子;所述泛素-蛋白酶体系统识别的蛋白水解靶向分子的氨基酸序列如SEQ ID No.1~52所示。所述TEVp识别位点的氨基酸序列如SEQ ID No.53所示,所述M1蛋白的C端和TEVp识别位点之间通过柔性接头1连接,所述柔性接头1的氨基酸序列如SEQ ID No.54所示,所述TEVp识别位点和蛋白水解靶向分子之间通过柔性接头2连接,所述柔性接头2的氨基酸序列如SEQ ID No.55所示;从M1蛋白的C端到终止密码子的氨基酸序列结构为:M1蛋白的C端-柔性接头1-TEVp识别位点-柔性接头2-蛋白水解靶向分子。
SEQ ID No.53的氨基酸序列为ENLYFQG;
SEQ ID No.54的氨基酸序列为GSGG;
SEQ ID No.55的氨基酸序列为GSG。
所述泛素-蛋白酶体系统识别的蛋白水解靶向分子的氨基酸序列如表1所示。
表1
所述蛋白水解靶向流感病毒中的流感病毒为A型病毒,所述流感病毒的亚型包括H1N1、H1N2、H1N3、H1N8、H1N9、H2N2、H2N3、H2N8、H3N1、H3N2、H3N8、H4N2、H4N4、H4N6、H4N8、H5N1、H5N2、H5N3、H5N6、H5N8、H5N9、H6N1、H6N2、H6N4、H6N5、H6N6、H6N8、H7N1、H7N2、H7N3、H7N7、H7N8、H7N9、H8N4、H9N1、H9N2、H9N5、H9N8、H10N3、H10N4、H10N7、H10N8、H10N9、H11N2、H11N6、H11N9、H12N1、H12N3、H12N5、H13N6、H13N8、H14N5、H15N2、H15N8、H16N3、H17N10和H18N11。
所述蛋白水解靶向流感病毒的制备过程示意图如图1所示,所述蛋白水解靶向流感病毒的制备方法包括如下步骤:
(1)构建用于制备蛋白水解靶向流感病毒的表达载体:
以流感病毒拯救系统中表达流感病毒M基因的质粒为模板,通过定点突变的方法在表达M1蛋白的基因序列的C端、终止密码子之前,插入可表达“柔性接头1(GSGG)-TEVp识别位点(ENLYFQG)-柔性接头2(GSG)-蛋白水解靶向分子”的氨基酸序列的基因序列。当所述蛋白水解靶向分子的氨基酸序列为PTD3时,构建出来的载体命名为M1-PTD3,当所述蛋白水解靶向分子的氨基酸序列为PTD4时,构建出来的载体命名为M1-PTD4,载体的命名规则,以此类推。获得的目标载体,经过测序验证,表达载体构建成功。
(2)用步骤(1)所得的表达载体替换流感病毒拯救系统中表达流感病毒M基因的质粒,将替换后的流感病毒拯救系统与细胞系中共转染,得到所述蛋白水解靶向流感病毒:
使用WSN流感病毒的12质粒拯救系统对WSN病毒进行拯救。当拯救蛋白水解靶向流感病毒时,只需要将该12质粒拯救系统中用于表达M基因的质粒用步骤(1)中构建的表达载体进行替换即可,拯救所得的病毒株用步骤(1)中对应的载体的名称进行命名。
将表达TEVp的细胞接种在6孔板中;第二天,将步骤(1)中的M1-PTD3载体与WSN流感病毒拯救系统中的另外11个质粒共同转染表达TEVp蛋白的细胞系(HEK293T细胞系),对应于6孔板的每个孔,每种质粒加0.2μg。转染6h后,将培养基换成新的含有0.5%FBS、1μg/mL TPCK-trypsin和双抗的DMEM培养基。之后,每天观察细胞的病变情况,当细胞病变达到80%时,收集病毒上清,即得到蛋白水解靶向病毒,命名为M1-PTD3。
依照同样的方法,可获得其他的蛋白水解靶向流感病毒,并按照同样的规则进行命名,所得的蛋白水解靶向流感病毒如表2所示:
表2
序号 | 病毒名称 | 序号 | 病毒名称 | 序号 | 病毒名称 |
1 | M1-PTD3 | 19 | M1-PTD49 | 37 | M1-PTD86 |
2 | M1-PTD4 | 20 | M1-PTD51 | 38 | M1-PTD87 |
3 | M1-PTD5 | 21 | M1-PTD52 | 39 | M1-PTD88 |
4 | M1-PTD6 | 22 | M1-PTD53 | 40 | M1-PTD89 |
5 | M1-PTD7 | 23 | M1-PTD54 | 41 | M1-PTD90 |
6 | M1-PTD8 | 24 | M1-PTD55 | 42 | M1-PTD91 |
7 | M1-PTD9 | 25 | M1-PTD56 | 43 | M1-PTD92 |
8 | M1-PTD10 | 26 | M1-PTD57 | 44 | M1-PTD93 |
9 | M1-PTD11 | 27 | M1-PTD75 | 45 | M1-PTD94 |
10 | M1-PTD12 | 28 | M1-PTD77 | 46 | M1-PTD95 |
11 | M1-PTD14 | 29 | M1-PTD78 | 47 | M1-PTD96 |
12 | M1-PTD41 | 30 | M1-PTD79 | 48 | M1-PTD97 |
13 | M1-PTD43 | 31 | M1-PTD80 | 49 | M1-PTD98 |
14 | M1-PTD44 | 32 | M1-PTD81 | 50 | M1-PTD99 |
15 | M1-PTD45 | 33 | M1-PTD82 | 51 | M1-PTD100 |
16 | M1-PTD46 | 34 | M1-PTD83 | 52 | M1-PTD101 |
17 | M1-PTD47 | 35 | M1-PTD84 | / | / |
18 | M1-PTD48 | 36 | M1-PTD85 | / | / |
将获得的病毒上清感染新的表达TEVp蛋白的细胞系,按照能否引起细胞病变的标准对构建的蛋白水解靶向病毒进行考察:
如果可以引起表达TEVp蛋白的细胞系病变(如表达TEVp的HEK293T细胞和/或表达TEVp的MDCK细胞),说明该蛋白水解靶向病毒拯救成功;
如果不能引起表达TEVp的细胞(如表达TEVp的HEK293T细胞和/或表达TEVp的MDCK细胞)病变,说明该蛋白水解靶向病毒拯救失败。
结果表明,所有蛋白水解靶向流感病毒均拯救成功。
测试例1
对所述蛋白水解靶向流感病毒进行制备效率和安全性评价:
(1)蛋白水解靶向流感病毒在细胞水平的制备效率和安全性评价:
对M1-PTD3至M1-PTD101流感病毒株在表达TEVp的MDCK细胞系(MDCK-TEVp细胞系)和正常MDCK细胞系中引起细胞病变的情况和生长曲线进行考察,考察毒株的制备效率和安全性。野生型流感病毒作为对照。
毒株的安全性的判断标准如下所示:
基于细胞病变的观察确定:野生型流感病毒在MDCK-TEVp细胞和MDCK细胞中均可以引起完全的细胞病变(100%)。如果毒株在MDCK-TEVp细胞系中可以引起明显的细胞病变(细胞病变达到50-100%),说明该毒株的制备效率较高;与野生型病毒相比,如果毒株在正常MDCK细胞系中不能引起细胞病变或者引起较少的细胞病变(细胞病变低于野生型病毒引起的100%细胞病变),说明该毒株是安全的。
基于生长曲线的考察确定:与野生型病毒相比,如果毒株在MDCK-TEVp细胞系中可以高度复制(病毒滴度高于野生型病毒、与野生型病毒相当,或者不低于野生型病毒的千分之一),说明该毒株的制备效率较高;与野生型病毒相比,如果毒株在正常MDCK细胞系中复制能力减弱甚至不复制(病毒滴度低于野生型病毒的滴度),说明该毒株是安全的。
(a)基于细胞病变检测的步骤如下所示:
将制备的蛋白水解靶向流感病毒和野生型流感病毒分别按照MOI=0.01的比例感染MDCK-TEVp细胞系和正常的MDCK细胞系,每天观察细胞的病变情况并记录,持续4天。
测试结果表明,在MDCK-TEVp细胞系中,所有的蛋白水解靶向流感病毒和野生型流感病毒均可以引起显著的细胞病变;而在正常MDCK细胞系中,只有野生型流感病毒可以引起显著的细胞病变,而蛋白水解靶向流感病毒引起的病变减少甚至没有病变。该结果说明制备的蛋白水解靶向流感病毒在细胞水平具有安全性。
(b)基于生长曲线检测的步骤如下所示:
将制备的蛋白水解靶向流感病毒和野生型流感病毒按照MOI=0.001的比例分别感染MDCK-TEVp细胞系和正常的MDCK细胞系,感染后的24h、48h、72h和96h,取细胞培养上清,分别用TCID50实验和噬斑实验检测上清中的病毒滴度,从而可知病毒在两种细胞中的复制能力。
结果表明,在MDCK-TEVp细胞系中,所有的蛋白水解靶向流感病毒和野生型流感病毒均具有良好的复制能力;而在正常MDCK细胞系中,只有野生型流感病毒展示了良好的复制能力,而蛋白水解靶向流感病毒的复制能力减弱甚至复制缺陷。该结果说明制备的蛋白水解靶向流感病毒在细胞水平具有安全性。
(2)蛋白水解靶向流感病毒在动物水平的安全性评价:
使用BALB/c和C57BL/6J小鼠对所述蛋白水解靶向流感病毒在动物水平的安全性进行评价。选择M1-PTD3作为蛋白水解靶向流感病毒的代表,进行病毒的安全性评价。
安全性评价的具体步骤如下所示:
(a)将30只7周的雌性BALB/c小鼠或C57BL/6J小鼠,分成3组,每组10只;
(b)第一组每只小鼠滴鼻接种PBS,第二组每只小鼠滴鼻接种1×105PFU M1-PTD3,第三组每只小鼠滴鼻接种1×105PFU野生型WSN流感病毒;
(c)接种三天后,每组取5只小鼠,取其肺组织,检测其中的病毒滴度;
(d)继续观察监测每组剩余5只小鼠的体重和死亡情况,持续14天。
结果表明:野生型WSN流感病毒可以在小鼠的肺中高度复制,并引起小鼠体重明显下降和小鼠死亡。而所述蛋白水解靶向流感病毒在小鼠肺中的复制能力很弱,并且不会引起小鼠体重下降,也不会引起小鼠死亡。因此所述蛋白水解靶向病毒疫苗具有良好的安全性。
测试例2
对蛋白水解靶向流感病毒的复制能力进行考察:
(1)使用Western Blot检测所述蛋白水解靶向流感病毒的M1蛋白表达水平,选择5株蛋白水解靶向病毒为代表性毒株,考察所述蛋白水解靶向流感病毒在正常细胞中的复制能力。
将蛋白水解靶向流感病毒和野生型流感病毒分别感染正常的MDCK细胞系(MOI=0.1),在培养基中分别补充25nM、50nM和100nM蛋白酶体抑制剂MG-132,以DMSO(与病毒相同稀释比例)与正常的MDCK细胞系的混合溶液作为对照。分别在感染24h、48h和72h后,收集细胞样品,用Western Blot检测病毒M1蛋白表达水平。
(2)通过免疫荧光实验检测所述蛋白水解靶向流感病毒M1蛋白的表达水平,考察所述蛋白水解靶向流感病毒的复制能力。
将所述蛋白水解靶向流感病毒和野生型流感病毒分别感染MDCK-TEVp细胞系和正常的MDCK细胞系(MOI=0.01),在培养基中分别补充0nM、25nM、50nM和100nM的蛋白酶体抑制剂MG-132,以DMSO(与病毒相同稀释比例)与正常的MDCK细胞系的混合溶液作为对照。在感染48h后,将细胞用4%PFA固定,通过免疫荧光实验检测所述蛋白水解靶向流感病毒M1蛋白的表达水平。
实验结果显示,蛋白水解靶向流感病毒在感染MDCK-TEVp细胞后可以大量复制,并合成大量的病毒蛋白。而蛋白水解靶向流感病毒感染正常的MDCK细胞后,无法大量复制,因此检测到较少的病毒蛋白M1的信号;而当细胞的蛋白酶体系统被抑制后,病毒蛋白M1的信号增加,说明当蛋白酶体系统被抑制后,病毒的复制能力增强。检测结果与测试例2中的Western Blot检测结果相符,进一步证明蛋白水解靶向分子的引入能介导细胞的蛋白酶体对病毒蛋白的降解,进而抑制病毒的复制能力;当细胞的蛋白酶体系统被抑制后,病毒的复制能力会恢复,这与所述蛋白水解靶向流感病毒的设计原理一致。
测试例3
蛋白水解靶向流感病毒在动物水平的免疫原性和保护性考察:
对所述蛋白水解靶向流感病毒在动物水平上的免疫原性和保护性进行评价。以灭活流感疫苗(IIV)为对照(灭活流感病毒疫苗为发明人根据中国药典提供的方法用同源的流感病毒颗粒制备而成),选择M1-PTD3作为蛋白水解靶向流感病毒的代表,进行蛋白水解靶向流感病毒的免疫原性和保护性评价。
免疫原性和保护性考察的具体步骤如下所示:
(1)将60只7周的雌性BALB/c小鼠或C57BL/6J小鼠,分成3组,每组20只;
(2)第一组每只小鼠滴鼻接种PBS,第二组每只小鼠滴鼻接种1×105PFU M1-PTD3,第三组每只小鼠滴鼻接种1×105PFU灭活流感疫苗;
(3)接种一周后,每组取5只小鼠,取其肺组织和脾脏,检测其中的T细胞的免疫反应;
(4)接种三周后,每组取5只小鼠,取血,分别用于血凝抑制(HI)试验、中和(NT)抗体检测和ELISA检测,检测其中的抗体免疫反应;
(5)接种三周后,每组小鼠滴鼻接种2×105PFU的野生型WSN流感病毒;
(6)接种野生型WSN流感病毒三天后,每组取5只小鼠,取其肺组织,检测其中的病毒滴度;
(7)继续观察监测每组剩余5只小鼠的体重和死亡情况,持续14天。
结果表明,M1-PTD3病毒可以在动物体内诱导高水平的血凝抑制抗体滴度、中和抗体滴度、anti-NP IgG和anti-NP IgA等。M1-PTD3病毒诱导的血凝抑制抗体、中和抗体、anti-NP IgG和anti-NP IgA水平,显著高于由灭活流感疫苗诱导的抗体水平。M1-PTD3病毒疫苗的接种可以显著减少动物肺组织中的野生型WSN流感病毒滴度;M1-PTD3病毒疫苗提供的保护性显著地优于灭活流感疫苗。因此所述蛋白水解靶向流感病毒疫苗具有更优异的免疫原性和保护效果。
测试例4
考察蛋白水解靶向流感病毒作为溶瘤病毒的潜能:
使用C57BL/c的黑色素瘤荷瘤模型,对所述蛋白水解靶向流感病毒的溶瘤效果进行评价。
具体试验步骤如下所示:
(1)在小鼠的背部皮下注射黑色素瘤,饲养9天,当瘤体的体积达到约100mm3时,继续实验操作;
(2)向瘤体内分别注射50μL蛋白水解靶向流感病毒M1-PTD3和PBS,每间隔1天注射1次,共注射4次;
(3)每天检测瘤体的体积。
结果表明,所述蛋白水解靶向流感病毒可以有效地抑制肿瘤体积的增加,证明所述蛋白水解靶向流感病毒具备成为溶瘤病毒的潜能。
综上,本发明提供的蛋白水解靶向流感病毒能被泛素-蛋白酶体系统识别并水解,复制能力弱,具有较高的安全性,可以作为活疫苗、减毒疫苗用于流感的预防;所述蛋白水解靶向流感病毒可以在肿瘤治疗中作为溶瘤病毒使用。本发明提供的蛋白水解靶向流感病毒的亚型种类丰富,对于流感病毒疫苗的研发和肿瘤治疗具有重要作用。
申请人声明,以上所述仅为本发明的具体实施方式,但本发明的保护范围并不局限于此,所属技术领域的技术人员应该明了,任何属于本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到的变化或替换,均落在本发明的保护范围和公开范围之内。
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<110> 中国科学院深圳先进技术研究院
<120> 一种蛋白水解靶向流感病毒及其制备方法和应用
<130> 2022
<160> 55
<170> PatentIn version 3.3
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Claims (10)
1.一种蛋白水解靶向流感病毒,其特征在于,所述蛋白水解靶向流感病毒含有水解靶向的M1蛋白;
所述水解靶向的M1蛋白的C端顺次插入了TEVp识别位点和被泛素-蛋白酶体系统识别的蛋白水解靶向分子;
所述泛素-蛋白酶体系统识别的蛋白水解靶向分子包括SEQ ID No.1~52所示的氨基酸序列。
2.根据权利要求1所述的蛋白水解靶向流感病毒,其特征在于,所述TEVp识别位点包括SEQ ID No.53所示的氨基酸序列;
优选地,所述M1蛋白的C端和TEVp识别位点之间通过柔性接头1连接;
优选地,所述柔性接头1包括SEQ ID No.54所示的氨基酸序列;
优选地,所述TEVp识别位点和蛋白水解靶向分子之间通过柔性接头2连接;
优选地,所述柔性接头2包括SEQ ID No.55所示的氨基酸序列。
3.根据权利要求1或2所述的蛋白水解靶向流感病毒,其特征在于,所述蛋白水解靶向流感病毒为A型病毒;
优选地,所述蛋白水解靶向流感病毒的亚型包括H1N1、H1N2、H1N3、H1N8、H1N9、H2N2、H2N3、H2N8、H3N1、H3N2、H3N8、H4N2、H4N4、H4N6、H4N8、H5N1、H5N2、H5N3、H5N6、H5N8、H5N9、H6N1、H6N2、H6N4、H6N5、H6N6、H6N8、H7N1、H7N2、H7N3、H7N7、H7N8、H7N9、H8N4、H9N1、H9N2、H9N5、H9N8、H10N3、H10N4、H10N7、H10N8、H10N9、H11N2、H11N6、H11N9、H12N1、H12N3、H12N5、H13N6、H13N8、H14N5、H15N2、H15N8、H16N3、H17N10或H18N11中任意一种或至少两种的组合。
4.一种核酸分子,其特征在于,所述核酸分子编码权利要求1~3中任一项所述的水解靶向的M1蛋白。
5.一种重组载体,其特征在于,所述重组载体含有至少一个拷贝的权利要求4所述的核酸分子。
6.一种权利要求1~3中任一项所述的蛋白水解靶向流感病毒的制备方法,其特征在于,所述制备方法包括如下步骤:
(1)构建用于制备蛋白水解靶向流感病毒的表达载体;
(2)用步骤(1)所得的表达载体替换流感病毒拯救系统中表达流感病毒M基因的质粒,于细胞系中共转染,得到所述蛋白水解靶向流感病毒。
7.根据权利要求6所述的蛋白水解靶向流感病毒的制备方法,其特征在于,步骤(1)中,所述表达载体中包括所述水解靶向的M1蛋白的编码序列;
优选地,步骤(2)中,所述细胞系为过表达TEVp的人工细胞系;
优选地,步骤(2)中,所述流感病毒拯救系统包括WSN流感病毒的12质粒拯救系统。
8.根据权利要求6或7所述的蛋白水解靶向流感病毒的制备方法,其特征在于,所述制备方法还包括将所述蛋白水解靶向流感病毒在过表达TEVp的人工改造的细胞系中进行复制,大规模生产的步骤。
9.一种流感病毒疫苗,其特征在于,所述流感病毒疫苗包括权利要求1~3中任一项所述的蛋白水解靶向流感病毒;
优选地,所述流感病毒疫苗为减毒疫苗、复制缺陷活病毒疫苗或复制可控活病毒疫苗中的任意一种。
10.权利要求1~3中任一项所述的蛋白水解靶向流感病毒、权利要求4所述的核酸分子、权利要求5所述的重组载体、权利要求6~8中任一项所述的蛋白水解靶向流感病毒的制备方法或权利要求9所述的流感病毒疫苗中任意一种或至少两种的组合在制备治疗流感的药物和/或溶瘤药物中的应用。
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