CN111825763B - 流感病毒ha蛋白茎部特异性单克隆抗体及其制备方法和应用 - Google Patents
流感病毒ha蛋白茎部特异性单克隆抗体及其制备方法和应用 Download PDFInfo
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Abstract
本发明属于生物技术领域，涉及一种识别流感病毒HA茎部的单克隆抗体、抗原表位、编码基因及其应用。本发明提供一株流感病毒HA茎部特异性单克隆抗体，包括重链可变区和轻链可变区，重链可变区的核苷酸与氨基酸序列分别如SEQ ID NO.1‑2所示；轻链可变区的核苷酸与氨基酸序列分别如SEQ ID NO.6‑7所示。该单克隆抗体特异性识别HA蛋白茎部以第431位W、433位Y、437位L为核心基序的抗原表位。该单克隆抗体由H7N9亚型禽流感病毒HA蛋白作为抗原免疫小鼠得到，亲和力高、敏感性强，与group 1和group 2的流感病毒具有良好的交叉反应性，在流感病毒的广谱免疫学检测方面具有较高的应用价值。
Description
技术领域
本发明涉及一种流感病毒HA茎部特异性单克隆抗体及其制备方法和应用，属于生物技术领域。
背景技术
A型流感病毒(以下称流感病毒)是造成人与动物流感的重要病原。流感病毒可分为两个群(group 1与group 2)，包含的亚型众多。根据病毒囊膜表面糖蛋白血凝素(HA)与神经氨酸酶(NA)的抗原性差异分为18个HA亚型(H1-H18)与11个NA亚型(N1-N11)。流感病毒为单股正链RNA病毒，基因组分为8个节段。因此，在流感病毒的感染过程中，不同亚型病毒之间的基因重配频繁发生，且在机体的免疫压力下，病毒的HA蛋白会发生位点突变，这两个因素共同造成流感病毒变异速度快、变异特征复杂、基因型与亚型众多等特性。
HA蛋白是位于流感病毒囊膜表面的糖蛋白，在病毒与受体结合与诱导保护性免疫等方面发挥重要的作用。HA蛋白分为头部与茎部两个主要的结构域，头部含有受体结合位点与大部分的抗原表位，在不同亚型病毒之间变异较大，属于HA蛋白的高变区；茎部主要介导细胞融合，在不同亚型病毒之间较为保守，是HA蛋白的保守区。因此，HA茎部也是通用流感疫苗与广谱病毒检测制剂的主要靶点。
单克隆抗体在流感病毒的免疫学检测中发挥越来越重要的作用。由于HA头部含有大部分免疫优势表位，因此目前针对HA蛋白的单克隆抗体大多识别HA头部的单一抗原表位。针对HA头部的单克隆抗体只能特异性识别某一特定亚型的病毒，不具备对其他亚型的交叉反应性，因此检测覆盖面较窄；而HA茎部特异性单克隆抗体识别的是保守表位，能够与不同亚型的病毒反应，在流感病毒的广谱检测方面更有优势。但是，HA茎部含有的抗原表位较少，且大多不是免疫优势表位，因此，获得HA茎部特异性单克隆抗体有一定难度。
发明内容
本发明解决了HA茎部特异性抗体的制备与临床上流感病毒的广谱检测问题，提供一种流感病毒HA茎部特异性单克隆抗体及其制备方法和应用，选用H7N9亚型禽流感病毒的HA蛋白作为免疫原，获得了一株针对HA茎部的单克隆抗体，定位了其识别的抗原表位，测定了编码抗体重链与轻链可变区的序列，且证实该抗体与group 1及group 2的不同亚型流感病毒具有良好的交叉反应性。
为解决上述技术问题，本发明提供一株流感病毒HA茎部特异性单克隆抗体，该抗体包含重链与轻链；所述的重链与轻链均包括可变区，所述的重链可变区的核苷酸序列为以下任一种：
(1)如SEQ ID NO.1所示；
(2)SEQ ID NO.1所示序列的互补序列；
(3)在SEQ ID NO.1所示序列及其互补序列的基础上，经一个或多个核苷酸的缺失、突变、插入得到的编码相似功能蛋白的核苷酸序列；
(4)与SEQ ID NO.1所示序列及其互补序列具有至少70％同源性且编码相同功能蛋白质的核苷酸序列；
所述的重链可变区的氨基酸序列为以下任一种：
(1)如SEQ ID NO.2所示；
(2)在SEQ ID NO.2所示序列的基础上，经一个或多个氨基酸的缺失、替换、插入得到的具有相似功能的氨基酸序列；
(3)与SEQ ID NO.2所示序列具有至少70％同源性且具有相同功能的氨基酸序列；
所述的轻链可变区的核苷酸序列为以下任一种：
(1)如SEQ ID NO.6所示；
(2)SEQ ID NO.6所示序列的互补序列；
(3)在SEQ ID NO.6所示序列及其互补序列的基础上，经一个或多个核苷酸的缺失、突变、插入得到的编码相似功能蛋白的核苷酸序列；
(4)与SEQ ID NO.6所示序列及其互补序列具有至少70％同源性且编码相同功能蛋白质的核苷酸序列；
所述的轻链可变区的氨基酸序列为以下任一种：
(1)如SEQ ID NO.7所示；
(2)在SEQ ID NO.7所示序列的基础上，经一个或多个氨基酸的缺失、替换、插入得到的具有相似功能的氨基酸序列；
(3)与SEQ ID NO.7所示序列具有至少70％同源性且具有相同功能的氨基酸序列。
优选地，所述的重链可变区核苷酸与氨基酸序列分别如SEQ ID NO.1与SEQ IDNO.2所示，所述的轻链可变区核苷酸与氨基酸序列分别如SEQ ID NO.6与SEQ ID NO.7所示。
优选地，所述的重链可变区包括互补决定区HCDR1、HCDR2、HCDR3，其氨基酸序列分别如SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5所示；所述的轻链可变区包括互补决定区LCDR1、LCDR2、LCDR3，其氨基酸序列分别如SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10所示。
优选地，通过抗体序列测定，鉴定所述抗体的重链为IgG1亚型，轻链为Kappa亚型。
本发明还提供产生上述单克隆抗体的杂交瘤细胞。
本发明还提供上述的单克隆抗体识别的抗原表位，用噬菌体表面展示随机多肽文库筛选技术，鉴定所述单克隆抗体识别的是包含H7 HA蛋白第431位W、433位Y、437位L(及不同亚型流感病毒中的相应位点)的表位。
本发明还提供上述的单克隆抗体的制备方法，在杆状病毒表系统中表达H7N9 HA蛋白，并以其作为抗原免疫小鼠，利用常规的杂交瘤细胞技术制备HA特异性单克隆抗体。
本发明用ELISA与IFA方法，鉴定所述单克隆抗体与group 1及group 2中不同亚型的流感病毒具有良好的交叉反应性。
本发明还提供上述的单克隆抗体在制备A型流感病毒检测试剂中的应用。
本发明还提供上述的抗原表位在制备流感疫苗与病毒检测试剂中的应用。
本发明所达到的有益效果：
本发明用H7N9 HA蛋白作为抗原免疫小鼠，获得了HA茎部特异性单克隆抗体；鉴定了HA蛋白茎部一个新的保守的抗原表位，对表位疫苗与检测制剂研制有参考意义；获得了所述单克隆抗体的轻重链序列，证明该抗体序列的唯一性，且为生产基因重组抗体奠定基础；所述单克隆抗体对group 1与group 2流感病毒显示广谱反应性，可作为通用性流感病毒检测的制剂。
附图说明
图1为表达H7N9 HA蛋白重组杆状病毒构建结果，右图为重组杆状病毒感染的sf9细胞，左图为野生型杆状病毒感染的sf9细胞；
图2为纯化的HA蛋白SDS-PAGE结果；
图3为HA蛋白的免疫印迹检测结果；
图4为不同浓度抗体与H7N9 HA蛋白反应性的ELISA结果；
图5为单克隆抗体与H7N9病毒感染细胞的IFA结果；
图6为单克隆抗体识别抗原表位的鉴定结果；
图7为抗体与流感病毒HA蛋白反应的ELISA结果；
图8为单克隆抗体与不同亚型流感病毒HA蛋白最低结合浓度测定结果；
图9为单克隆抗体与不同亚型流感病毒的IFA检测结果。
具体实施方式
下面结合具体的实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外，应理解，阅读了本发明讲授的内容之后，本领域的技术人员可以对本发明作各种改动或修改，这些等价形式同样落于申请所附权利要求书所限定的范围。
实施例1.HA蛋白特异性单克隆抗体的制备
1.重组H7N9 HA蛋白的表达
1.1表达H7N9 HA基因的重组杆状病毒构建
PCR扩增H7N9(A/chicken/Guangdong/GD15/2016,GD15)HA基因的胞外区，在C-端添加6×His标签，两端添加NotI与XbaI酶切位点，并连接至转移载体pVL1393中，构建转移质粒(pVL-GDHA)。中间转移质粒经酶切、测序验证后，与苜蓿银纹夜蛾多核型多角体病毒线性化基因组DNA共转染昆虫细胞sf9，拯救重组杆状病毒。
病毒拯救的简要程序为：
(1)转染复合物配制：在一个离心管中，加入0.5mL Sf900 III SFM无血清培养基与5μL DNA shuttle转染试剂，混合均匀；在另一个离心管中，加入30μL sapphirebaculovirus DNA，2μgpVL-GDHA质粒与0.5mL Sf900 III SFM无血清培养基，混合均匀；将两管合并为一管，轻轻混匀，室温静置20min，形成转染复合物。
(2)细胞转染：在6孔细胞板中加入sf9细胞，1×106细胞/孔，27℃培养1h；去除培养液，沿孔壁缓缓加入1mL Sf900 III SFM无血清培养基洗涤细胞单层；加入转染复合物，27℃孵育4～5h；去除转染复合物，每孔加2mL含5％血清的Excell-420培养基，继续培养5天。
(3)产物收获与鉴定：5天后，收获细胞培养上清；同时，将转染细胞转移至96孔板，进行间接免疫荧光试验(IFA)检测杆状病毒是否拯救成功。拯救成功的重组杆状病毒命名为rBac-GDHA，结果如图1所示，右图为重组杆状病毒感染的sf9细胞，可见绿色荧光，左图为野生型杆状病毒感染的sf9细胞，检测不到荧光。
1.2 HA蛋白的大量表达与纯化
(1)按照1.0×106/mL的密度接种sf9细胞至培养瓶，培养体积为500mL，120rpm振摇培养。
(2)按0.5mL病毒粒子/50mL细胞悬液的比例接种病毒粒子，27℃培养4天；
(3)4000rpm离心10min，去上清，加PBS洗涤细胞三遍。
(4)加入裂解液4℃裂解30min，11000rpm离心20min，分离上清及沉淀。
(5)用浓HCl调节上清pH至8.0。
(6)用镍柱纯化HA蛋白。
(7)分别以不同浓度的咪唑洗脱，分段收集洗脱液至G250检测液不变色。
(8)以3倍柱体积去离子水洗涤柱料，以20％乙醇封柱。
(9)对收集的洗脱液进行SDS-PAGE和Western blot检测，结果如图2和图3所示，图2为纯化的HA蛋白SDS-PAGE结果，可见分子量约为66kDa的目的条带；图3为HA蛋白的免疫印迹检测，可见HA蛋白以单体、二聚体与三聚体形式存在。
2.HA单克隆抗体制备
用纯化的HA蛋白免疫小鼠进行单克隆抗体制备。10μg纯化的HA蛋白与佐剂配伍，免疫8只SPF级小鼠，并间隔2周进行加强免疫，共3次。定期监测血清抗体效价并据此进行加强免疫。利用常规的杂交瘤细胞技术进行单克隆抗体的制备。用筛选的杂交瘤细胞注射小鼠制备腹水，用于抗体的纯化。
这部分的实验结果显示，成功构建了一株表达H7N9 HA蛋白的重组杆状病毒，并进行了HA蛋白的大量表达与纯化。用HA蛋白作为抗原免疫小鼠，用杂交瘤技术获得了一株与HA蛋白反应的单克隆抗体(5D3 1B5)。
实施例2.单克隆抗体的性质鉴定
1.单克隆抗体与HA蛋白的亲和力测定
将纯化的单克隆抗体进行倍比稀释(500、1000、5000、10000、100000、1000000倍)，用ELISA方法检测不同稀释度的抗体与HA蛋白的反应性，简要流程为：
(1)用纯化的HA蛋白(0.25μg/mL)包被ELISA板，4℃孵育过夜。
(2)倾去包被液，每孔加200μL的含5％脱脂乳的PBST，37℃封闭1h。
(3)PBST洗涤5遍，每孔加100μL倍比稀释的抗体，37℃孵育1h。
(4)PBST洗涤5遍，每孔加100μL HRP标记的羊抗鼠IgG二抗(1:2000)，37℃孵育1h。
(5)PBST洗涤5遍，每孔加100μL TMB底物，室温避光显色10min。
(6)每孔加50μL终止液，酶标仪读取OD450。
2.间接免疫荧光试验(IFA)检测抗体与病毒的反应性
(1)用H7N9 GD15株接种鸡胚成纤维细胞(CEF)，培养24h。
(2)用甲醇固定细胞，每孔加倍比稀释的单克隆抗体，37℃孵育1h。
(3)PBS洗涤3遍，每孔加羊抗鼠绿色荧光二抗，37℃孵育1h。
(4)PBS洗涤3遍，荧光显微镜下观察。
这部分的实验结果说明：图4为不同浓度抗体与H7N9 HA蛋白反应性的ELISA结果，5D3 1B5与H7N9 HA蛋白的最小结合浓度在ng级别，显示其亲和力较强；图5为单克隆抗体与H7N9病毒感染细胞的IFA结果，抗体(1:8000)与病毒感染的细胞反应，显示其能识别病毒感染状态下的HA蛋白且反应性较强。
实施例3.单克隆抗体的抗原表位鉴定
用噬菌体表面展示随机多肽文库筛选5D3 1B5识别的表位，所用文库为ph.D.TM-12Phage Display Peptide Library(NEB)，按照试剂盒说明书进行操作。提取阳性噬菌体克隆的DNA，用引物-96gIII(5’-CCCTCATAGTTAGCGTAACG-3’)进行测序，推导噬菌体中插入的12肽序列。通过与HA基因序列的比对，定位吻合位点，确定抗体识别的模拟表位。
图6为单克隆抗体识别抗原表位的鉴定结果，结果显示，选取10个阳性噬菌体克隆测序，推导的模拟多肽为AWQYSLTLPDVL，其中与HA蛋白第431位W、433位Y与437位L完全吻合，提示这三个氨基酸是5D3 1B5识别表位的核心基序。
实施例4.单克隆抗体可变区的序列测定
(1)取状态良好的杂交瘤细胞(106个)加1mL RNAiso Plus处理，再加入三氯甲烷反应后取上清液，上清液用异丙醇沉淀，沉淀干燥后用加水溶解，琼脂糖电泳检测RNA条带。
(2)根据TaKaRa反转录试剂盒操作过程进行反转录。
(3)使用百英生物杂交瘤细胞测序PCR试剂盒进行PCR，扩增轻链与重链可变区，琼脂糖电泳检测条带。
(4)PCR产物连接至pUC-19载体，转化TOP10感受态细胞。
(5)挑取过夜平板中单菌落，用M13+/M13-引物进行菌落PCR，电泳鉴定阳性克隆。
(6)随机选择6个阳性菌，阳性菌液用M13+引物测序。
结果显示，成功从杂交瘤细胞中扩增出了抗体可变区的基因条带。序列分析显示，重链可变区为IgG1亚型，轻链可变区为Kappa亚型。重链可变区中的HCDR1、HCDR2、HCDR3序列分别为GYAFSNYL(SEQ ID NO.3)、IYPGSDGT(SEQ ID NO.4)、ARGGITTGFAY(SEQ ID NO.5)；轻链可变区中的LCDR1、LCDR2、LCDR3序列分别为SSVNY(SEQ ID NO.8)、DTS(SEQ ID NO.9)、QQWSSNPPIT(SEQ ID NO.10)。
实施例5.单克隆抗体与不同亚型流感病毒的反应性
1.ELISA测定抗体与不同亚型流感病毒HA蛋白的反应性
用ELISA方法检测5D3 1B5与group 1代表亚型(H1、H2、H5、H9)与group 2代表亚型(H3、H4、H7、H15)HA蛋白的反应性，实验程序参照2.1所述。
图7为抗体与流感病毒HA蛋白反应的ELISA结果，可见其与group 1(除H2N2)及group 2流感病毒的HA蛋白均反应；图8为单克隆抗体与不同亚型流感病毒HA蛋白最低结合浓度测定结果，可见其与所测HA蛋白结合浓度均在ng级别。
2.IFA测定抗体与不同亚型流感病毒的反应性
(1)CEF细胞接种12孔板(5x106个/孔)，过夜培养至单层完全汇合。
(2)将H1N1、H5N6、H9N2与H3N2、H7N9、H15N1亚型流感病毒接种CEF，培养48h。
(3)倾去培养基，PBS清洗3遍，每孔加400μL冷甲醇，-20℃孵育10min，固定细胞。
(4)倾去固定液，室温风干，每孔加400μL 5D3 1B5(500x)，37℃孵育1h。
(5)去除一抗，PBS洗3遍，每孔加400μL羊抗鼠IgG荧光二抗(1000x)，37℃孵育1h。
(6)PBS洗3遍，荧光显微镜观察。
图9为单克隆抗体与不同亚型流感病毒的IFA检测结果，可见抗体与不同亚型流感病毒感染的细胞均反应，表明抗体能识别不同亚型的流感病毒，提示其具有良好的交叉反应性。
综上所述，本发明用H7N9 HA蛋白免疫小鼠，利用杂交瘤技术获得了一株HA蛋白特异性的单克隆抗体。该单克隆抗体的重链鉴定为IgG1亚型，轻链鉴定为Kappa亚型。该抗体特异性识别HA蛋白茎部以431W-433Y-437L为核心基序的抗原表位，说明其是茎部特异性抗体。本发明还公开了编码所述单克隆抗体重链与轻链可变区的序列，确认了单克隆抗体核苷酸与氨基酸序列的唯一性。用ELISA与IFA证实所述单克隆抗体与group 1及group 2的流感病毒具有广谱反应性。因此，本发明公开的单克隆抗体及其表位与基因序列在流感病毒广谱检测与通用疫苗研发方面均有较大的应用价值。
本发明所述的实例是对本发明的详细说明但不能限制本发明，在与本发明相当的含义和范围内的任何改变和调整，都应认为是在本发明的范围内。
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Claims (4)
1.一株流感病毒HA茎部特异性单克隆抗体，该抗体包含重链与轻链；所述的重链与轻链均包括可变区，其特征是，所述的重链可变区的氨基酸序列如SEQ ID NO. 2所示，所述的轻链可变区的氨基酸序列如SEQ ID NO. 7所示，编码所述的重链可变区的核苷酸序列如SEQ ID NO.1所示，编码所述的轻链可变区的核苷酸序列如SEQ ID NO. 6所示。
2.根据权利要求1所述的流感病毒HA茎部特异性单克隆抗体，其特征是，所述的重链可变区包括互补决定区HCDR1、HCDR2、HCDR3，其氨基酸序列分别如SEQ ID NO. 3、SEQ ID NO.4、SEQ ID NO.5所示；所述的轻链可变区包括互补决定区LCDR1、LCDR2、LCDR3，其氨基酸序列分别如SEQ ID NO. 8、SEQ ID NO. 9、SEQ ID NO. 10所示。
3.根据权利要求1所述的流感病毒HA茎部特异性单克隆抗体，其特征是，所述抗体的重链为IgG1亚型，轻链为Kappa亚型。
4.权利要求1-3任意一项所述的单克隆抗体在制备A型流感病毒检测试剂中的应用。
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| WO2017122130A1 (en) * | 2016-01-11 | 2017-07-20 | Novartis Ag | Immune-stimulating humanized monoclonal antibodies against human interleukin-2, and fusion proteins thereof |
| CN107449912A (zh) * | 2017-08-29 | 2017-12-08 | 扬州大学 | 抗h7n9亚型禽流感病毒单克隆抗体抗原表位及其筛选方法和应用 |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102164613A (zh) * | 2008-07-25 | 2011-08-24 | 生物医学研究所 | 中和抗甲型流感病毒抗体及其用途 |
| WO2017122130A1 (en) * | 2016-01-11 | 2017-07-20 | Novartis Ag | Immune-stimulating humanized monoclonal antibodies against human interleukin-2, and fusion proteins thereof |
| CN108473569A (zh) * | 2016-01-11 | 2018-08-31 | 诺华有限公司 | 针对人白介素-2的免疫刺激性人源化单克隆抗体及其融合蛋白 |
| CN107449912A (zh) * | 2017-08-29 | 2017-12-08 | 扬州大学 | 抗h7n9亚型禽流感病毒单克隆抗体抗原表位及其筛选方法和应用 |
Non-Patent Citations (2)
| Title |
|---|
| Structure and Function Analysis of an Antibody Recognizing All Influenza A Subtypes;Nicole L Kallewaard等;《Cell》;20160728;第166卷(第3期);596-608 * |
| 抗A型流感病毒广谱中和抗体及广谱通用疫苗研究进展;冯道斌等;《中华微生物学和免疫学杂志》;20181031;第38卷(第10期);782-789 * |
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