CN111440228B - 多种亚型流感病毒ha2蛋白共同抗原表位、抗体、鉴定方法和应用 - Google Patents
多种亚型流感病毒ha2蛋白共同抗原表位、抗体、鉴定方法和应用 Download PDFInfo
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Abstract
本发明涉及多种亚型流感病毒HA2蛋白共同抗原表位、抗体、鉴定方法和应用,该抗原表位能够与H1、H3、H4、H5、H6、H7、H8、H9和H10亚型流感病毒的免疫血清结合。选择一株H5N1亚型流感毒株S,根据其HA2蛋白的序列设计长度为20个氨基酸且互相重叠10个氨基酸的18条多肽,人工合成后点样至改性硅胶摸制成多肽芯片,使用针对不同亚型IAV的抗血清筛选通用表位。构建该表位突变的重组病毒,通过蛋白质印迹分析表位与免疫血清的结合能力。结果表明,HA2蛋白上485‑FYHKCDNECME‑495具有与H1,H3,H4,H5,H6,H7,H8,H9和H10亚型IAV抗血清的广谱结合活性。重组病毒中的K/D突变导致血清结合活性降低,表明它们是血清结合活性的关键氨基酸。该肽段还包含HLA‑DR1限制性CD4 T细胞表位。
Description
技术领域
本发明涉及生物技术领域,具体涉及多种亚型流感病毒HA2蛋白共同抗原表位、抗体、鉴定方法和应用。
背景技术
A型流感病毒(Influenza Avirus,IAV)由8个分节段的单股负链RNA组成,易发生抗原漂移和抗原转变。目前已发现有18种HA亚型和11种NA亚型,其中禽流感病毒(Avianinfluenza virus,AIV)包括16种HA亚型和9种NA亚型。根据遗传进化分析,这18种HA亚型可被分为2组,Group 1为H1、H2、H5、H6、H8、H9、H11、H12、H13、H16和H17;Group 2为H3、H4、H7、H10、H14和H15。由于持续存在的地方性和季节性流行,同一亚型的IAV的抗原性也发生了较大差异,逐渐衍生出不同的谱系。IAV的监测和防控也变得越来越困难。
IAV从动物传播给人类事件的不断发生以及流感疫苗短暂的时效性,促进了流感疫苗的开发、检测方法的改进和新型抗病毒治疗方法的发展。近年来,研究团队鉴定了许多新的、高保守表位,用于诱导产生中和IAV的抗体或建立能够检测不同亚型IAV的方法。Dreyfus等在H2亚型IAV的HA2蛋白中鉴定了能够产生广泛中和H1,H2,H5,H6和H9亚型IAV抗体的表位。Friesen等则是发现位于HA茎区的CR8043表位能够刺激机体产生中和Group 2的IAV的抗体。Velumani等发现一个能与人源和禽源H5亚型IAV都特异性结合的HA蛋白表位CNTKCQTP,并以此建立了针对H5N1流感病毒抗体的高度特异性的检测方法。A型流感病毒HA1蛋白上鉴定出的抗原表位往往是亚型特异性的,不具有广谱性。
发明内容
有鉴于此,本发明实施例提供一种多种亚型流感病毒HA2蛋白共同抗原表位、抗体、鉴定方法和应用,主要目的是鉴定多种亚型流感病毒HA2蛋白共同抗原。
而HA2蛋白在不同亚型流感病毒间比较保守,所获得的抗原表位可作为广谱性靶标。抗原表位鉴定可使用生物信息学法、单克隆抗体反向识别法、多肽微阵列芯片技术等方法。其中多肽芯片技术常与肽序列扫描法相结合,即通过将互相重叠部分氨基酸的多肽与血清抗体反应,筛选出所需的优势表位。如Liu等运用肽序列扫描法成功鉴定了克里米亚刚果出血热病毒NP2蛋白的5个优势表位。因此,本申请通过多肽芯片技术和肽序列扫描法对H5亚型IAVHA2蛋白进行鉴定,以期获得新的广谱性的抗原表位。
为实现本发明的目的,本发明采用的技术方案是:
第一方面,提供多种亚型流感病毒HA2蛋白共同抗原表位,所述的多种亚型流感病毒HA2蛋白共同优势抗原表位的氨基酸序列如:FYHKCDNECME。
具体的,所述的多种亚型流感病毒的毒株类型包括H1、H3、H4、H5、H6、H7、H8、H9、H10。
第二方面,提供多种亚型流感病毒HA2蛋白共同抗原表位的鉴定方法,包括:
(1)初步确定HA2蛋白表位区域:根据H5N1亚型流感毒株S的HA2蛋白序列设计并合成长度为20个氨基酸且互相重叠10个氨基酸的18条多肽,合成后点样至改性硅胶膜,制成多肽芯片,通过血清学方法筛选目的肽段,使用针对不同亚型IAV的抗血清筛选通用表位,并且通过蛋白印迹分析能够验证H1、H3、H4、H5、H6、H7、H8、H9和H10亚型流感病毒的共同抗原表位位于H5-14肽(KELGNGCFEFYHKCDNECME);
(2)确定优势表位序列:将所述14th肽(KELGNGCFEFYHKCDNECME)细切为4条互相重叠8个氨基酸的多肽14-1(KELGNGCFEFY)、14-2(GNGCFEFYHKC)、14-3(CFEFYHKCDNE)和14-4(FYHKCDNECME),点样至改性硅胶膜,制成多肽芯片;
构建通用表位突变的重组病毒,通过蛋白质印迹分析表位与免疫血清的结合能力,确定出最短优势表位序列,所述最短优势表位序列为序列:FYHKCDNECME;
结果表明,HA2蛋白上485-FYHKCDNECME-495具有与H1,H3,H4,H5,H6,H7,H8,H9和H10亚型IAV抗血清的广谱结合活性。
进一步的,所述多肽芯片试验即将不同亚型的IAV血清(H1、H3、H4、H5、H6、H7、H8、H9、H10)与抗体稀释液以1:50的比例稀释后,将其加入至多肽芯片中,于恒温振荡器中进行孵育(37℃,500r/min,30min),用PBST洗涤后加羊抗鸡IgY酶标二抗和化学发光底物,检测化学发光强度计算信噪比(SNR)。SNR≥2时为阳性响应。
进一步的,根据多肽分段截短后的芯片检测结果,选择SNR值较高肽段,设计不同的突变和缺失模式构建转录表达载体,通过反向遗传技术拯救病毒。将病毒以MOI=0.1的剂量感染鸡胚成纤维细胞,12h收样处理后经12%SDS-PAGE电泳,通过Western-blotting确定抗血清与相应位点的反应性。
在一些实施例中,构建表位突变的重组病毒,包括:
通过序列分析,根据14th肽(FYHKCDNECME)中相对保守的一段序列“YHKCD”分别设计16种缺失和突变模式,并将改造后的HA基因克隆至pHW2000转录表达载体,同时以S毒株的其余7个基因(PB2、PB1、PA、NP、NA、M和NS)为骨架,拯救重组病毒。
在所拯救的重组病毒中只有S-Y-G、S-H-G、S-K-G和S-D-G具有HA效价并能够在10d的SPF鸡胚中稳定复制;拯救的病毒S-Y-G、S-H-G、S-K-G和S-D-G分别以MOI=0.1的剂量感染原代鸡胚成纤维细胞12h,其制成的蛋白样通过Western-Blotting可验证,14th肽偶联BSA的高免血清与S-K-G和S-D-G病毒的HA2蛋白的结合能力显著下降;
用于S毒株HA基因突变/缺失的引物分别为Y-G F、Y-G R、H-G F、H-G R、K-G F、K-GR、C-G F、C-G R、D-G F、D-G R、R△YHKCD、F△YHKCD、R△HKCD、F△HKCD、R△YKCD、F△YKCD、R△YHCD、F△YHCD、R△YHKD、F△YHKD、R△YHKC、F△YHKC、R△Y、F△Y、R△H、F△H、R△K、F△K、R△C、F△C、R△D和F△D,其序列分别如表4所示;
在一些实施例中,通过表位数据库分析预测,所述优势表位序列(FYHKCDNECME)包含HLA-DR1限制性CD4 T细胞表位。
第三方面,提供多种亚型流感病毒抗体,所述抗体是所述的多种亚型流感病毒HA2蛋白共同抗原表位序列偶联BSA,按照免疫程序免疫动物获得的抗血清,所述抗血清即为所述多种亚型流感病毒抗体。
第四方面,多种亚型流感病毒抗体和抗原的鉴别方法,包括采用所述的多种亚型流感病毒HA2蛋白共同抗原表位序列作为抗原去检测多种亚型流感病毒抗体;采用权利要求6所述的抗体检测多种亚型流感病毒。
第五方面,多种亚型流感病毒疫苗,所述疫苗包括所述的多种亚型流感病毒HA2蛋白共同抗原表位经过包括缺失或插入修饰构建的标记疫苗。
第六方面,所述的多种亚型流感病毒HA2蛋白共同抗原表位在制备多种亚型流感病毒抗原、疫苗中的应用。
所述的多种亚型流感病毒HA2蛋白共同抗原表位在鉴定多种亚型流感病毒抗原、抗体中的应用。
相对于现有技术,本申请取得了以下有益效果:常见的鉴定HA蛋白抗原表位方法有氨基酸突变方法、噬菌体展示肽、生物酶消化法,这些方法通常需要配备有相应的单克隆抗体。本申请在多肽芯片技术与肽序列扫描法的基础上,建立了HA2蛋白的优势抗原表位筛选方法,提高了灵敏度,缩减了制备单克隆抗体的步骤。在目前已知的HA蛋白广谱性抗原表位的研究中,大部分表位只能结合某一个Group或者某几个亚型的流感病毒。本申请中所鉴定的广谱性抗原表位FYHKCDNECME,同时能够结合H1、H3、H4、H5、H6、H7、H8、H9和H10亚型的流感病毒免疫血清。
附图说明
图1为芯片点样和检测示意图;
图2不同亚型流感病毒免疫血清与多肽结合能力分析(虚线表示SNR=2。H5-01:A/Mallard/Huadong/S/2005高免血清;H5-02:A/Chicken/Huadong/yz1111/2016高免血清;H5-03:A/Chicken/Huadong/dt0303/2016高免血清;H5-04:Re-11血清;H5-04:Re-12血清;H7-01:A/Chicken/Jiangsu/W1-8/15高免血清;H7-02:A/Chicken/Huadong/JD/17高免血清;H9-01:A/Chicken/Shanghai/F/98高免血清;H9-02:A/Chicken/Taixing/10/2010高免血清);
图3为不同亚型流感病毒与14th肽免疫血清的结合能力分析(H5-01:A/Mallard/Huadong/S/2005;H5-02:A/Chicken/Huadong/yz1111/2016;H5-03:A/Chicken/Huadong/dt0303/2016;H7-01:A/Chicken/Jiangsu/W1-8/15;H7-02:A/Chicken/Huadong/JD/17;H9-01:A/Chicken/Shanghai/F/98;H9-02:A/Chicken/Taixing/10/2010);
图4为14th和14-4肽在H5亚型流感病毒S株模似血凝素蛋白晶体结构中的定位;
图5为突变后重组病毒HA2蛋白的蛋白印迹分析。
具体实施方式
实施例
1.血清制备
表1 13株流感病毒的背景信息及其免疫血清的血凝抑制效价
全病毒免疫鸡血清的制备:IAV用甲醛灭活后,与Tween 80和白油司本混合后制成油乳剂灭活苗,以0.3mL/只的剂量免疫3周龄的SPF鸡。2周后以相同的剂量进行二次免疫。二次免疫后的第二周采集血清测,血凝抑制(HI)效价大于等于6log2备用(详见表1)。
多肽免疫鸡血清的制备:将100μg偶联BSA的肽段(上海GL Biochem公司合成)溶于0.1mL生理盐水中与等量弗氏完全佐剂(美国Sigma公司)混合后,皮下免疫21日龄的SPF鸡。2周后,将上述BSA偶联的肽段溶于0.1mL生理盐水中与等量弗氏不完全佐剂混合后进行二次免疫。二次免疫后的第2周后收集血清,芯片点样,阳性,收集血清,-70℃冰箱保存备用。
2.多肽芯片制备
将H5亚型流感病毒A/Mallard/Huadong/S/2005(S,GenBank ID:EU195389-EU195396)的HA2蛋白按照推导出的氨基酸序列合成互相重叠的多肽(相邻的2条多肽重叠10个氨基酸,于上海GL Biochem公司合成),点样至改性硅胶摸(modified silica gelfilm,iPDMS,由苏州偲聚生物材料有限公司制备),制成多肽芯片(图1)。除H5第11th多肽不能合成外,一共合成18条多肽。为验证其他亚型的流感病毒相应位置的14th多肽是否具有相似的血清结合特性,随机选取一株H7亚型流感病毒(A/Chicken/Huadong/JD/17)的氨基酸序列,合成其HA2蛋白上的14th多肽。为进一步验证广谱性表位的关键氨基酸,14th多肽被细切为互相重叠8个氨基酸的4条短肽。多肽信息详见表2。
表2合成的流感病毒HA2多肽的序列
3.芯片实验
将血清样品在血清稀释缓冲液(GuardianTM Peroxidase ConjugateStabilizer/Diluent)中按1:50的比例稀释;在每个微阵列孔中加入200μL稀释液,在振荡器上孵育30min(500r/min,37℃);用TBST(20mM的Tris-HCl,pH 6.8,137mM的NaCl,0.1%Tween20)洗涤芯片板3次,将芯片板在吸水纸上拍干;加入100μL1:10000稀释的辣根过氧化物酶(HRP)标记的羊抗鸡IgY,在振荡器上孵育30min(500r/min,37℃);重复上述洗涤步骤;将15μL化学发光底物加入到微阵列孔中,使用LAS4000成像系统的CCD相机捕获微阵列孔中每个多肽点的化学发光信号;将捕获到的信号保存为TIFF格式的图像,然后使用GenePixPro 6.0软件处理每个多肽点化学发光强度;将化学发光强度转化为信噪比(signal-to-noise ratio,SNR)。SNR=(信号强度-背景强度)/背景强度(SNR≥2视为阳性响应)。
在所有肽中,H5亚型流感病毒14th肽能与所有亚型流感病毒的免疫血清结合,其SNR值均在2以上。而以H7亚型流感病毒相应14th肽仅能与少数亚型流感病毒的免疫血清结合,说明并不是所有亚型流感病毒相应多肽都具有广谱的结合特性。在进一步的验证过程中,14th肽的14-4肽与不同亚型血清之间的SNR值均大于或等于2,表明广谱性血清结合作用的关键氨基酸存在于14-4肽中(图2)。
4.流感病毒14th肽的广谱性鉴定
分别将H1、H3、H4、H5、H6、H7、H8、H9和H10亚型流感病毒以MO1=0.1的剂量接种CEF细胞,置于37C温箱1h。而后用PBS(pH 7.2)洗涤细胞2次,然后加入含有1%FBS的M199培养基。12h后,将细胞用预冷的PBS(4℃)洗涤一次,并加以适量的RIPA Lysis Buffer(strong),于冰上裂解15-20min。将细胞样收集于指形管中,在4C环境中离心10min(12000r/min)。取适量上清,加入蛋白质上样缓冲液,于100℃下煮沸6-8min,然后于-40℃保存。将制备好的蛋白质样于12%SDS-PAGE电泳,并转膜至PVDF膜上,而后用5%的脱脂奶常温封闭1.5h。用TBST洗3次,每次5min,再加入一抗(免疫14th-BSA的鸡血清,1:1000稀释),4℃过夜。重复洗涤步骤,加入HRP标记的羊抗鸡二抗(1:5000稀释),常温作用1h。与此同时,相应位置的β-actin条带用一抗(monoclonal anti-β-actin clone,1:3000稀释)和二抗(HRP标记的羊抗鼠IgG,1:5000稀释)进行孵育。最后使用化学发光成像分析系统分析蛋白印迹。实验结果表明,14th肽的免疫血清能与上述不同亚型的流感病毒的HA2蛋白均产生特异性反应(图3)。
5.流感病毒14th肽广谱性表位的关键氨基酸鉴定
表3 14-4肽在不同亚型A型流感病毒中氨基酸序列变化情况
a氨基酸变化频率。
b氨基酸变化模式。
通过比对不同亚型IAV HA2蛋白序列(表3),14-4肽的序列在不同亚型流感病毒中不完全保守,其中“--HKC---CM-”这5个氨基酸相对比较保守。在HA蛋白3D模拟图中(图4),α-螺旋和无规则卷曲结构组成了整个肽段。“CM”位于不易形成表位的α螺旋区域,因此排除。基于连续氨基酸(YHKCD),设计16种突变/缺失模式。
根据说明书,使用Mut Express II Fast Mutagenesis Kit V2将S株的HA基因的5个氨基酸(YHKCD)分别突变为甘氨酸(G)。使用Overlap方法将S株的HA基因的5个氨基酸进行全缺和部分缺失。将改造后的HA基因插入pHW2000转录/表达载体中。通过测序(上海BGI公司)确认构建成功。引物信息见表4。
重组病毒通过Hoffmann等建立的8质粒转染法进行拯救。转染前18-24h将HEC293T和M90细胞按1.5:1的比例铺在六孔板中,并在含有10%FBS的DMEM培养基中培养。转染前1h更换新鲜培养基,具体转染步骤参照PolyJetTM转染试剂说明书。转染后15h,将细胞培养液更换为含有2μg/mL的DMEM基础培养基。转染后48-72小时,将细胞于-70C冻融3次,以0.25mL/胚的剂量接种7日龄的SPF鸡胚。72h后,通过血凝试验(HA)测定鸡胚尿囊液效价。若有效价,提取病毒尿囊液的RNA,反转录为cDNA,而后通过PCR扩增相关基因并送至测序验证病毒序列,测序结果与目标序列相一致则证明重组病毒构建成功。将重组病毒尿囊液以0.25mL/胚的剂量接种10日龄鸡胚扩增病毒。结果表明,只有将“YHKCD”中的“YHKD”分别突变为G时,才能获得重组病毒,而其他任何单突变组合、组合突变、缺失突变均不能成功拯救重组病毒。所获提的重组病毒能够在10日龄鸡胚中稳定复制,效价为6log2,将其分别命名为S-Y-G、S-H-G、S-K-G和S-D-G。详见表5。
将提前制备的CEF用0.25%胰酶消化后均匀铺至96孔板,每孔约4-5×104个细胞。用PBS清洗96板内的细胞3次。与此同时,将病毒用M199基础培养基进行10倍稀释,共稀释10个梯度(10-1~10-10)。将10-3~10-10梯度,以100μL/孔的剂量接种于96孔板内,每个梯度做8个重复,于37℃的温箱放置1h。而后,用PBS洗涤3次,加入含有2μg/mL的M199基础培养基,每孔100μL。72h后,测定每个孔的上清液的HA效价,统计阳性感染孔的数量。TCID50根据Reed-Muench等建立的方法计算。详见表5。
将重组病毒以MOI=0.1的剂量感染CEF,12h后收集相应的细胞蛋白样品进行Western-blotting分析(具体步骤参照实施方式4)。结果显示当氨基酸K/D分别突变为G时,重组病毒与14th肽免疫血清的结合活性显着降低(图5),表明这两个氨基酸为关键的氨基酸位点。尽管H5-14肽和H7-14肽均具有核心氨基酸HKCD,但H5的14th肽比H7的14th肽具有更广谱的血清结合活性。这些数据表明,14-4肽广谱表位由HKCD和相邻氨基酸组成。
6细胞表位预测
通过免疫表位数据库(IEDB)预测HA2蛋白的T/B细胞表位。结果表明,本研究所鉴定的肽段FYHKCDNECME包含具有HLA-DR1限制性的CD4 T细胞表位和B细胞表位,而B细胞表位已通过测定血清结合活性被证明。
综上所述,肽段FYHKCDNECME被鉴定为具有与不同亚型IAVs血清结合能力的广谱性表位,为B细胞表位,且还预测包含HLA-DR1限制性的T细胞表位。该表位可以用作诊断或疫苗开发的新型通用靶标。
表4定点突变和缺失的引物序列
F为上游引物;R为下游引物;△为缺失。
表5重组病毒血凝效价和TCID50测定
序列表
<110> 扬州大学
<120> 多种亚型流感病毒HA2蛋白共同抗原表位、抗体、鉴定方法和应用
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Claims (8)
1.多种亚型流感病毒HA2蛋白共同抗原表位肽,其特征在于,所述的多种亚型流感病毒HA2蛋白共同优势抗原表位肽的氨基酸序列为SEQ ID NO:1。
2.多种亚型流感病毒HA2蛋白共同抗原表位肽的鉴定方法,其特征在于,包括:
(1)初步确定HA2蛋白表位区域:根据H5N1亚型流感毒株S的HA2蛋白序列设计并合成长度为20个氨基酸且互相重叠10个氨基酸的18条多肽,合成后点样至改性硅胶膜,制成多肽芯片,通过血清学方法筛选目的肽段,使用针对不同亚型IAV的抗血清筛选通用表位,并且通过蛋白印迹分析能够验证H1、H3、H4、H5、H6、H7、H8、H9和H10亚型流感病毒的共同抗原表位肽位于H5-14肽序列为SEQ ID NO:2,即14th肽;
(2)确定优势表位序列:将所述14th肽细切为4条互相重叠8个氨基酸的多肽,4个多肽序列分别为SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:1,点样至改性硅胶膜,制成多肽芯片;
构建通用表位突变的重组病毒,通过蛋白质印迹分析表位与免疫血清的结合能力,确定出最短优势表位序列,所述最短优势表位序列为SEQ ID NO:1;
结果表明,HA2蛋白上485至495位序列SEQ ID NO:1具有与H1,H3,H4,H5,H6,H7,H8,H9和H10亚型IAV抗血清的广谱结合活性。
3.根据权利要求2所述的多种亚型流感病毒HA2蛋白共同抗原表位肽的鉴定方法,其特征在于,构建表位突变的重组病毒,包括:
通过序列分析,根据序列SEQ ID NO:1中相对保守的一段序列“YHKCD”分别设计16种缺失和突变模式,并将改造后的HA基因克隆至pHW2000转录表达载体,同时以S毒株的其余7个基因PB2、PB1、PA、NP、NA、M和NS为骨架,拯救重组病毒。
4.根据权利要求3所述的多种亚型流感病毒HA2蛋白共同抗原表位肽的鉴定方法,其特征在于,在所拯救的重组病毒中只有S-Y-G、S-H-G、S-K-G和S-D-G具有HA效价并能够在10 d的SPF鸡胚中稳定复制;拯救的病毒S-Y-G、S-H-G、S-K-G和S-D-G分别以MOI=0.1的剂量感染原代鸡胚成纤维细胞12 h,其制成的蛋白样通过Western-Blotting可验证,14th肽偶联BSA的高免血清与S-K-G和S-D-G病毒的HA2蛋白的结合能力显著下降;
和/或,用于S毒株HA基因突变/缺失的引物分别为Y-G F、Y-G R、H-G F、H-G R、K-G F、K-G R、C-G F、C-G R、D-G F、D-G R、R△YHKCD、F△YHKCD、R△HKCD、F△HKCD、R△YKCD、F△YKCD、R△YHCD、F△YHCD、R△YHKD、F△YHKD、R△YHKC、F△YHKC、R△Y、F△Y、R△H、F△H、R△K、F△K、R△C、F△C、R△D和F△D,其序列分别为SEQ ID NO:6至SEQ ID NO:37;
和/或,通过表位数据库分析预测,优势表位序列SEQ ID NO:1包含HLA-DR1限制性CD4T细胞表位。
5.多种亚型流感病毒抗体,其特征在于,所述抗体是采用权利要求1所述的多种亚型流感病毒HA2蛋白共同抗原表位肽序列偶联BSA,按照免疫程序免疫动物获得的抗血清,所述抗血清即为所述多种亚型流感病毒抗体。
6.多种亚型流感病毒疫苗,其特征在于,所述疫苗包括权利要求1所述的多种亚型流感病毒HA2蛋白共同抗原表位肽。
7.根据权利要求1所述的多种亚型流感病毒HA2蛋白共同抗原表位肽在制备多种亚型流感病毒抗原、疫苗中的应用。
8.根据权利要求1所述的多种亚型流感病毒HA2蛋白共同抗原表位肽在制备鉴定多种亚型流感病毒抗原、抗体的试剂中的应用。
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