CN117679491B - IFITM3抗病毒蛋白在制备抗PDCoV增殖药物中的应用 - Google Patents
IFITM3抗病毒蛋白在制备抗PDCoV增殖药物中的应用 Download PDFInfo
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Abstract
本发明公开了IFITM3抗病毒蛋白在制备抗PDCoV增殖药物中的应用。属于抗病毒药物技术领域。本发明构建慢病毒稳定表达IFITM3的ST细胞株,并发现IFITM3对PDCoV病毒增殖有显著的抑制作用。该细胞系不仅可以用于猪源IFITM3的制备,还可用于PDCoV与IFITM3相互作用的机制研究,并以此为基础为抗PDCoV新药的筛选和研发提供思路。
Description
技术领域
本发明涉及抗病毒药物技术领域,更具体的说是涉及IFITM3抗病毒蛋白在制备抗PDCoV增殖药物中的应用。
背景技术
猪德尔塔冠状病毒(PDCoV)属于冠状病毒科和德尔塔冠状病毒属,在猪中引起疾病,影响畜牧业,具有严重的社会经济影响。PDCoV是一种包膜病毒,含有单链阳性RNA,编码4种结构蛋白,刺突(S)、包膜(E)、膜(M)、核衣壳(N)和3种辅助蛋白NS6、NS7、NS7a。
干扰素(IFN)作为宿主先天免疫系统的一部分,诱导多种ISGs的表达干扰病毒的生命周期,从而在细胞内建立抗病毒状态。IFN诱导的跨膜蛋白3(IFITM3)是诱导量最大的ISG之一,可阻断多种病毒的感染和增殖,如埃博拉病毒(EBOV)、登革热病毒(DENV)、西尼罗河病毒(WNV)和其他冠状病毒,如严重急性呼吸综合征(SARS-CoV-2)、中东呼吸综合征冠状病毒(MERS-CoV)、人类冠状病毒(HCoV-OC43)、猪流行性腹泻病毒(PEDV)和传染性胃肠炎病毒(TGEV),但对PDCoV增殖的影响尚不清楚。
综上,探究猪源IFITM3对PDCoV增殖的影响是本领域技术人员亟需解决的问题。
发明内容
有鉴于此,本发明提供了IFITM3抗病毒蛋白在制备抗PDCoV增殖药物中的应用。
为了实现上述目的,本发明采用如下技术方案:
IFITM3抗病毒蛋白在制备抗PDCoV增殖药物中的应用。
进一步的,过表达IFITM3抗病毒蛋白用于抑制PDCoV增殖。
进一步的,所述IFITM3抗病毒蛋白为猪源IFITM3抗病毒蛋白。
进一步的,所述IFITM3抗病毒蛋白的氨基酸序列如SEQ ID NO:1所示;
MNCASQPFFTGAHGGPPTYEMLKEEHEVAVLGAPQTSAPVATTVINIRSETSVPDHVVWSLFNTLFMNWCCLGFVAFAYSVKARDRKMVGDIIGAQSYASTAKCLNIWALVLGLLLIIAFIIVCTTGSLVIFQAVLQLIKDYRGY,SEQ ID NO:1。
经由上述的技术方案可知,与现有技术相比,本发明取得的有益效果为:
在细胞内证明了猪源IFITM3的过表达能显著抑制PDCoV的增殖,有望成为增强猪天然免疫的新靶点,在作为抗病毒感染药物及疫苗增强剂方面有良好的应用前景。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据提供的附图获得其他的附图。
图1附图为本发明实施例1中IFITM3在猪细胞中的普遍表达;
图2附图为本发明实施例1中构建的载体图谱(a)及酶切电泳图片(b)酶切位点:ApaLI+NheI(2105,3985,1246,2521);
图3附图为本发明实施例1中稳定过表达猪源IFITM3细胞系构建及鉴定,其中,a为空白ST细胞在荧光显微镜下观察结果;b为CMV在荧光显微镜下观察结果;c为CMV-IFITM3在荧光显微镜下观察结果;d为细胞增殖活力结果;e为mRNA表达水平结果;f为westernblotting结果;
图4附图为本发明实施例1中IFITM3限制PDCoV的mRNA表达结果;
图5附图为本发明实施例1中Flag-His-TAP串联亲和纯化IFITM3相互作用蛋白复合物的鉴定,第一步纯化(第2道和第5道)与第二步纯化(第3道和第6道)的考马斯亮蓝染色的蛋白电泳图;其中,图中显示了总细胞裂解物(CMV-IFITM3通道1和PDCoV感染CMV-IFITM3通道4)、通过His标签第一步纯化样本(CMV-IFITM3通道2和PDCoV感染CMV-IFITM3通道5)和通过Flag标签第二步纯化样本(CMV-IFITM3通道3和PDCoV感染CMV-IFITM3通道6),白色椭圆形代表Flag-His-IFITM3融合蛋白,分子质量标记显示在左侧;
图6附图为本发明实施例1中LC-MS/MS鉴定的CMV-IFITM3组和CMV-IFITM3+PDCoV组Flag-His-IFITM3相互作用蛋白的韦恩图;
图7附图为本发明实施例2中瞬时转染3.1-Flag-IFITM3过表达真核载体限制PDCoV的mRNA表达结果;
图8附图为本发明实施例3中瞬时转染shIFITM3干扰载体促进PDCoV的mRNA表达结果。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
本发明所需药剂为常规实验药剂,采购自市售渠道;未提及的实验方法为常规实验方法,在此不再一一赘述。
实施例1
1材料与方法
1.1载体构建、细胞培养及过表达IFITM3细胞系的建立
根据猪IFITM3全基因序列(JQ315416.1)设计引物:
F1:5-’ATCGCGTCTCGGGCTGCCACCATGGTGAGCAAGGGCGAGGAGGA-3’,SEQ ID NO:2;
R1:5-’ATCGCGTCTCGGGGTTTACTTGTACAGCTCGTCCATGCC-3’,SEQ ID NO:3。
以ST细胞总RNA为模板,扩增获得N’端串连6×His和Flag标签的猪IFITM3片段,连接到目标载体上,获得猪IFITM3双标签融合过表达慢病毒载体CMV-IFITM3。克隆载体酶切鉴定后送金唯智生物科技有限公司测序。
提前进行HEK293T细胞的复苏,于37℃、5%CO2培养箱中培养。同时克隆包装辅助质粒pGAG、pREV和pVSV-G。转染前24h消化对数生长期的HEK293T细胞,当细胞密度达70%~80%时可进行转染,取包装质粒pGAG、pREV、pVSV-G各0.5μg和目的质粒CMV、CMV-IFITM3各1.5μg,借助转染试剂LipofectamineTM3000转染至细胞中。继续培养16h后,加入AdvancedDMEM、20mL/L FBS、L-Glu、0.01mmol/L胆固醇、0.01mmol/L蛋黄卵磷脂和chemicallydefined lipid concentrate(CD)。转染培养48h后,取上清得到病毒液,此上清液即为包装好的慢病毒液,将上清液冻存于-80℃冰箱中备用。
以收集好的病毒液感染ST细胞。用嘌呤霉素(1μg/mL)筛选获得ST细胞系。用先前已经培养好的ST细胞作为感染用细胞。细胞密度为70%时开始,设常规试验分组。加入慢病毒液,37℃培养箱中培养6h~10h后进行1次换液;孵育24~48h后,在荧光倒置显微镜下观察发出荧光的阳性细胞的荧光表达情况,以嘌呤霉素筛选阳性细胞选。反复筛选4~5代后,可得到稳定表达细胞系。为保证建立的细胞株可用于后续试验,需要确定细胞株的细胞活力,选用细胞计数试剂盒-8(CCK-8)检测细胞活力。试验步骤依照CKK-8试剂盒。使用450nm的微孔板读取器进行验证。按公式计算:
细胞生长活性=[(对照组OD-试验组OD)/对照组OD]×100%;
绘制细胞生长活性率柱状图。
1.2RNA提取及实时荧光定量PCR
使用TRIzol试剂(Thermo Fisher Scientific,Waltham,MA,USA)从ST细胞中提取总RNA,并根据制造商的说明使用PrimeScriptTMRTase(Takara Bio,Dalian,China)反转录成cDNA。RT-qPCR使用SYBR Premix Ex TaqTMⅡ(TakaraBio,大连,中国),按照制造商的方案进行。引物如下:
F2:5-’TGGCTTTCGCCTACTCCG-3’,SEQ ID NO:4;
R2:5-’ACAGTGGCTCCGATGGTCAG-3’,SEQ ID NO:5。
IFITM3的相对表达量变化采用2-ΔΔCt方法以β-actin为内参进行归一化分析。所有分析均采用三次技术和生物学重复进行。
1.3免疫印迹法
如前所述,收集细胞,裂解并处理以进行western blot分析。用12%SDS-PAGE分离细胞提取物,然后将蛋白质转移到PVDF膜(Millipore)上。用5%脱脂乳封闭,一抗和二抗先后孵育后,用ECL试剂(ZATA LIFE)检测蛋白。以β-actin蛋白为对照。将β-actin(兔)单抗(Abcam,ab8227)、抗ifitm3(兔)单抗(Proteintech,11714-1-AP)、抗flag小鼠单抗(ab克隆,AE005)和抗6x His tag(兔)单抗(Abcam,ab213204)与山羊抗兔IgG抗体(AS003)偶联抗体进行稀释和检测。
1.4TAP-LC-MS/MS分离鉴定IFITM3相互作用蛋白
收集CMV-IFITM3细胞和感染的CMV-IFITM3细胞的全细胞裂解液,裂解并根据anti-FLAG M2琼脂糖珠手工程序(Sigma,Missouri,USA)进行处理。将未结合的蛋白离心并洗涤三次。收集以上洗脱液后,根据PurKineTM His-TagNi-NTA色谱柱说明书(Abbkine,武汉,中国)进一步纯化融合蛋白。杂质蛋白被平衡溶液冲走。洗脱后,纯化蛋白用SDS-loading缓冲液煮沸,用考马斯亮蓝染色的SDS-PAGE凝胶(Solarbio,Beijing,China)进行分析。将每条通道的可见蛋白切成1mm2的凝胶片段,仅除去IFITM3融合蛋白的区域。然后将样品送至中国北京Novogene公司进行LC-MS/MS分析。采用常规方法对Q Exactive HFX与EASY-nLCTM(Thermo fisher Scientific,USA)耦合进行分析。为了提高分析结果的质量,降低假阳性率,Proteome Discoverer2.5软件对搜索结果进行了进一步的过滤:置信水平大于99%的Peptide Spectrum Matches(psm)被认为是可信的psm,含有至少一个独特肽段(特异性肽段)的蛋白质被认为是可信的蛋白质。只保留可信的肽和蛋白质,并进行错误发现率(FDR)验证,以去除FDR大于1%的肽和蛋白质。LC-MS/MS鉴定的蛋白使用猪数据库(Sus_scrofa_uniprot_2023_3_13)进行查询。Fasta,326225序列,https://www.uniprot.org/)。
2结果
2.1IFITM3在猪细胞中的普遍表达
为了探究IFITM3在不同猪细胞中的存在,我们检测了IFITM3在猪肠上皮细胞系J2(IPEC-J2)、猪肺泡巨噬细胞细胞系3D4/21、猪睾丸细胞系ST和两种猪肾细胞系PK-15和LLC-PK-1中的蛋白表达水平(图1)。这表明IFITM3在猪的肠、睾丸、肾、肺等组织中广泛表达,可能在猪的组织中发挥重要的保守作用。
2.2真核表达载体构建及稳定过表达细胞株筛选和培育
从NCBI的GenBank上查询公布的猪源IFITM3基因序列。以ST细胞总RNA反转的cDNA为模板,用高保真酶扩增IFITM3基因,将IFITM3片段连接之前改造好的慢病毒包装载体上,转化克隆得到CMV-IFITM3质粒。载体采用ApaLI+NheI限制性内切酶进行双酶切鉴定结果如图2b,83号泳道切出2015bp、3985bp、1246bp和2521bp四条带,大小正确。将质粒送金唯智生物科技有限公司测序,没有插入、缺失或突变。质粒CMV-IFITM3可用于后续试验。
将过表达载体CMV-IFITM3及其对照空白载体CMV经慢病毒包装系统包装为慢病毒,滴度测定结果为:CMV-IFITM3病毒滴度为1.74×108TU/mL,CMV病毒滴度为2.39×108TU/mL。在荧光显微镜下观察CMV和CMV-IFITM3细胞的绿色荧光显示转染效率高,空白ST细胞未观察到绿色荧光(图3a~图3c)。对同时将细胞系细胞进行细胞活力检测,检测结果显示猪IFITM3的瞬时过表达对细胞活性不产生显著影响(P>0.05)(图3d)。收取转染细胞总RNA,反转录成cDNA后,RT-PCR检测猪IFITM3的转录水平的过表达情况。如图3e,与空白ST细胞和对照细胞系CMV相比,CMV-IFITM3 ST细胞系中猪IFITM3的mRNA水平显著上调(P<0.001)。CMV-IFITM3细胞的Western blotting分析显示(图3f),IFITM3水平升高,在18kDa左右明显出现Flag-tag蛋白或His-tag蛋白。
2.3IFITM3限制PDCoV的mRNA表达表达
以CMV细胞株为对照组,用PDCoV(MOI=1)感染过表达IFITM3的ST细胞株(CMV-IFITM3)。分别于24h和48h收取转染细胞总RNA,通过反转PCR获得cDNA,以cDNA为模板进行RT-PCR以测定PDCoV的基因组RNA表达水平(图4)。检测结果显示,稳定过表达猪源IFITM3能显著抑制PDCoV的mRNA表达表达。
2.5利用Flag-His-TAP串联亲和纯化IFITM3相互作用蛋白复合物
为了明确PDCoV感染过程中与IFITM3相互作用的其他蛋白,我们使用了基于串联亲和纯化耦合质谱的蛋白质组学技术。CMV-IFITM3 ST细胞株与PDCoV孵育,MOI为0.01(CMV-IFITM3+PDCoV)。以未感染的CMV-IFITM3ST细胞系为对照(CMV-IFITM3)。用CMV-IFITM3组和CMV-IFITM3+PDCoV组感染后24h(hpi)总细胞裂解液进行两步纯化。对于第一步纯化,用抗flag M2琼脂糖珠纯化flag融合蛋白的总裂解物。纯化后的样品用抗His蛋白纯化镍柱孵育。分别用SDS-PAGE梯度分离法和考马斯亮蓝染色法对感染或未感染PDCoV细胞的纯化蛋白复合物进行分离,以监测两种纯化质量。如图5所示,通过两步串联亲和纯化,大部分非特异性或非相互作用蛋白被去除和减少。
2.6LC-MS/MS鉴定PDCoV感染过程中与IFITM3相互作用的细胞蛋白
将CMV-IFITM3和PDCoV感染的CMV-IFITM3的SDS凝胶切割成部分,不包括黑框标记的IFITM3融合蛋白相关条带。凝胶组分被烷基化,用胰蛋白酶消化,并通过质谱分析,如先前在材料和方法中所述。所有检测到的蛋白质都使用材料和方法中描述的特定数据库和程序,以高度置信度的肽序列进行验证。仅考虑从CMV-IFITM3裂解物中与IFITM3共纯化的病毒和细胞蛋白。正如预期的那样,在CMV-IFITM3和PDCoV感染的CMV-IFITM3样本中,诱饵蛋白IFITM3丰富。在CMV-IFITM3和PDCoV感染的CMV-IFITM3样品中,基于至少2个高质量独特肽(>95%)与同一蛋白匹配的标准,共鉴定出449种蛋白,置信度高(>99%)。在所有细胞蛋白中,169种蛋白存在于CMV-IFITM3和PDCoV感染的CMV-IFITM3样品中(图6)。
实施例2
瞬时转染3.1-Flag-IFITM3过表达真核载体抑制PDCoV的增殖
以不同剂量(0.5、1、2μg/L)的3.1-Flag-IFITM3瞬时转染ST细胞48h,分别以PDCoV感染ST,分析过表达IFITM3对PDCoV感染的影响。载体构建过程详见《Antiviral Role ofIFITM Proteins in Classical Swine Fever Virus Infection》。以转染空载体pcDNA3.1的ST为阴性对照。采用RT-qPCR检测感染后24h PDCoV mRNA水平。
如图7所示,转染3.1-Flag-IFITM3后,PDCoV的mRNA水平明显受到抑制,且呈剂量依赖性。说明瞬时转染过表达IFITM3载体能显著抑制PDCoV增殖。
实施例3
瞬时转染shIFITM3干扰载体促进PDCoV的增殖
分别将随机干扰载体shN和shIFITM3干扰载体转染ST细胞48h,分别以PDCoV感染ST,分析干扰IFITM3表达对PDCoV感染的影响。载体构建过程详见《Antiviral Role ofIFITM Proteins in Classical Swine Fever Virus Infection》。以转染随机干扰载体shN的ST为阴性对照。采用RT-qPCR检测感染后24h PDCoV mRNA水平。
如图8所示,转染shIFITM3后,PDCoV的mRNA水平上升。说明瞬时转染干扰IFITM3载体能显著促进PDCoV增殖。
本说明书中各个实施例采用递进的方式描述,每个实施例重点说明的都是与其他实施例的不同之处,各个实施例之间相同相似部分互相参见即可。
对所公开的实施例的上述说明,使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (2)
1.IFITM3抗病毒蛋白在制备抗PDCoV增殖药物中的应用,其特征在于,
过表达IFITM3抗病毒蛋白用于抑制PDCoV增殖;
所述IFITM3抗病毒蛋白的氨基酸序列如SEQ ID NO:1所示;
MNCASQPFFTGAHGGPPTYEMLKEEHEVAVLGAPQTSAPVATTVINIRSETSVPDHVVWSLFNTLFMNWCCLGFVAFAYSVKARDRKMVGDIIGAQSYASTAKCLNIWALVLGLLLIIAFIIVCTTGSLVIFQAVLQLIKDYRGY,SEQID NO:1。
2.如权利要求1所述的应用,其特征在于,所述IFITM3抗病毒蛋白为猪源IFITM3抗病毒蛋白。
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