CN113527446A - 一种MERS-CoV S-RBD线性B细胞表位及其特异性识别单克隆抗体和应用 - Google Patents
一种MERS-CoV S-RBD线性B细胞表位及其特异性识别单克隆抗体和应用 Download PDFInfo
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Abstract
本发明涉及一种MERS‑CoV S‑RBD线性B细胞表位及其特异性识别单克隆抗体和应用。利用HEK 293F细胞表达系统表达MERS‑CoV S1重组蛋白，经纯化获得高纯度、具有生物学活性和免疫原性的目的蛋白。随后免疫小鼠获得单克隆抗体；利用ELISA鉴定其中一株单克隆抗体6E8可特异性识别RBD‑rFc蛋白；利用真核细胞HEK‑293T重组表达MERS‑CoVS‑RBD重组蛋白不同片段，通过Dot‑blot鉴定获得的6E8单克隆抗体特异性识别MERS‑CoV S‑RBD N端区域；利用ELISA进一步鉴定N端区域合成多肽，鉴定6E8单克隆抗体特异性识别序列为FTCSQIS的线性B细胞表位。
Description
技术领域
本发明涉及一种MERS-CoV S-RBD线性B细胞表位及其特异性识别单克隆抗体和应用，属于分子生物学和免疫学领域。
背景技术
中东呼吸综合征(Middle East Respiratory Syndrome，MERS)是由中东呼吸综合征冠状病毒(Middle East Respiratory Syndrome Coronavirus，MERS-CoV)引起的一种传染性和高致死性的呼吸系统疾病，主要表现为非典型性肺炎和急性呼吸综合征，严重者可引发肾衰竭而死亡。中东呼吸综合征冠状病毒S蛋白(棘突糖蛋白，spike glycoprotein)是三聚体状态的I型跨膜糖蛋白，位于病毒膜表面，介导病毒附着宿主细胞及病毒-细胞膜的融合，是细胞靶向性和发病机制的主要决定因子。S蛋白包含S1亚基和S2亚基两个功能子单元，其中S1亚基包含N端结构域(N-terminal domain，NTD)和独立折叠的受体结合域(receptor-binding domain，RBD)，RBD与细胞受体二肽激肽酶4(DPP4)结合，介导病毒颗粒附着于细胞。研究发现，全长S蛋白、S1以及RBD均可作为开发MERS-CoV疫苗和治疗抗体的靶点。因此S-RBD目前成为许多科研工作者的主要研究对象。然而，对于MERS-CoV S-RBD抗原表位鉴定及其免疫识别等问题尚有待全面解决。
发明内容
针对现有技术的不足，本发明的目的是提供一种MERS-CoV S-RBD线性B细胞表位及其特异性识别单克隆抗体和应用。
为了实现上述目的，本发明所采用的技术方案是：
一种MERS-CoV S-RBD线性B细胞表位肽，所述表位肽的氨基酸序列为：FTCSQIS。
一种单克隆抗体6E8，特异性识别含有所述的MERS-CoV S-RBD线性B细胞表位序列肽或蛋白。
所述单克隆抗体6E8是由保藏编号为CCTCC NO:C2021159的杂交瘤细胞株6E8分泌，该杂交瘤细胞株6E8保藏于中国典型培养物保藏中心，保藏地址为武汉大学，保藏时间为2021年7月6日。
所述的MERS-CoV S-RBD线性B细胞表位肽在制备MERS诊断试剂或药物中的应用。
编码所述MERS-CoV S-RBD线性B细胞表位肽的核苷酸序列。
所述核苷酸序列为TTTACATGTAGCCAGATCTCT。
本发明有益效果：
本发明利用HEK 293F细胞表达系统表达MERS-CoV S1重组蛋白，经镍填料亲和层析和凝胶过滤层析两步纯化步骤，获得了高纯度、具有生物学活性和免疫原性的目的蛋白。随后免疫BALB/c小鼠，获得单克隆抗体；利用酶联免疫吸附试验(ELISA)鉴定其中一株单克隆抗体6E8可特异性识别RBD-rFc蛋白；利用真核细胞HEK-293T重组表达MERS-CoV S-RBD重组蛋白不同片段，通过Dot-blot鉴定获得的6E8单克隆抗体特异性识别MERS-CoV S-RBD N端区域，序列为TKLLSLFSVNDFTCSQISPAAI；利用酶联免疫吸附试验(enzyme-linkedimmunosorbent assay，ELISA)进一步鉴定MERS-CoV S-RBD N端区域合成多肽，鉴定获得的6E8单克隆抗体特异性识别序列为FTCSQIS的线性B细胞表位。
本发明综合利用分子生物学和免疫学等技术，鉴定了一种MERS-CoV S-RBD的全新线性B细胞表位，利用本发明制备的6E8单克隆抗体对含有MERS-CoV S-RBD线性B细胞表位的多肽、蛋白进行识别，表明该MERS-CoV S-RBD线性B细胞表位可被特异性识别。
本发明鉴定的MERS-CoV S-RBD线性B细胞表位丰富了MERS-CoV S-RBD免疫学功能，为后续研究S蛋白抗原漂移提供参考，可应用于抗病毒药物研发。
附图说明
图1是MERS-CoV S1重组蛋白纯化结果。
图中，M：蛋白marker，1：MERS-CoV S1蛋白。
图2是MERS-CoV S-RBD重组蛋白纯化结果。
图中，M：蛋白marker，1：MERS-CoV S-RBD蛋白。
图3是ELISA鉴定制备的MERS-CoV S-RBD单克隆抗体的结果。
图4是Dot-blot鉴定制备的MERS-CoV S-RBD单克隆抗体识别的MERS-CoV S-RBD片段。
图中，A:MERS-CoV S-RBD截短片段示意图，B:Dot-blot鉴定制备的MERS-CoV S-RBD单克隆抗体识别的截短体片段，C：Dot-blot鉴定制备的MERS-CoV S-RBD单克隆抗体识别的N1截短体片段。
图5是ELISA鉴定制备的MERS-CoV S-RBD单克隆抗体识别的MERS-CoV S-RBD线性B细胞表位的结果。
具体实施方式
以下结合实施例对本发明的具体实施方式作进一步详细说明。
实施例1.MERS-CoV S1、S-RBD重组蛋白的制备与纯化
1.1MERS-CoV S1蛋白克隆构建及重组蛋白在HEK-293F细胞中的表达及纯化
将MERS-CoV S1基因序列(GenBank accessionAFS88936.1)交由生工生物工程(上海)股份有限公司进行合成，氨基端引入CD5信号肽，羧基端引入6×His标签，序列插入pcDNA3.1载体，得到重组质粒S1-His。
将重组质粒S1-His转染人胚肾上皮细胞293F(HEK-293F)，扩大培养转染细胞。将上清通过经裂解液平衡的镍离子亲和柱(GE Healthcare)，经BufferA(20mM Tris-HCl pH7.5，150mM NaCl，20mM咪唑)进行漂洗，随后用Buffer B(20mM Tris-HCl pH 7.5，150mMNaCl，200mM咪唑)进行洗脱，收集洗脱液即为蛋白粗纯溶液。
将蛋白粗纯溶液通过分子筛Superdex 200Increase 10/300GL(GE Healthcare)进一步纯化，使用Buffer C(20mM Tris-HCl pH 7.5，150mM NaCl)，收集蛋白峰组分并经SDS-PAGE检测蛋白样品的纯度，通过图1可得，经过分子筛纯化后得到纯度较高的重组S1-His蛋白，分子量约为120kD。S1-His重组蛋白将用于后续抗体制备。
1.2MERS-CoV S-RBD蛋白克隆构建及重组蛋白在昆虫细胞中的表达及纯化
S-RBD编码基因(GenBank accessionAFS88936.1,367-606aa)交由生工生物工程(上海)股份有限公司进行合成，同时羧基端引入兔源Fc(rabbit IgG Fc)序列、6×His标签，随后将RBD-rFc基因经PCR扩增，上游引入BamHⅠ酶切位点，下游引入XhoⅠ酶切位点，得到PCR产物。将gp67信号肽基因通过同源重组方法插入pFastBac1载体，得到重组载体pFastBac1-gp67。将PCR产物及pFastBac1-gp67载体经BamHⅠ、XhoⅠ双酶切后回收，经DNA连接酶连接，转化大肠杆菌TOP10，经过基因测序鉴定，成功构建重组质粒pFastBac1-gp67-RBD-rFc。
将重组质粒pFastBac1-gp67-RBD-rFc转化至DH10Bac感受态细胞，经蓝白斑筛选，挑选白色菌落于LB液体培养基，振荡培养过夜，随后使用QIAGEN试剂盒提取重组黏粒。以0.8×105～1×106/孔的细胞数将处于对数生长期的sf21细胞加入六孔板，静置15min以上使细胞充分贴壁。在CellfectinⅡ介导下，将重组黏粒转染至上述贴壁sf21昆虫细胞，观察细胞病变情况。细胞发生典型病变后，收取细胞培养上清作为第一代重组杆状病毒P1。在10cm无菌培养皿中加入无血清培养基10mL，随后加入sf21细胞，使细胞总数在6×106个左右，轻晃培养皿使细胞均匀分布，静置10min，待细胞贴壁后，向其中加入800μL左右的P1病毒，将培养皿放入28℃培养箱，避光静置培养3～4d，收获细胞培养上清，即为2代病毒P2。使用同样方法扩增，收集P3代杆状病毒。以5个感染复数的P3代杆状病毒接种至对数生长期的sf21细胞，细胞振荡培养72h，4℃条件下3000rpm离心30min，收集细胞培养上清。
将上清通过经裂解液平衡的镍离子亲和柱(GE Healthcare)，经BufferA(20mMTris-HCl pH 7.5，150mM NaCl，20mM咪唑)进行漂洗，随后用Buffer B(20mM Tris-HCl pH7.5，150mM NaCl，200mM咪唑)进行洗脱，收集洗脱液即为蛋白粗纯溶液。将蛋白粗纯溶液通过分子筛Superdex 200Increase 10/300GL(GE Healthcare)进一步纯化，使用Buffer C(20mM Tris-HCl pH 7.5，150mM NaCl)，收集蛋白峰组分并经SDS-PAGE检测蛋白样品的纯度，通过图2可得，经过分子筛纯化后得到纯度较高的重组RBD-rFc蛋白，分子量约为50kD。RBD-rFc蛋白将用于后续抗体筛选。
实施例2.MERS-CoV S-RBD重组蛋白单克隆抗体制备与鉴定
2.1MERS-CoV S-RBD重组蛋白单克隆抗体制备
用实施例1中纯化所得重组S1-His蛋白作为抗原免疫3只8周龄的SPF级BALB/c雌性小鼠，抗原与等体积完全弗氏佐剂(首免)或不完全弗氏佐剂(加强免疫)混合并进行乳化，充分混合至油包水状态进行颈背部皮下多点注射，3次加强免疫，每次免疫间隔周期2周，之后进行效价检测，高于>1:10000后1周内进行腹腔冲击，直接将免疫剂量的抗原溶于250μL的PBS中，具体免疫次数及免疫剂量如表1所示：
表1.免疫次数及免疫剂量
| 免疫次数 | 免疫原制备 | 免疫途径 | 免疫剂量 |
| 一免 | 抗原+完全弗氏佐剂+PBS | 皮下 | 1μg/只 |
| 二免 | 抗原+不完全弗氏佐剂+PBS | 皮下 | 1μg/只 |
| 三免 | 抗原+不完全弗氏佐剂+PBS | 皮下 | 1μg/只 |
| 四免 | 抗原+不完全弗氏佐剂+PBS | 皮下 | 1μg/只 |
| 冲击加强 | 抗原+PBS | 腹腔 | 2μg/只 |
免疫示例：一免中，将1μg的抗原溶于250μLPBS，然后与佐剂按体积1:1混合。
腹腔冲击3天后，无菌取小鼠脾脏，制备成单细胞悬液；处于对数期的SP2/0细胞处理后，与脾细胞以体积比1:5比例混合，50％(wt)PEG1500作用1min，以基础培养基DMEM稀释终止，低速离心后，再用含20％(wt)胎牛血清的HAT培养基轻轻悬浮并混匀，按照2×107/板铺至预先准备好的饲养层细胞板里，置于5％CO2，37℃下培养。细胞融合10天后，融合的杂交瘤细胞形成克隆并且占据一定面积的细胞培养孔。经酶联免疫吸附试验(ELISA)初步鉴定后，将阳性杂交瘤细胞克隆转移到24孔细胞培养板中，通过有限稀释法进行单克隆化，确保获得稳定分泌单克隆抗体的杂交瘤细胞株，并将有效的阳性单克隆(单克隆抗体6E8)进行小鼠腹水制备，备用。
该单克隆抗体6E8由保藏编号为CCTCC NO:C2021159的杂交瘤细胞株6E8分泌，该杂交瘤细胞株6E8保藏于中国典型培养物保藏中心，保藏地址为武汉大学，保藏时间为2021年7月6日。
2.2酶联免疫吸附试验(ELISA)鉴定MERS-CoV S-RBD单克隆抗体
将实施例1中纯化所得RBD-rFc蛋白溶于碳酸盐-碳酸氢盐缓冲液(CBS,pH 9.6)，随后以RBD-rFc蛋白(2.5μg/mL,100μL/孔)包被96孔板，37℃孵育2h，PBST漂洗五次后加入5％(w/v)脱脂奶粉，37℃孵育2h，弃掉，将杂交瘤细胞(单克隆抗体6E8)上清液按50μL/孔加至96孔板，以阴性杂交瘤细胞上清液作对照，37℃孵育1h，PBST漂洗五次后加入辣根过氧化物酶标记的羊抗鼠IgG抗体作为二抗，37℃孵育1h，PBST漂洗五次后加入3,3',5,5'-四甲基联苯胺(TMB，美国Sigma公司)，室温避光反应5min，随后用2M H2SO4终止反应，使用酶标仪读取每孔在450nm的吸光度值。如图3所示，经ELISA鉴定，单克隆抗体6E8的OD450读值大于3，远远大于阴性对照，表明单克隆抗体6E8可以识别RBD-rFc蛋白。
实施例3.Dot-blot鉴定单克隆抗体识别MERS-CoV S-RBD区域
3.1MERS-CoV S-RBD片段重组表达载体构建
表2.MERS-CoV S-RBD截短片段引物序列
注：下划线表示引入的限制性内切酶切割位点。
以MERS-CoV S-RBD cDNA作为PCR模板，以表2中引物进行PCR扩增S-RBD不同片段基因(具体蛋白区域序列见表2)，在PCR产物两端分别引入EcoR I和Nco I酶切位点。PCR目的产物经1％琼脂糖凝胶电泳鉴定并回收，经双酶切获得的目的片段插入表达载体pFuse-hIgG1-Fc2载体(美国InvivoGen公司)相应酶切位点，将重组表达质粒转化大肠杆菌克隆菌株JM109感受态细胞(TaKaRa公司)，挑取单克隆菌落经PCR鉴定阳性后交由生工生物工程(上海)股份有限公司鉴定，根据鉴定结果提取鉴定正确的重组表达质粒。
3.2MERS-CoV S-RBD片段重组表达
将上步鉴定正确的重组表达质粒转染人胚肾上皮细胞293T(HEK-293T)。扩大培养转染细胞，转染48h后离心收取细胞上清液。上清液经ProteinA亲和层析纯化后，在硝酸纤维素膜(美国Millipore公司)上点印重组表达的不同MERS-CoV S-RBD片段，以人源IgG1型抗体的Fc区段为对照，依次加入兔抗人源IgG Fc抗体和辣根过氧化物酶标记的羊抗兔IgG抗体进行孵育，使用ECL试剂显色检测目的蛋白。如图4B结果显示，同人源IgG1型抗体的Fc区段(hFc)阳性对照一样，MERS-CoV S-RBD不同片段均有表达(呈现黑色颜色反应阳性点)。
3.3Dot-blot鉴定MERS-CoV S-RBD单克隆抗体识别区域
确认目的蛋白表达后，在硝酸纤维素膜上点印重组表达的不同MERS-CoV S-RBD片段。自然干燥后，将印迹膜37℃封闭1h。随后依次加入制备的MERS-CoV S-RBD单克隆抗体6E8、辣根过氧化物酶标记的羊抗鼠IgG抗体进行孵育。使用ECL试剂显色判定，以阳性点呈现黑色颜色反应，阴性点不出现颜色反应进行判定。如图4B结果显示，N-N1区域可被制备的单克隆抗体6E8特异性识别。
根据N1区域序列，合成表3中多肽，合成多肽经鉴定序列正确，纯度均可达到95％以上。使用DMSO溶解合成多肽，加入0.01M盐酸将pH值调至4-5；加入含1-(3-二甲氨基丙基)-3-乙基碳二亚胺(EDC，购自美国Thermo公司)反应液，室温反应15min；稀释牛血清白蛋白(BSA)，逐滴加入到合成多肽反应液中，混匀后室温反应2h，形成BSA-多肽备用。在硝酸纤维素膜上点印BSA-多肽，自然干燥后，重复上述dot-blot步骤，如图4C结果显示，序列为TKLLSLFSVNDFTCSQISPAAI的N1-4区域可被制备的单克隆抗体6E8特异性识别，我们将这一区域重命名为P1。
表3.N1区域多肽序列合成
| 多肽名称 | 多肽氨基酸序列 |
| N1-1 | EAKPSGSVVEQAEGVECDFS |
| N1-2 | ECDFSPLLSGTPPQVYNFKR |
| N1-3 | YNFKRLVFTNCNYNLTKLLS |
| N1-4(P1) | TKLLSLFSVNDFTCSQISPAAI |
实施例4.酶联免疫吸附试验(ELISA)鉴定MERS-CoV S-RBD单克隆抗体识别的线性B细胞表位
根据实施例3鉴定的区域P1，分别通过N端截短和C端截短合成表4中多肽。合成多肽与BSA偶联得到BSA-多肽备用。以5mg/mL浓度将BSA-多肽按100μL/孔包被96孔酶标板，4℃孵育过夜后，加入含5％(w/v)脱脂奶粉封闭液封闭。加入制备的MERS-CoV S-RBD单克隆抗体6E8的杂交瘤细胞上清液(体积比1:100稀释，每孔加100μL)，37℃孵育1h，加入辣根过氧化物酶标记的羊抗鼠IgG抗体和3,3',5,5'-四甲基联苯胺(TMB，美国Sigma公司)，室温避光反应5min，使用2M浓硫酸终止反应，并读取450nm的吸光度值，检测MERS-CoV S-RBD单克隆抗体6E8与酶标板上BSA-多肽的结合，同时以不结合小鼠IgG的BSA为阴性对照。样品OD450值与阴性对照OD450值的比值大于或等于2.2时可判定为阳性，其它则为阴性。
表4.截短多肽序列
*N端引入Cysteine以增加与BSA的偶联反应效率。
经ELISA鉴定，单克隆抗体6E8能与N端截短片段P1-N1至P1-N11结合，OD450值与阴性对照OD450值的比值均大于2.2，表明单克隆抗体6E8可以和N端截短至P1-N11(FTCSQISPAAI)的片段结合；而单克隆抗体6E8仅与C端截短片段中P1-C1至P1-C4结合，OD450值与阴性对照OD450值的比值大于2.2，表明这株单克隆抗体结合的位点位于P1-C4的C端(图5)。综上所述，制备的这株单克隆抗体6E8识别的B细胞表位序列为FTCSQIS(SEQ IDNO.13)，为线性表位，其编码序列为TTTACATGTAGCCAGATCTCT(SEQ ID NO.14)。
以上所述仅为本发明较佳的实施例，对于本领域的技术人员来说，本发明可以有各种更改和变化。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。
序列表
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<120> 一种MERS-CoV S-RBD线性B细胞表位及其特异性识别单克隆抗体和应用
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tttacatgta gccagatctc t 21
Claims (6)
1.一种MERS-CoV S-RBD线性B细胞表位肽，其特征在于，所述表位肽的氨基酸序列为：FTCSQIS。
2.一种单克隆抗体6E8，其特征在于，特异性识别含有权利要求1所述的MERS-CoV S-RBD线性B细胞表位序列肽或蛋白。
3.如权利要求2所述单克隆抗体6E8，其特征在于，所述单克隆抗体6E8是由保藏编号为CCTCC NO:C2021159的杂交瘤细胞株6E8分泌，该杂交瘤细胞株6E8保藏于中国典型培养物保藏中心，保藏地址为武汉大学，保藏时间为2021年7月6日。
4.一种权利要求1所述的MERS-CoV S-RBD线性B细胞表位肽在制备MERS诊断试剂或药物中的应用。
5.编码如权利要求1所述MERS-CoV S-RBD线性B细胞表位肽的核苷酸序列。
6.根据权利要求5所述的编码MERS-CoV S-RBD线性B细胞表位肽的核苷酸序列，其特征在于，所述核苷酸序列为TTTACATGTAGCCAGATCTCT。
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