CN107050425A - 新型抗病毒蛋白C19orf66在制备抗寨卡病毒药物中的应用 - Google Patents
新型抗病毒蛋白C19orf66在制备抗寨卡病毒药物中的应用 Download PDFInfo
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Abstract
本发明公开了新型抗病毒蛋白C19orf66在制备抗寨卡病毒药物中的应用,所述新型抗病毒蛋白C19orf66通过干扰素刺激及寨卡病毒感染,能有效刺激其表达水平上升,同时稳定高表达的C19orf66可以特异性地抑制寨卡病毒的感染,同时,本发明还发现,沉默内源性C19orf66可促进寨卡病毒的感染。本发明证明了C19orf66具有高效的抗寨卡病毒感染和增殖的活性,为抗寨卡病毒的药物开发提供新的思路及方向。
Description
技术领域
本发明属于蛋白质工程技术领域,更具体地,涉及新型抗病毒蛋白C19orf66在制备抗寨卡病毒药物中的应用。
背景技术
寨卡病毒属于黄病毒科(Flaviviridae)黄病毒属(Flavivirus),病毒颗粒呈球状,直径约为40~70nm。寨卡病毒为单股正链RNA病毒,基因组长约10.8kb,含一条单一开放读码框,病毒蛋白由一个单一的多蛋白前体,经宿主蛋白酶和病毒蛋白酶切而成,包括3个结构蛋白(C、prM/M、E)和7个非结构蛋白(NS1、NS2A、NS2B、NS3、NS4A、NS4B、NS5),结构蛋白位于氨基端,非结构蛋白位于羧基端,具有丝氨酸蛋白酶、RNA解旋酶和RNA依赖RNA聚合酶(RdRP)功能。其中,衣壳蛋白和单股正链RNA基因组构成20面体对称的核衣壳,外层脂质包膜。黄病毒科病毒基因组RNA具有mRNA,复制模板RNA和遗传物质RNA三种作用,病毒大多通过受体介导的内吞作用进入细胞,在低pH值环境下病毒包膜与细胞膜融合,释放病毒核酸。RNA复制在细胞质内进行,合成全长负链RNA,形成中间体,子代病毒出芽到细胞内膜结构(ER)中,然后通过宿主细胞分泌通路,最终转运至包膜,成熟释放。分子生物学和生物信息学分析表明,寨卡病毒目前主要存在非洲型和亚洲型两个亚型,在系统发生树上与同为黄病毒属的登革病毒、日本脑炎病毒及西尼罗病毒相近。
寨卡病毒主要通过病毒感染的伊蚊类蚊媒叮咬传播,与登革病毒及基孔肯雅病毒传播方式相似,埃及伊蚊(Aedes aegypti)为主要传播媒介。寨卡病毒还可通过母婴、血液和性接触传播,以患者、隐性感染者和感染寨卡病毒的非人类灵长动物作为传染源。包括孕妇在内的各类人群对寨卡病毒普遍易感。
寨卡病毒于1947年首次在乌干达被发现,科学家们在用于黄热病监测的恒河猴体内分离到一种病毒,命名为“寨卡病毒”。1952年,乌干达及坦桑尼亚发现人感染寨卡病毒,随后仅有该病的零星报告或小规模流行。直至2007年,位于南太平洋地区密克罗尼西亚(Micronesia)联邦雅浦(Yap)岛发生寨卡病毒暴发流行,病毒由东南亚输入,也是首次发现寨卡病毒在亚非大陆以外传播。2013年,法属波利尼西亚发生寨卡病毒暴发。2015年以前,暴发疫情主要分布在非洲、东南亚和太平洋岛国。2015年5月,巴西发现首例确诊寨卡病例,泛美卫生组织(PAHO)发出警告,巴西政府估计有44~150万人感染寨卡病毒。随后,美洲多个国家相继发生寨卡病毒感染病例。迄今,全世界已有30多个国家报道寨卡病毒感染并引发多个国家输入性病例。截止2016年9月12日,中国大陆已发现16例输入性寨卡病毒病病例。寨卡病毒病流行范围不断扩大,已成为国际社会关注的焦点,WHO于2016年2月1日针对寨卡病毒召开紧急委员会会议,并宣布寨卡病毒的扩散已构成“全球关注的紧急公共卫生事件”。
寨卡病毒典型的临床表现为急性发热伴斑丘疹、关节痛或结膜炎。其他常见的症状可包括肌痛和头痛。但是,有病例在寨卡病毒感染后出现格林-巴利综合征(Guillain-Barré综合征)及自身免疫病症状。同时,在巴西,卫生部门发现寨卡病毒感染与小头畸形患儿增加有关。根据世界卫生组织的报道,2015年巴西的寨卡暴发流行中发现了很多小头畸形的新生儿(出生的新生儿头围与匹配的相同性别和孕龄的孩子比,低于平均值超过了两个标准差)。对寨卡疫情开展调查发现,越来越多的证据表明寨卡病毒与小头症之间存有关联。目前,尚无特异性的寨卡疫苗及抗病毒药物上市,因此,开发具有寨卡病毒特异性的实验室诊断检测工具以解决有效的疫苗和特异性治疗药物的研制工作已成为当前的关键科学问题及重大需求。
干扰素(interferon,IFN),是由英国科学家Isaacs于1957年利用鸡胚绒毛尿囊膜研究流感病毒干扰现象时首先发现的。IFN是一种广谱抗病毒剂,并不直接杀伤或抑制病毒,而主要是通过细胞表面受体作用使细胞产生抗病毒蛋白,从而抑制病毒的复制,同时还可增强自然杀伤细胞(NK细胞)、巨噬细胞和T淋巴细胞的活力,从而起到免疫调节作用,并增强抗病毒能力。是目前最主要的抗病毒感染的生物制品。
最近的研究表明,C19orf66具有拮抗登革病毒复制的功能,可能通过与登革病毒RNA及细胞mRNA结合蛋白相互作用而干扰登革病毒的翻译。目前尚无研究证明C19orf66具有抗寨卡病毒感染的功能。
发明内容
本发明的目的在于根据现有技术中的不足,提供了新型抗病毒蛋白C19orf66在制备抗寨卡病毒药物中的应用。
本发明的目的通过以下技术方案实现:
本发明提供了抗病毒蛋白C19orf66在制备抗寨卡病毒药物中的应用。
进一步地,上述抗病毒蛋白C19orf66是在制备抑制寨卡病毒在宿主细胞增殖的药物中的应用。
进一步地,所述抗病毒蛋白C19orf66是在制备抗寨卡病毒感染药物中的应用。
I型及III型干扰素刺激人肝癌细胞系(Huh7)基因动态表达分析中,在IFN-α及IFN-β的刺激下,C19orf66的RNA表达量上升,提示其为可能的ISG。C19orf66位于19号染色体短臂1区3带2亚带,cDNA全长768bp,有两种转录剪接体,表达的蛋白产物含有255位氨基酸残基。
本研究通过探索新型抗病毒蛋白C19orf66,是否可以特异性地抑制寨卡病毒复制,为抗寨卡病毒药物的开发提供科学依据,并为临床抗病毒治疗提供新思路。
具体验证过程包括:
1、分别采用I型干扰素IFN-α和IFN-β刺激A549细胞,鉴定细胞内源性C19orf66表达水平:
2、构建高表达pMSCV-C19orf66重组质粒,并鉴定外源性C19orf66的细胞株蛋白表达水平;
3、采用寨卡病毒GZ01株感染高表达C19orf66的A549细胞株,鉴定C19orf66抗寨卡病毒活性;
4、采用基因沉默技术,鉴定沉默内源性C19orf66的活性。
与现有技术相比,本发明具有以下有益效果:
本发明所提供的新型抗病毒蛋白C19orf66蛋白,能高效地抑制寨卡病毒株在宿主细胞中的增殖。这开拓了人源化抗病毒蛋白在抗病毒防治领域的临床应用新领域,从而为抗寨卡病毒的药物开发提供新的思路及方向。
附图说明
图1是Real time RT‐PCR方法检测IFN‐α、IFN‐β刺激A549细胞后,0.5h,1h,3h,6h,24h,48h各时间点C19orf66mRNA表达变化的结果图。
图2是寨卡病毒GZ01株感染A549细胞0h,3h,6h,12h,24h,48h各时间点细胞裂解液中内源性C19orf66mRNA表达变化的结果。
图3是稳定高表达C19orf66的A549细胞裂解液中C19orf66蛋白表达变化的Western blotting鉴定结果图。
图4是实时荧光定量RT‐PCR检测寨卡病毒GZ01株感染高表达C19orf66的A549细胞后,A549细胞内及上清中的寨卡病毒RNA结果图。
图5是沉默内源性C19orf66的Huh7细胞裂解液中,C19orf66蛋白表达变化的Western blotting鉴定结果图。
图6是实时荧光定量RT‐PCR检测寨卡病毒GZ01株感染沉默内源性C19orf66的Huh7细胞后,胞内及上清中的寨卡病毒RNA结果图。
具体实施方式
以下结合具体实施例和附图来进一步说明本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明,本发明所用试剂和材料均为市购。
实施例1:I型干扰素IFN-α、IFN-β刺激A549细胞,细胞内源性C19orf66表达水平的鉴定
1.以A549为细胞模型,用IFN-α、IFN-β两种I型干扰素,在500U/ml的浓度下刺激细胞。
2.分别收取0.5h,1h,3h,6h,24h,48h时间点的细胞,提取细胞总RNA。
贴壁细胞Trizol法总RNA提取具体操作为:
(1)去RNase处理:RNA提取所用玻璃器皿、耗材均需经使用0.1%DEPC水浸泡过夜后,高温高压蒸汽灭菌2次进行RNA酶灭活处理。涉及RNA操作所需配制的试剂均使用高压灭菌0.1%DEPC水配制;
(2)细胞培养后,弃去原培养液,用PBS洗涤细胞2次;
(3)往培养血中加入l ml TRlzol,吹打混匀(每10cm2面积,(即3.5cm直径的培养板加1ml TRlzol);
(4)用细胞刮从培养皿中刮取收集细胞,将细胞裂解液转移至1.5ml EP营,静置5分钟;
(5)加入200μl三氯甲烷,震荡混匀1min,室温静置5分钟;
(6)4℃12000r/min,离心15min,小心吸取上清约200μl;
(7)加入与上清等体积的异丙醇,充分混匀后,-40℃静置30分钟;
(8)4℃12000r/min,离心15分钟,小心弃去上清;
(9)加入500μl 70%预冷酒精洗涤沉淀物;
(10)4℃12000r/min.离心5分钟;
(11)弃去上清,自然晾干;
(12)加入50μl DEPC水溶解,Nanodrop定量提取的RNA浓度。
3.利用TOYOBO试剂盒将提取的细胞总RNA逆转录为cDNA
(1)在无RNA酶的0.2ml离心管中加入RNA进行预变性;
预变性体系2μl(RNA总量为1μg):
Total RNA 2μl(500μg/μl)
在PCR仪上,65℃反应5min后速置于冰上;
(2)逆转录体系:
反应条件:
反应结束后稀释至80μl。
4.用针对C19orf66的特异性引物,用real-time PCR的方法检测刺激之后C19orf66的表达水平。
Real-time PCR具体反应体系:
具体反应条件:
结果如图1所示,IFN-α、IFN-β刺激可以诱导A549细胞C19orf66mRNA水平上调。
实施例2:pMSCV-C19orf66重组质粒的构建、鉴定及提取
1.引物设计:从GenBank数据库下载C19orf66基因序列,利用PrimerPremier 5.0软件自行设计一对引物扩增全长C19orf66基因,引物由上海英俊生物公司合成,引物序列如下:
Forward:GGAAGATCTGCCATGTCTCAGGAAGGTGTGGAGCTGG
Reverse:CCGCTCGAGCTACTCCCTGGGCCCGCCCTC
2.获取目的片段:采用逆转录反应获得C19orf66基因片段,以其cDNA进行PCR扩增,并将所有扩增产物用1.5%琼脂糖凝胶(含终浓度为0.5μg/ml的EB)电泳并切胶回收纯化。
3.用QIAprep spin miniprep kit抽提pMSCV质粒
4.pMSCV质粒DNA与C19orf66基因片段PCR产物的双酶切反应,并进行连接。
5.将连接产物10μl加入200μl DH5α感受态细菌中,进行pMSCV-C19orf66重组质粒的转化。
6.挑取单菌落接种于3ml含氨苄青霉素(50μg/ml)的LB培养液中,37℃300rpm摇菌过夜,用QIAprep spin miniprep kit抽提pMSCV-C19orf66重组质粒DNA。
7.pMSCV-C19orf66重组质粒的双酶切鉴定和目的基因序列鉴定。
8.氯化铯梯度离心法大量提取pMSCV-C19orf66重组质粒DNA。
实施例3:稳定高表达外源性C19orf66细胞系的建立及鉴定
1.磷酸钙法转染制备逆转录病毒
在293T细胞中用磷酸钙法瞬时共转染重组质粒pMSCV-puro、pMSCV-C19orf66以及包装质粒pIK。
具体实验步骤如下:
(1)取对数期生长的293T细胞株,消化后并进行细胞计数,按每孔1×106个细胞的密度接种于100mm培养皿内,加入10ml DMEM完全培养基,置于37℃,5%CO2细胞培养箱培养(细胞准备数量根据具体情况而定);
(2)24h后,在1.5ml离心管中,分别将20μg的各种重组质粒、20μg包装质粒pIK、380μl 1×TE、60μl 2M CaCl2混匀后,再逐滴缓慢加入480μl 2×HEPES,室温静止30min;
(3)将混合液均匀加入的HEK-293T的培养基内,同时加入10μl氯喹(100μM)并轻柔混匀,将细胞置于37℃,5%CO2细胞培养箱培养;
(4)6h后,用1×Solution A洗涤细胞3次,更换成DMEM完全培养基,置入37℃,5%CO2细胞培养箱培养,而后按时收集含病毒液培养基。
2收集病毒并感染A549细胞
次日8时、12时、16时、20时、24时收集病毒液,并用0.22μm孔径滤器过滤。其中8时、12时、16时、20时收集的病毒液加入Polybrane(40μg/ml)立即感染A549细胞,24时收集的病毒液冻存于-80℃备用。24时将感染的细胞的培养基换为正常培养基过夜。连续感染三天。
3嘌呤霉素筛选阳性细胞
感染结束后,更换成含有嘌呤霉素(0.5μg/ml)的培养基筛选阳性细胞。
4.细胞生长稳定后收集细胞的总蛋白,Western blotting检测其各种C19orf66的表达情况。
Western blotting具体操作为:
(1)蛋白表达量的测定
1)绘制标准曲线。
2)将待测蛋白取2μl,加入23μl三蒸水。分别加入96孔板各孔中。
3)配置工作反应液:A液:B液为50:1,然后每孔加入200μl工作液,摇动30秒以混匀,37℃孵育30min。
4)应用酶标仪测定吸光度,然后根据标准曲线和吸光度计算蛋白的含量。
(2)将蛋白质样品进行非还原性SDS-PAGE,待溴酚蓝跑出胶后停止电泳;准备好两张3M滤纸和一张PVDF膜,浸入甲醇去离子水5分钟,然后与专用滤纸和纤维垫浸泡于1×转膜缓冲液中;剥下凝胶,去掉浓缩胶部分,并把滤纸和PVDF膜裁成凝胶大小;按照三明治夹心法转膜,安置次序如下:负极-纤维垫-1张专用滤纸-凝胶-PVDF膜-1张专用滤纸-纤维垫正极板,将其放入转膜槽中;冰浴,300mA转膜1小时;取出PVDF膜,封闭液封闭2小时;一抗孵育3小时;回收一抗,TBST洗膜,每次15min,重复3次;二抗孵育45-60min;回收二抗,TBST洗膜,每次15min,重复3次;在暗房用ECL显色液显色。
实施例4:高表达C19orf66的抗寨卡病毒活性鉴定
1.ZIKV-GZ01感染稳定高表达C19orf66的A549细胞株,48h后提取细胞及上清RNA。细胞总RNA提取方法见实施例1步骤2,利用LogPure Viral DNA/RNAKits提取细胞上清病毒RNA方法如下:
(1)转移200μl细胞上清至装有20μl Proteinase K的离心管中,振荡混匀5s;
(2)转移200μl Buffer AL/Carrier RNA至样品中,涡旋混匀20s;
(3)55℃水浴10min消化样品;
(4)加入250μl无水乙醇至裂解液中,涡旋混匀20s,室温静置5min,短暂离心收集管壁液滴;
(5)把HiPure Viral Micro Column装在2ml收集管中,转移所有混合液至柱子中,10000×g离心30~60s;
(6)弃去滤液把柱子装回收集管中,加入500μl Buffer VHB(已用无水乙醇稀释)至柱子中,10000×g离心30~60s;
(7)弃去滤液把柱子装回收集管中,加入650μl Buffer RW2(已用无水乙醇稀释)至柱子中,10000×g离心30~60s;
(8)倒弃滤液把柱子套回收集管,10000×g离心空柱3min,将柱子转移至新的1.5ml离心管;
(9)加入15~50μl RNase Free Water至柱子的膜中央,静置1min,10000×g离心1min;
(10)弃去柱子,保存病毒RNA。
2.应用实时荧光定量RT-PCR检测细胞内及上清中病毒的RNA的拷贝数,RT-PCR方法详见实施例1步骤4。
结果见图2~4所示,寨卡病毒感染可诱发宿主细胞内的C19orf66mRNA的水平明显升高(图2)。通过Western Blotting检测,以Actin作为内源性对照,发现A549-C19orf66细胞中C19orf66蛋白表达量明显高于对照组细胞A549-Vector,表明稳定高表达外源性C19orf66蛋白的易感细胞系A549-C19orf66构建成功(图3)。同时,外源性稳定高表达C19orf66蛋白能较强的抑制寨卡病毒的感染(图4)。
实施例5:沉默内源性C19orf66及鉴定
1.针对C19orf66设计两个寡核苷酸片段C19orf66-siRNAi-1、C19orf66-siRNAi-2及Scramble-RNAi对照寡核苷酸片段,siRNA由广州市锐博生物科技有限公司合成。
2.选取一株内源性表达C19orf66蛋白较高的易感细胞Huh7,接种1×105~5×105个细胞至含有适量完全培养基的24孔板培养孔中,使转染时的细胞密度达到30~50%。
3.将Scramble-RNAi对照寡核苷酸片段(对照组)、C19orf66-siRNAi-1以及C19orf66-siRNAi-2转染至细胞,转染浓度为50nM,具体步骤为:
(1)用30μl 1×riboFECTTM CP Buffer稀释1,25μl的20μM siRNA储存液,轻轻混匀。
(2)加入3μl riboFECTTM CP Reagent,轻轻吹打混匀,室温孵育0~15min。
(3)将riboFECTTM CP混合液加入到细胞培养基中,轻轻混匀。
(4)将培养板置于37℃的CO2培养箱中培养24~96h。
4.转染完成后,应用Western Blotting检测细胞裂解液中内源性C19orf66的表达变化,并以Actin作为内源性对照,Western Blotting操作方法详见实施例3步骤4。
结果见图5和图6所示,Huh7中C19orf66蛋白表达量明显低于对照组细胞,表明沉默易感细胞Huh7的C19orf66蛋白表达成功(图5,图中C19-1和C19-2分别表示实施例5所述针对C19orf66设计的两个寡核苷酸片段C19orf66-siRNAi-1和C19orf66-siRNAi-2)。沉默内源性C19orf66蛋白表达能较强的促进寨卡的感染(图6)。
最后说明的是,以上实施例仅用以说明本发明的技术方案而非限制,尽管参照较佳实施例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本技术方案的宗旨和范围,其均应涵盖在本发明的权利要求范围当中。
SEQUENCE LISTING
<110> 中山大学
<120> 新型抗病毒蛋白C19orf66在制备抗寨卡病毒药物中的应用
<130>
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 37
<212> DNA
<213> Forward
<400> 1
ggaagatctg ccatgtctca ggaaggtgtg gagctgg 37
<210> 2
<211> 30
<212> DNA
<213> Reverse
<400> 2
ccgctcgagc tactccctgg gcccgccctc 30
Claims (3)
1.抗病毒蛋白C19orf66在制备抗寨卡病毒药物中的应用。
2.根据权利要求1所述的应用,其特征在于,所述抗病毒蛋白C19orf66是在制备抑制寨卡病毒在宿主细胞增殖的药物中的应用。
3.根据权利要求1所述的应用,其特征在于,所述抗病毒蛋白C19orf66是在制备抗寨卡病毒感染药物中的应用。
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CN110408622A (zh) * | 2019-05-31 | 2019-11-05 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | 抗猪irav蛋白的单克隆抗体及其应用 |
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CN111000981B (zh) * | 2019-12-12 | 2021-08-13 | 中山大学 | 抗病毒蛋白C19orf66在靶向寨卡病毒非结构蛋白NS3抗病毒药物中的应用 |
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