CN113817050A - 一种抗SARS-CoV-2的单克隆抗体1H8 - Google Patents
一种抗SARS-CoV-2的单克隆抗体1H8 Download PDFInfo
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- CN113817050A CN113817050A CN202010566581.0A CN202010566581A CN113817050A CN 113817050 A CN113817050 A CN 113817050A CN 202010566581 A CN202010566581 A CN 202010566581A CN 113817050 A CN113817050 A CN 113817050A
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Abstract
本发明涉及一种抗SARS‑CoV‑2的单克隆抗体1H8,该抗体的六个CDR区为:(1)重链CDR1(VHCDR1)包含如SEQ ID NO.1所示的氨基酸序列;(2)重链CDR2(VHCDR2)包含如SEQ ID NO.2所示的氨基酸序列;(3)重链CDR3(VHCDR3)包含如SEQ ID NO.3所示的氨基酸序列;(4)轻链CDR1(VLCDR1)包含如SEQ ID NO.4所示的氨基酸序列;(5)轻链CDR2(VLCDR2)包含如SEQ ID NO.5所示的氨基酸序列;(6)轻链CDR3(VLCDR3)包含如SEQ ID NO.6所示的氨基酸序列。
Description
技术领域
本发明属于生物医药技术领域,具体的,涉及一种抗SARS-CoV-2的单克隆抗体1H8。
背景技术
含有高浓度的抗SARS-CoV-2抗体的康复期血浆已在治疗中显示出积极的效果,说明抗SARS-CoV-2特异性抗体能有效阻断病毒与细胞的结合。另外,在SARS、MERS等烈性传染病爆发期间研发了一系列的具有中和活性的单克隆抗体,已被证明在疾病的预防和治疗中具有安全性和有效性。这些都提示在应对SARS-CoV-2病毒时,可考虑制备抗SARS-CoV-2单克隆抗体,尤其是全人源单克隆抗体,该种抗体不仅能通过阻断SARS-CoV-2与受体细胞的结合从而避免病毒的侵入,达到保护的作用,而且相对人源化或人鼠嵌合抗体具有副作用更小的优点,将为COVID-19的特异性预防和治疗提供新的手段。
发明内容
本发明首先涉及针对SARS-CoV-2病毒的单克隆抗体1H8,其特征在于,该抗体的六个CDR区为:
(1)重链CDR1(VHCDR1)包含如SEQ ID NO.1所示的氨基酸序列:EITVSSNYMN;
(2)重链CDR2(VHCDR2)包含如SEQ ID NO.2所示的氨基酸序列:VIYSGGTTYYADSVKG;
(3)重链CDR3(VHCDR3)包含如SEQ ID NO.3所示的氨基酸序列:DLMEVGGMDV;
(4)轻链CDR1(VLCDR1)包含如SEQ ID NO.4所示的氨基酸序列:RASQGVSNYLA;
(5)轻链CDR2(VLCDR2)包含如SEQ ID NO.5所示的氨基酸序列:AASTLQS;
(6)轻链CDR3(VLCDR3)包含如SEQ ID NO.6所示的氨基酸序列:QNYNSAPFA。
进一步的,
所述的单克隆抗体1H8的重链可变区全长包含如SEQ ID NO.7所示的氨基酸序列:EVQLVESGGGLVQPGGSLRLSCAASEITVSSNYMNWVRQAPGKGLEWVSVIYSGGTTYYADSVKGRFTISRDNSENTLYLQMNSL RAEDTAVYYCARDLMEVGGMDVWGQGTTVTVSS;
所述的单克隆抗体1H8的轻链可变区全长包含如SEQ ID NO.8所示的氨基酸序列:DIVMTQSPSSLSASVGDRVTITCRASQGVSNYLAWYQQKPGKVPKLLIYAASTLQSGVPSRFSGSRSGTDFTLTISSLQPEDVATYYC QNYNSAPFAFGPGTKVEIK。
进一步的,所述的单克隆抗体1H8为人IgG型抗体。
进一步的,所述的单克隆抗体1H8结合的抗原为SARS-CoV-2病毒的刺突蛋白S1,具体的,所述的单克隆抗体1H8结合的抗原结构区域为SARS-CoV-2病毒的刺突蛋白S1中的RBD结构域。
最优选的,所述的单克隆抗体1H8的轻链可变区和重链可变区分别是SEQ ID NO.8和SEQ ID NO.7所示的氨基酸序列。
本发明还涉及编码所述单克隆抗体1H8的核酸片段。
本发明还涉及一种抗体,所述的抗体的轻链为:
(1)SEQ ID NO.8所示的氨基酸序列经替换、缺失或添加一个或几个氨基酸后,形成的具有同等功能的序列;
或(2)与SEQ ID NO.8所示的氨基酸序列具有95%以上同源性的氨基酸序列;
所述的抗体的重链为:
(1)SEQ ID NO.7所示的氨基酸序列经替换、缺失或添加一个或几个氨基酸后,形成的具有同等功能的序列;
或(2)与SEQ ID NO.7所示的氨基酸序列具有95%以上同源性的氨基酸序列。
本发明还包括所述的单克隆抗体1H8在制备检测SARS-CoV-2病毒的试剂中的应用。
本发明还包括所述的单克隆抗体1H8在制备抑制SARS-CoV-2病毒的试剂中的应用。
本发明还包括单克隆抗体1H8在制备药物中的应用,所述的药物为预防和/或治疗因SARS-CoV-2病毒感染导致的疾病的药物。
本发明的有益效果:由于目前针对SARS-CoV-2病毒引起的疾病仍无上市的疫苗和特异药物,虽然康复期血浆治疗已被证明是一种有希望的治疗方法,但是由于大规模制备受限,且该方法主要针对重症和危重症患者,而单克隆抗体则具有纯度高,靶向性强、副作用小、可大量制备等优点。本发明所涉及的1H8抗体的实验结果表明,该单克隆抗体1H8中和活性和阻断活性均较高。因此,本发明获得的人源单克隆抗体 1H8为COVID-19的特异性预防和治疗提供了新的候选药物。
附图说明
图1、单克隆抗体1H8的SDS-PAGE电泳检测结果:泳道1为非还原SDS-PAGE电泳,泳道2为还原性 SDS-PAGE电泳。
图2、单克隆抗体1H8的SEC-HPLC检测结果。
图3、单克隆抗体1H8结合活性检测结果。A为针对SARS-CoV-2刺突蛋白S1的结合活性;B为针对 SARS-CoV-2受体结合区蛋白RBD的结合活性。
图4、单克隆抗体1H8阻断活性检测结果。A为针对SARS-CoV-2刺突蛋白S1的阻断活性;B为针对 SARS-CoV-2受体结合区蛋白RBD的阻断活性。
图5、单克隆抗体1H8亲和力检测结果。
图6、单克隆抗体1H8的活病毒中和实验检测结果。
具体实施方式
若未特别说明,以下实施例中所用的技术手段均为本领域技术人员所熟知的常规手段,所有试剂耗材均为市售商品。
实施例1、单克隆抗体1H8表达载体的构建、表达与纯化
将含1H8抗体重链和轻链可变区的编码基因片段分别整合入含人IgG1抗体重轻链恒定区序列的 pcDNA3.4表达载体中,得到能分别表达目标抗体重链和轻链的重组表达载体。
细胞的转染、抗体表达和纯化
1、转染:采用Gibco公司转染试剂盒并按照说明书进行转染操作,步骤简述为:
将两种重组表达载体总DNA与转染试剂混合以形成DNA ExpiFectamineTM 293复合物;
随后加入含40mL、2.94x106个/mL的Expi293细胞培养物中;
最后置于37℃,125rpm,8%CO2培养。
2、纯化:
培养5天后,4000rpm,25℃离心10分钟收集上清,随后用MabSelect Sure亲和层析柱进行纯化。纯化步骤简述为:
用pH7.0的0.1M Tris缓冲液平衡;
上样后,用pH7.0的0.1M Tris缓冲液淋洗;
随后用pH8.0的1.0M Tris缓冲液进行洗脱。
收集洗脱液进一步于PBS缓冲液中透析。取纯化后的抗体进行SDS-PAGE和HPLC-SEC检测分析。
3、结果分析:SDS-PAGE结果见图1,显示在非还原条件下,人源SARS-CoV-2抗体呈现分子量约为150kDa 的条带;在还原条件下呈现分子量约为50kDa和25kDa的两条带,分别对应抗体的重链和轻链。SEC-HPLC结果见图2,显示纯化后的单克隆抗体纯度达到98%以上。纯化后的单克隆抗体经肽图分析后得到的氨基酸序列与预期的氨基酸序列一致。具体的,轻链可变区和重链可变区的序列分别与SEQ ID NO.8、SEQ ID NO.7相同。
实施例2、抗体的功能分析
1、人源抗SARS-CoV-2抗体1H8与抗原的结合活性检测
采用ELISA法测定抗体与SARS-CoV-2(2019-nCoV)病毒的刺突蛋白S1和RBD结构域结合能力。步骤简述为:
(1)以SARS-CoV-2刺突S1-His重组蛋白(Sino公司,货号:40591-V08H)或RBD结构域重组蛋白(Sino 公司,货号40592-V05H)为包被抗原,用重碳酸盐缓冲液将1.0μg/mL抗原包被于酶标板,4℃过夜;
(2)用酪蛋白缓冲液于37℃封闭1h;加入系列稀释后的待检抗体,37℃1h;
(3)加入1:10000稀释后的羊抗人IgG-HRP(Bethyl公司,货号:A80-304P),37℃1h;
(4)用显色液显色后,2M HCl终止反应;酶标仪检测吸光度A450和A540值。
结果:
1H8抗体与抗原结合活性检测结果见图3,可知单克隆抗体1H8针对S1蛋白的结合活性(图3A)为 EC50=0.054nM;针对RBD结构域的结合活性((图3B)为EC50=0.035nM。
2、人源抗SARS-CoV-2抗体1H8的阻断活性检测
采用ELISA检测法检测抗体阻断ACE2重组蛋白(带His标签)结合SARS-CoV-2(2019-nCoV)病毒的刺突蛋白S1和RBD结构域的能力。步骤简述为:
(1)以SARS-CoV-2刺突S1亚基蛋白(Sino公司,货号:40591-V02H)或RBD重组蛋白(Sino公司,货号40592-V05H)为包被抗原,用重碳酸盐缓冲液将1.0μg/mL抗原包被于酶标板,4℃过夜;
(2)用酪蛋白缓冲液于37℃封闭1h;加入系列稀释后的待检抗体和浓度为0.5μg/mL或0.02μg/mL的 ACE重组蛋白进行共孵育,37℃1h;
(3)加入1:3000稀释后的生物素标记的小鼠抗His标签抗体(GenScript公司,货号:A00613),37℃ 1h;
(4)加入1:20000稀释的链霉亲和素-HRP(Thermofisher公司,货号:SNN1004),37℃1h;用显色液显色后,2M HCl终止反应;酶标仪检测吸光度A450和A540值。
结果:
1H8抗体阻断活性检测结果见图4,可知单克隆抗体1H8针对ACE2与S1结合的阻断活性(图4A)为 IC50=5.76nM,最大阻断率为93.76%;针对ACE2与RBD结合的阻断活性(图4B)为IC50=3.6nM,最大阻断率为96.11%。
3、人源抗SARS-CoV-2抗体1H8的亲和力检测
利用GE公司的Biacore 8K仪器进行表面等离子共振实验,检测抗体亲和力。
单克隆抗体1H8亲和力检测结果见图5,可知1H8结合常数ka值为7.36E+04 1/Ms,解离常数kd值为 1.38E-03 1/s,亲和力常数KD值为1.87E-08M。
4、人源抗SARS-CoV-2抗体活病毒中和试验
病毒蚀斑减少中和试验采用SARS-CoV-2病毒BetaCoV/IVDC-HB-envF13/2020株进行。步骤简述为:将定量的SARS-CoV-2病毒和系列倍比稀释的单克隆抗体混合后孵育,然后加入到预先准备好的含Vero细胞的检测平板中,培养后观察记录病毒蚀斑数,并计算病毒中和活性(以IC50表示)。
结果:
抗体1H8的活病毒中和实验检测结果见图6,显示该单克隆抗体能较好的中和SARS-CoV-2病毒 (IC50<0.98ng/ml)。
最后需要说明的是,以上实施例仅用作帮助本领域技术人员理解本发明的实质,不用于限定本发明的保护范围。
SEQUENCE LISTING
<110> 武汉生物制品研究所有限责任公司
<120> 一种抗SARS-CoV-2的单克隆抗体1H8
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Glu Asp Val Ala Thr Tyr Tyr Cys Gln Asn Tyr Asn Ser Ala Pro Phe
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Ala Phe Gly Pro Gly Thr Lys Val Glu Ile Lys
100 105
Claims (8)
1.一种抗SARS-CoV-2病毒的单克隆抗体1H8,其特征在于,该抗体的六个CDR区为:
(1)重链CDR1(VHCDR1)包含如SEQ ID NO.1所示的氨基酸序列:EITVSSNYMN;
(2)重链CDR2(VHCDR2)包含如SEQ ID NO.2所示的氨基酸序列:VIYSGGTTYYADSVKG;
(3)重链CDR3(VHCDR3)包含如SEQ ID NO.3所示的氨基酸序列:DLMEVGGMDV;
(4)轻链CDR1(VLCDR1)包含如SEQ ID NO.4所示的氨基酸序列:RASQGVSNYLA;
(5)轻链CDR2(VLCDR2)包含如SEQ ID NO.5所示的氨基酸序列:AASTLQS;
(6)轻链CDR3(VLCDR3)包含如SEQ ID NO.6所示的氨基酸序列:QNYNSAPFA。
2.根据权利要求1所述的抗体,其特征在于,
所述的单克隆抗体1H8的重链可变区全长包含如SEQ ID NO.7所示的氨基酸序列:
所述的单克隆抗体1H8的轻链可变区全长包含如SEQ ID NO.8所示的氨基酸序列。
3.根据权利要求1或2所述的抗体,其特征在于,所述的单克隆抗体1H8为人IgG型抗体。
4.根据权利要求1或2所述的抗体,其特征在于,所述的单克隆抗体1H8结合的抗原为SARS-CoV-2病毒的刺突蛋白S1,具体的,所述的单克隆抗体1H8结合的抗原结构区域为SARS-CoV-2病毒的刺突蛋白S1中的RBD结构域。
5.编码权利要求1-4任一所述的抗体的核酸片段。
6.权利要求1-4任一所述的单克隆抗体1H8在制备检测SARS-CoV-2病毒的试剂中的应用。
7.权利要求1-4任一所述的单克隆抗体1H8在制备抑制SARS-CoV-2病毒的试剂中的应用。
8.权利要求1-4任一所述的单克隆抗体1H8在制备药物中的应用,所述的药物为预防和/或治疗因SARS-CoV-2病毒感染导致的疾病的药物。
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