CN117886950A - 呼吸道合胞病毒假病毒及其制备方法 - Google Patents
呼吸道合胞病毒假病毒及其制备方法 Download PDFInfo
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- CN117886950A CN117886950A CN202311839808.4A CN202311839808A CN117886950A CN 117886950 A CN117886950 A CN 117886950A CN 202311839808 A CN202311839808 A CN 202311839808A CN 117886950 A CN117886950 A CN 117886950A
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Abstract
本发明公开了呼吸道合胞病毒假病毒及其制备方法,涉及生物技术领域。本发明提供了一种呼吸道合胞病毒融合前F蛋白,所述的呼吸道合胞病毒融合前F蛋白与野生型相比,含有以下突变中至少两种:N67I、N116Q、S215P、D486N,所述的呼吸道合胞病毒融合前F蛋白的氨基酸序列如SEQ ID NO:1所示。本发明的呼吸道合胞病毒融合前F蛋白的构象使其稳定在融合前的构象进而增加F蛋白的感染力度,并且实现了其高效表达。本发明还提供了包含上述呼吸道合胞病毒融合前F蛋白的呼吸道合胞病毒假病毒。
Description
技术领域
本发明涉及生物技术领域,具体涉及呼吸道合胞病毒假病毒及其制备方法。
背景技术
呼吸道合胞病毒(Respiratory Syncytial Virus,RSV)是一种非节段性单股负链RNA病毒,属于副黏病毒科(Paramyxoviridae)、肺炎病毒亚科(Pneumovirinae)、肺病毒属(Pneumovirus)。病毒粒子外层为脂质囊膜,内部是病毒核衣壳。病毒颗粒由包膜、核衣壳和核心组成。呼吸道合胞病毒包膜上镶嵌着三个关键的蛋白分别是SH蛋白、F蛋白和G蛋白。其中F蛋白有融合前构象(pre-fusion)和融合后构象(post-fusion)两种形式,核衣壳由壳粒组成,病毒核心是由病毒基因组RNA和N、P、L等非结构蛋白组成的复合体。
RSV是婴幼儿、免疫力低下者和老年人急性呼吸道感染的重要病原,具有易反复感染、传播快、病情进展迅速等特点。在感染早期,病毒主要在上呼吸道复制,引起鼻塞,可伴有发热、乏力和头痛等临床症状,较少部分患者会出现下呼吸道感染,引起毛细血管炎和肺炎,表现为头痛、乏力和发热等症状,重症患者易发生严重并发症,包括呼吸功能不全、肠胃功能不全、呼吸暂停及病毒性心肌炎等相关症状。
F蛋白是一种Ⅰ型跨膜糖蛋白,包含信号肽、信号肽切割位点、F2区、多肽裂解区和F1区。F蛋白三聚体包含融合前(Pre-fusion F protein,Pre-F)和融合后(Post-fusion Fprotein,Post-F)两种构象。Pre-F是一种亚稳定结构,当病毒与细胞发生融合后,Pre-F转变为稳定的Post-F。融合前构象的F蛋白感染力度更强,融合后构象的F蛋白更加稳定但是感染力度弱。以往研究主要通过呼吸道合胞病毒野毒进行抗体中和实验和利用其感染宿主进行感染机制的研究,但呼吸道合胞病毒野毒的使用本身存在安全问题且对实验室的环境要求较高。
中国专利CN116284266A中公开了突变型呼吸道合胞病毒融合前F蛋白及其应用,所述的突变型呼吸道合胞病毒融合前F蛋白,相较于野生型呼吸道合胞病毒融合前F蛋白存在以下突变:(a)F1亚基和/或F2亚基中至少一个氨基酸残基被半胱氨酸取代,(b)野生型呼吸道合胞病毒融合前F蛋白第104-144位氨基酸残基被连接肽部分或全部取代,所述连接肽的氨基酸残基长度至少两个。该发明的突变型呼吸道合胞病毒融合前F蛋白相比于同类品具有较高的中和抗体结合活性和热稳定性。但其感染力度并没有明显改善。
因此,在本领域如何改造F蛋白以实现其高效表达和提高感染力度是亟待解决的问题。
发明内容
本发明的目的是提供一种呼吸道合胞病毒融合前F蛋白,改造呼吸道合胞病毒融合前F蛋白的构象使其稳定在融合前的构象进而增加F蛋白的感染力度。
为实现上述发明目的,本发明的技术方案如下:
一方面,本发明提供了一种呼吸道合胞病毒融合前F蛋白,所述的呼吸道合胞病毒融合前F蛋白与氨基酸序列如SEQ ID NO:6相比,含有以下突变中至少两种:N67I、N116Q、S215P、D486N。
优选地,所述的呼吸道合胞病毒融合前F蛋白的氨基酸序列如SEQ ID NO:1所示。
又一方面,本发明提供了一种核酸分子,所述的核酸分子编码上述的呼吸道合胞病毒融合前F蛋白。
具体地,所述的核酸分子包含一种或多种经密码子优化的核酸分子。
又一方面,本发明提供了一种载体,所述的载体包含上述的核酸分子。
优选地,所述的载体包括但不限于质粒、病毒。
进一步地,所述的载体是病毒载体。
进一步地,所述的病毒载体为流感病毒载体、逆转录酶病毒载体或腺病毒载体。
优选地,本发明提供了一种宿主细胞,所述的宿主细胞包含上述的呼吸道合胞病毒融合前F蛋白或上述的核酸分子或上述的载体。
优选地,所述的宿主细胞选自原核细胞或真核细胞。
进一步地,所述的原核细胞选自大肠杆菌细胞。
进一步地,所述的真核细胞包括哺乳动物细胞。
再进一步地,所述的哺乳动物细胞包括小鼠细胞和人细胞。
具体地,哺乳动物细胞为人细胞,所述的人细胞包括造血细胞、上皮细胞、肝细胞、神经细胞。
又一方面,本发明提供了一种呼吸道合胞病毒假病毒,包含上述的呼吸道合胞病毒融合前F蛋白。
又一方面,本发明提供了一种呼吸道合胞病毒假病毒的制备方法,包括以下步骤:
(1)质粒pcDNA3.1+作为载体,利用限制性内切酶BamHI和EcoRI酶切获得线性化载体,通过同源重组将线性化的pcDNA3.1+载体分别与SEQ ID NO.1、SEQ ID NO:2、SEQ IDNO:3连接,分别获得pcDNA3.1-pre F质粒、pcDNA3.1-G质粒、pcDNA3.1-ZsGreen-IRES-Luc2质粒;
(2)在293T细胞中进行假病毒的包装,加入pcDNA3.1-pre F质粒、pcDNA3.1-G质粒、pcDNA3.1-ZsGreen-IRES-Luc2质粒和psPAX2质粒混匀,再加入1xPEI混匀,获得转染混合物;
(3)转染混合物加入到培养皿中,进行转染;
(4)离心,收集细胞培养基上清,加入病毒保护液进行重悬,再加入全能核酸酶,获得呼吸道合胞病毒假病毒。
优选地,步骤(2)中293T细胞密度在90%左右、无支原体且细胞状态良好才可进行细胞转染。
优选地,步骤(2)转染前每皿换液2% FBS血清的DMEM培养基。
优选地,步骤(2)中质粒的加入量为psPAX2质粒2.5ug,pcDNA3.1-ZsGreen-IRES-Luc2质粒4.5ug,pcDNA3.1-pre F质粒2ug,pcDNA3.1-G质粒2ug。
优选地,步骤(3)中转染的时间为6-8h。
优选地,步骤(4)中离心的转速为50000xg,离心的时间为2h。
又一方面,本发明提供了上述的呼吸道合胞病毒融合前F蛋白或上述的核酸分子或上述的载体或上述的宿主细胞或上述的呼吸道合胞病毒假病毒或上述的制备方法制备得到的呼吸道合胞病毒假病毒在制备治疗、预防或辅助治疗呼吸道合胞病毒感染的药物中的应用。
又一方面,本发明提供了上述的呼吸道合胞病毒融合前F蛋白或上述的核酸分子或上述的载体或上述的宿主细胞或上述的呼吸道合胞病毒假病毒或上述的制备方法制备得到的呼吸道合胞病毒假病毒在制备预防呼吸道合胞病毒感染的疫苗中的应用。
又一方面,本发明提供了一种药物组合物,包含上述的呼吸道合胞病毒融合前F蛋白或上述的核酸分子或上述的载体或上述的宿主细胞或上述的呼吸道合胞病毒假病毒或上述的制备方法制备得到的呼吸道合胞病毒假病毒。
优选地,所述的药物组合物中还包括药学上可接受的载体。
进一步地,所述的药学上可接受的载体包括但不限于赋形剂、缓冲剂、乳化剂、稳定剂、稀释剂、粘合剂、防腐剂。
具体地,所述赋形剂选自微晶纤维素、乳糖、预胶化淀粉、环糊精、羧甲基纤维素、甘露醇中的至少一种。
具体地,所述缓冲剂选自磷酸二氢钠、碳酸氢钠、碳酸氢铵、醋酸钠、枸橼酸盐、组氨酸、琥珀酸盐中的至少一种。
具体地,所述乳化剂选自硬脂酸镁、硬脂酸锌、硬脂酸钙、硬脂酸甘油酯、山梨坦异硬脂酸酯、山梨坦油酸酯、甘油油酸酯、聚甘油-3聚蓖麻醇酸酯中的至少一种。
具体地,所述稳定剂选自金合欢胶、琼脂、藻酸、纤维素醚、羧甲基甲壳酯中的至少一种。
具体地,所述稀释剂选自赤藓糖醇、甘露醇、山梨糖醇、木糖醇、乳糖、蔗糖、玉米淀粉、马铃薯淀粉、磷酸钙、柠檬酸钙、结晶纤维素中的至少一种。
具体地,所述粘合剂选自乙醇、淀粉浆、糖浆、羟丙基甲基纤维素、羧甲基纤维素钠、海藻酸钠、聚乙烯吡咯烷酮中的至少一种。
具体地,所述防腐剂选自羟苯甲酸甲酯、对羟苯甲酸丙酯、尼泊金甲酯、尼泊金乙酯、尼泊金丙酯、三氯叔丁醇、硫柳汞、氧氰化汞、苯氧乙醇、氯己定、苯甲酸、苯甲酸钠、氯甲酚、苯扎溴铵、苯扎氯铵、羟苯乙酯中的至少一种。
优选地,所述的药物的剂型选自注射剂、片剂、胶囊剂、口服液体剂型、颗粒剂、软膏剂、混悬剂、散剂、乳剂、溶液剂、滴丸剂、栓剂、气雾剂中的任意一种。
又一方面,本发明提供了一种疫苗,所述的疫苗包含上述的呼吸道合胞病毒融合前F蛋白或上述的核酸分子或上述的载体或上述的宿主细胞或上述的呼吸道合胞病毒假病毒或上述的制备方法制备得到的呼吸道合胞病毒假病毒。
优选地,所述的疫苗还可以包含佐剂。
本发明的有益效果为:
本发明提供了一种呼吸道合胞病毒融合前F蛋白,其为在野生型呼吸道合胞病毒融合前F蛋白的基础上引入了四个突变假病毒,N67I、N116Q、S215P、D486N,改造的呼吸道合胞病毒融合前F蛋白的构象使其稳定在融合前的构象进而增加F蛋白的感染力度,并且实现了其高效表达。
附图说明
图1为呼吸道合胞病毒假病毒包装、转染和检测流程模式图。
图2为pcDNA3.1-pre F质粒图谱。
图3为pcDNA3.1-G质粒图谱。
图4为pcDNA3.1-ZsGreen-IRES-Luc2质粒图谱。
图5为呼吸道合胞病毒假病毒琼脂糖凝胶成像图。
图6为呼吸道合胞病毒假病毒qPCR检测拷贝数图。
图7为呼吸道合胞病毒假病毒感染A549和Hep-2细胞系荧光图。
图8为呼吸道合胞病毒假病毒蛋白凝胶成像图。
图9为呼吸道合胞病毒假病毒双荧光素酶检测数据分析柱状图。
具体实施方式
为了使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解,下面结合具体实施例,进一步阐明本发明,但下述实施例仅为本发明的优选实施例,并非全部。基于实施方式中的实施例,本领域技术人员在没有做出创造性劳动的前提下所获得其它实施例,都属于本发明的保护范围。下述实施例中,若无特殊说明,所用的操作方法均为常规操作方法,所用设备均为常规设备,各个实施例所用设备材料均相同。
本发明说明书中图1为呼吸道合胞病毒假病毒包装、转染和检测流程模式图。
实施例1呼吸道合胞病毒假病毒相关质粒构建
(1)全基因组合成的方法获得以下序列,具体如表1所示。
表1.
(2)质粒pcDNA3.1+作为载体,利用限制性内切酶BamHI和EcoRI酶切获得线性化载体,通过同源重组将线性化的pcDNA3.1+载体与合成的SEQ ID NO.1连接获得pcDNA3.1-preF质粒,图谱如图2所示。
(3)质粒pcDNA3.1+作为载体,利用限制性内切酶BamHI和EcoRI酶切获得线性化载体,通过同源重组将线性化的pcDNA3.1+载体与合成的SEQ ID NO.2连接获得pcDNA3.1-G质粒,图谱如图3所示。
(4)质粒pcDNA3.1+作为载体,利用限制性内切酶BamHI和EcoRI酶切获得线性化载体,通过同源重组将线性化的pcDNA3.1+载体与合成的SEQ ID NO.3接获得pcDNA3.1-ZsGreen-IRES-Luc2质粒,图谱如图4所示。
构建好的质粒取1ug跑1%的琼脂糖凝胶电泳,电压条件为120V 30min;电泳条带正确的质粒送第一代基因组测序鉴定,测序由天霖公司完成,琼脂糖凝胶电泳的结果见图5。
实施例2呼吸道合胞病毒假病毒包装实验
(1)转染前显微镜下观察293T细胞状态,密度在90%左右、无支原体且生长状态良好可进行细胞转染;
(2)转染前10cm dish细胞每皿换液10mL含2% FBS血清的DMEM培养基减少血清对质粒转染的影响;
(3)实验分组及质粒用量如下表(下表中psPAX2质粒购自addgene公司,货号为12260):
表2.
(4)按照上表的质粒和1xPEI需求量分别加入对应的含1mL 1xPBS/皿的1.5mL EP管中,先加入质粒混匀后再加入1xPEI(购自HARVEYBIO公司,货号为49553-93-7)进行混匀,静置15min获得转染混合物;
(5)转染混合物滴加到10cm dish中,转染8h小时后换液10mL含2% FBS血清的DMEM培养基;
(6)转染后48h后收集细胞培养基上清,离心机50000xg离心2h后用100ul1xPBS(购自Gibco公司,货号为70011044)进行重悬,同时按照1皿10cm dish加入25ul全能核酸酶(购自zrbiorise公司,货号为ZRXX12688)在37℃处理1h消除游离的DNA和RNA后过滤纯化获得纯化的呼吸道合胞病毒假病毒,分别命名为Pre-F+G virus、Pre-F virus和G virus。
实施例3呼吸道合胞病毒荷载量检测
(1)病毒核酸提取试剂盒(购自Beaverbio公司,货号为Cat.NO.70406)分别提取呼吸道合胞病毒假病毒RSV pre F&G、RSV pre F和RSV G携带的RNA,三种病毒携带的RNA相同(包含ZsGreen和dual Luciferase的mRNA),具体提取方法按照说明书进行。
(2)RNA利用一步法RT-qPCR试剂盒(购自诺唯赞公司,HiScript IIOne Step qRT-PCR SYBR Green Kit,货号:Q221-01)进行qPCR,该试剂盒可以同时实现RNA的逆转和qPCR。
(3)检测dual Luciferase mRNA的qPCR引物序列如下:
表3.
序列编号 | 合成序列 |
SEQ ID NO:4 | Forward Primer:CGCACATATCGAGGTGGACA |
SEQ ID NO:5 | Reverse Primer:GCAAGCTATTCTCGCTGCAC |
(4)RT-qPCR的20ul反应体系如下:
表4反应体系
按照上表反应体系在冰上配制并混匀。
(5)RT-qPCR的反应程序如下:
表5反应程序
按照上表程序设置qPCR反应程序进行qPCR反应,并导出数据进行分析,数据如图6,结果可知Pre-F virus的拷贝数相对于Pre-F+G virus和G virus较高。
实施例4呼吸道合胞病毒假病毒感染力度检测
(1)A549和Hep-2细胞系使用含10%血清的DMEM培养基进行培养,复苏细胞超过三代后观察细胞生长状态,待细胞生长至80%-90%密度且细胞状态良好时进行铺孔六孔板,共计6孔,铺孔密度30%-40%。
(2)第二天将20ul的呼吸道合胞病毒假病毒和1x助感染试剂(购自biosharp公司,货号为BL628A)混匀后加入六孔板中,六孔板按照十字摇匀法进行摇匀后置于含5% CO2的37℃细胞培养箱进行培养。
(3)感染12h左右换液2mL含10%血清的DMEM进行培养,感染72h时用荧光显微镜拍照,呼吸道合胞病毒假病毒的荧光图如图7所示,结果可知Pre-F+G virus的荧光强度相对于Pre-F virus和G virus较高。
实施例5呼吸道合胞病毒假病毒蛋白鉴定(WB)
(1)取60ul实施例2中的病毒液加上20ul的4x SDS loading buffer(购自Takara公司,货号为9173)混匀后100℃煮10min进行变性处理,变性完成后12000rpm离心2min去除杂质;
(2)上样量20ul进行聚丙烯酰胺凝胶电泳,电压条件为120V跑1h10min;
(3)电泳完成后进行转膜,使用的是PVDF膜,转膜条件为100mA转1h30min或者胶上无明显蛋白marker残留即可;
(4)转膜完成的PVDF膜使用5%的脱脂奶粉进行封闭,置于40rpm的平转摇床上孵育1h;
(5)孵育完成的PVDF用1xPBST(购自Solarbio公司,货号为P1031)清洗后分别用对应的F蛋白(购自abcam公司,货号为ab9110))和G蛋白抗体(购自abcam公司,货号为ab94966)进行4℃过夜孵育,F蛋白和G蛋白的稀释比例是1:1000;
(6)一抗孵育完成的PVDF膜使用1xPBST进行清洗,分三次进行,每次置于80rpm的平转摇床中清洗5min;
(7)清洗完成的PVDF膜加入对应抗性的二抗进行孵育,置于40rpm的平转摇床孵育1h,二抗的稀释比例是1:10000;
(8)二抗孵育完成的PVDF膜使用1xPBST进行清洗,分三次进行,每次置于80rpm的平转摇床中清洗5min;
(9)洗膜完成的PVDF膜均匀滴加发光液放入蛋白凝胶成像仪中进行发光拍摄图片,具体地蛋白凝胶成像图如图8所示,数据表明F蛋白和G蛋白的条带位置与实际的一致,证明了F蛋白和G蛋白成功表达。其中Control为重悬纯化病毒的1x病毒保护液。
实施例6呼吸道合胞病毒假病毒表达量
(1)A549和Hep-2细胞系使用含10%血清的DMEM培养基进行培养,复苏细胞超过三代后观察细胞生长状态,待细胞生长至80%-90%密度且细胞状态良好时进行铺孔96孔板,每种病毒3个复孔共计9个孔,铺孔密度30%-40%。
(2)第二天将10ul的呼吸道合胞病毒假病毒和1x助感染试剂混匀后加入96孔板中,孔板摇匀后置于含5% CO2的37℃细胞培养箱进行培养。
(3)感染72h小时后使用1xPBS(购自Gibco公司,货号为10-010-023)清洗细胞,胰酶消化后收集细胞进行1000rpm离心5min处理,离心完成后弃上清废液,细胞沉淀每个样品用双荧光素酶检测试剂盒(购自Promega公司,货号为E1910)中的50ul裂解液进行裂解后转移至酶标板,放入酶标仪中设置好程序进行检测,根据检测数据绘制柱状图进行分析,柱状图如图9所示,数据表明相对于对照来说病毒拥有较高的荧光强度。其中Control为未作处理的野生型细胞A549 WT/Hep-2 WT。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种呼吸道合胞病毒融合前F蛋白,其特征在于,所述的呼吸道合胞病毒融合前F蛋白与氨基酸序列如SEQ ID NO:6相比,含有以下突变中至少两种:N67I、N116Q、S215P、D486N。
2.根据权利要求1所述的呼吸道合胞病毒融合前F蛋白,其特征在于,所述的呼吸道合胞病毒融合前F蛋白的氨基酸序列如SEQ ID NO:1所示。
3.一种核酸分子,其特征在于,所述的核酸分子编码权利要求1-2任一项所述的呼吸道合胞病毒融合前F蛋白。
4.一种载体,其特征在于,所述的载体包含权利要求3所述的核酸分子。
5.一种呼吸道合胞病毒假病毒,其特征在于,包含权利要求1-2任一项所述的呼吸道合胞病毒融合前F蛋白。
6.权利要求5所述的呼吸道合胞病毒假病毒的制备方法,其特征在于,包括以下步骤:
(1)质粒pcDNA3.1+作为载体,利用限制性内切酶BamHI和EcoRI酶切获得线性化载体,通过同源重组将线性化的pcDNA3.1+载体分别与SEQ ID NO.1、SEQ ID NO:2、SEQ ID NO:3连接,分别获得pcDNA3.1-pre F质粒、pcDNA3.1-G质粒、pcDNA3.1-ZsGreen-IRES-Luc2质粒;
(2)在293T细胞中进行假病毒的包装,加入pcDNA3.1-pre F质粒、pcDNA3.1-G质粒、pcDNA3.1-ZsGreen-IRES-Luc2质粒和psPAX2质粒混匀,再加入1xPEI混匀,获得转染混合物;
(3)转染混合物加入到培养皿中,进行转染;
(4)离心,收集细胞培养基上清,加入病毒保护液进行重悬,再加入全能核酸酶,获得呼吸道合胞病毒假病毒。
7.权利要求1-2任一项所述的呼吸道合胞病毒融合前F蛋白或权利要求3所述的核酸分子或权利要求4所述的载体或权利要求5所述的呼吸道合胞病毒假病毒或权利要求6所述的制备方法制备得到的呼吸道合胞病毒假病毒在制备治疗、预防或辅助治疗呼吸道合胞病毒感染的药物中的应用。
8.权利要求1-2任一项所述的呼吸道合胞病毒融合前F蛋白或权利要求3所述的核酸分子或权利要求4所述的载体或权利要求5所述的呼吸道合胞病毒假病毒或权利要求6所述的制备方法制备得到的呼吸道合胞病毒假病毒在制备预防呼吸道合胞病毒感染的疫苗中的应用。
9.一种药物组合物,其特征在于,所述的药物组合物包含权利要求1-2任一项所述的呼吸道合胞病毒融合前F蛋白或权利要求3所述的核酸分子或权利要求4所述的载体或权利要求5所述的呼吸道合胞病毒假病毒或权利要求6所述的制备方法制备得到的呼吸道合胞病毒假病毒。
10.一种疫苗,其特征在于,所述的疫苗包含权利要求1-2任一项所述的呼吸道合胞病毒融合前F蛋白或权利要求3所述的核酸分子或权利要求4所述的载体或权利要求5所述的呼吸道合胞病毒假病毒或权利要求6所述的制备方法制备得到的呼吸道合胞病毒假病毒。
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