CN112786101A - 热稳定性抗体的制备方法 - Google Patents
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- CN112786101A CN112786101A CN201911083366.9A CN201911083366A CN112786101A CN 112786101 A CN112786101 A CN 112786101A CN 201911083366 A CN201911083366 A CN 201911083366A CN 112786101 A CN112786101 A CN 112786101A
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Abstract
本发明提供一种热稳定性抗体的制备方法，包括：1)构建抗体的空间模型，并对模型进行评价；2)确定关键氨基酸的位点并进行定点突变；3)通过软件模拟计算评估突变前后抗体结合能的变化。进一步地，本发明以磺胺类药物单链抗体scFv4C7为例，通过序列比对、结构模拟及优化、虚拟氨基酸突变等，从理论上探讨影响抗体热稳定性的关键因素，结合生物学实验结果显示，利用本方法获得的抗体突变体，其热稳定性均得到不同程度地提高，为抗体分子热稳定性的改造提供理论依据。
Description
技术领域
本发明涉及结构生物学、生物信息学及免疫学领域，具体地说，涉及一种热稳定性抗体的制备方法。
背景技术
随着分子生物学技术、抗体结构和功能研究的不断进展，基因重组抗体(第三代抗体)，已广泛应用于免疫诊断与治疗、药物开发和环境分析等领域。然而，在抗体改造过程中，提高其稳定性的难题尚未解决。蛋白质的稳定性及生物学效应不仅取决于一级序列，更主要的是取决于其空间构象。随着计算机化学的发展，已有采用计算机方法设计抗体，来增强其热稳定性和化学稳定性，并降低IgG恒定结构域的聚集领域的尝试。
发明内容
本发明的目的是提供一种热稳定性抗体的制备方法。
为了实现本发明目的，第一方面，本发明提供一种提高抗体热稳定性的方法，具体利用计算机图形学等技术提高抗体的热稳定性。所述方法包括以下步骤：
1)对于序列已知的抗体，利用生物学软件构建抗体的空间模型，并对模型进行评价(评价模型的合理性)；
2)确定关键氨基酸的位点并进行定点突变；
3)通过软件模拟计算评估突变前后抗体结合能的变化。
进一步地，所述抗体来源于人、小鼠、大鼠、兔、猪、羊、骆驼、羊驼、牛或鲨鱼等。
进一步地，所述抗体为IgG、Fab、scFv或Nanobody等。
进一步地，所选突变区域可以是抗体的可变区和/或恒定区。
第二方面，本发明提供一种磺胺类药物单链抗体scFv4C7的突变体，所述突变体至少包含单链抗体scFv4C7的如下①～⑤突变之一：
①P146W突变；
②G105I突变；
③V144W突变；
④A72W突变；
⑤S149C突变。
优选地，所述突变体包含①～⑤五个突变位点。
本发明中，磺胺类药物单链抗体scFv4C7的氨基酸序列如SEQ ID NO:1所示。包含上述五个突变位点的突变体的氨基酸序列如SEQ ID NO:2所示。
第三方面，本发明提供编码所述突变体的核酸分子。
第四方面，本发明提供含有所述核酸分子的生物材料，所述生物材料包括但不限于重组DNA、表达盒、转座子、质粒载体、噬菌体载体、病毒载体、工程菌或转基因细胞系。
第五方面，本发明提供一种磺胺类药物检测试剂或试剂盒，有效成分为上述突变体。
第六方面，本发明提供所述突变体的以下任一应用：
1)用于磺胺类药物检测；
2)用于制备磺胺类药物检测试剂或试剂盒。
本发明中，所述磺胺类药物包括氨苯磺胺、磺胺醋酰、磺胺胍、磺胺苯吡唑、酞磺胺噻唑、磺胺二甲异噁唑、磺胺噻唑、磺胺甲基异噁唑、磺胺二甲噁唑、磺胺甲二唑、磺胺硝苯、柳氮磺胺吡啶、磺胺吡啶、磺胺氯哒嗪、磺胺甲氧哒嗪、磺胺乙氧哒嗪、磺胺喹噁啉、磺胺氯吡嗪、磺胺多辛、磺胺苯酰、磺胺林、磺胺二甲基异嘧啶、磺胺间甲氧嘧啶、磺胺对甲氧嘧啶、磺胺嘧啶、磺胺二甲基嘧啶、磺胺间二甲氧嘧啶、磺胺甲基嘧啶、磺胺溴二甲嘧啶。
本发明以磺胺类药物的单链抗体(scFv4C7)为例，构建抗体的空间模型，评价模型的合理性，通过氨基酸定点突变确定非CDR区影响抗体热稳定性的关键氨基酸，然后根据突变氨基酸的极性、非极性、电荷相似原则，选择单链抗体scFv4C7上的A72、G105、V144、P146、S149、G198、I200七个氨基酸进行定点突变，通过突变能的变化确定提高抗体热稳定性的P146W(代表146位点由Pro突变为Trp)、G105I、V144W、A72W和S149C五个突变位点。突变后抗体的亲和力未受明显影响，突变能降低了9.95kcal/mol，Tm值提高了11.3℃。本发明提供的抗体稳定性进化方法适用于提高抗体的热稳定性。
借由上述技术方案，本发明至少具有下列优点及有益效果：
本发明提供一种精准、高效、用于提高单链抗体热稳定性的方法，以磺胺类药物单链抗体scFv4C7为例，通过序列比对、结构模拟及优化、虚拟氨基酸突变等，从理论上探讨影响抗体热稳定性的关键因素，结合生物学实验结果显示，利用本方法获得的抗体突变体，其热稳定性均得到不同程度地提高，为抗体分子热稳定性的改造提供理论依据。
附图说明
图1为本发明单链抗体scFv4C7的轻链VL和重链VH的氨基酸序列图。
图2为本发明单链抗体scFv4C7的空间模型图。
图3为本发明单链抗体scFv4C7空间构象的拉氏图。
图4为本发明单链抗体scFv4C7空间构象的Profile-3D分析结果。
图5为本发明突变前单链抗体scFv4C7的Tm曲线图。
图6为本发明突变后抗体MscFv4C7的Tm曲线图。
图7为本发明部分氨基酸突变能变化图。
图8为本发明单链抗体scFv4D7与突变后抗体MscFv4C7的结合能变化。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段，所用原料均为市售商品。
实施例1利用计算机图形学等技术提高抗体的热稳定性
本实施例利用已有磺胺类药物单链抗体scFv4C7(SEQ ID NO:1)的轻链可变区VL和重链可变区VH的氨基酸序列为模式抗体，利用Discovery Studio 2019软件中的ModelAntibody模块，可以对scFv4C7的氨基酸序列进行注释，如图1所示，VH的高变区为：CDR1:26GYSITSGYS；CDR2：52IHYSGST；CDR3：97ARYGGY，VL的高变区为：CDR1：27QSLLDSDGKTY；CDR2：55LVS；CDR3：94WQGTHFPQT。
经ExPASy数据库分析，scFv4C7共由245个氨基酸组成(表1)，分子量约为26KD，理论等电点为8.62，为碱性蛋白质，在中性环境中带正电荷。该scFv的具体氨基酸组成见表1。其中负电性氨基酸(Asp和Glu)共17个，正电性氨基酸(Arg和Lys)共20个。摩尔消系数为52370-52620M-1cm-1。在水中不稳定性系数为42.70，表明该scFv4C7属于不稳定蛋白。
表1单链抗体scFv4C7的氨基酸组成
基于NCBI、Kabat、IMGT等抗体结构信息数据库，将scFv4C7的VL和VH空间结构信息导入Discovery Studio 2019软件中的Molecular Builder模块进行分子展示，构建scFv4C7的空间模型，根据PDF值或者DOPE值挑选最优模型，如图2所示。
然后，利用Ramanchandran Plot和Profile-3D对scFv4C7的构象合理性进行评价。通过拉氏图(图3)对模型结构进行评估，245个氨基酸中91.02％的残基落在允许区或最适区，构象不合理的氨基酸不超过9％，表明scFv4C7的三维结构是合理可信的。Profile-3D图显示(图4)，仅有7个氨基酸Verify Score小于0，说明scFv4C7同源建模的可信度较高。
进一步通过定点氨基酸突变来提高scFv热稳定性是在不降低抗体和小分子药物的亲和力的基础上实现的，突变位点主要位于非CDR区，理论上在不影响抗体空间构象的前提下，这些残基的改变不影响与抗原的相互作用。
极性氨基酸包括：苏氨酸(Thr)丝氨酸(Ser)半胱氨酸(Cys)天冬酰胺(Asn)谷氨酰胺(Gln)酪氨酸(Tyr)。带正电氨基酸包括：赖氨酸(Lys)精氨酸(Arg)组氨酸(His)。带负电氨基酸包括：天冬氨酸(Asp)谷氨酸(Glu)。非极性氨基酸包括：甘氨酸(Gly)丙氨酸(Ala)缬氨酸(Val)亮氨酸(Leu)异亮氨酸(Ile)苯丙氨酸(Phe)色氨酸(Trp)甲硫氨酸(Met)脯氨酸(Pro)。一般来说，非极性氨基酸通常在蛋白内部，极性氨基酸通常在蛋白表面，因此，氨基酸突变应在同类氨基酸之间进行，以尽可能地降低突变对抗体构象的影响。
通过Discovery Studio 2019软件对单链抗体scFv4C7赋予CHARMm力场，然后对非CDR区氨基酸进行单点突变确定影响抗体热稳定性的关键氨基酸，分别为位于非CDR区的A72、G105、V144、P146、S149、G198、I200。
将这7个氨基酸位点命名为突变(Mutation)，应用Discovery Studio 2019软件中的Design Protein模块中的Calculate Mutation Energy(Stability)突变为其他19种氨基酸。结果显示(表2，图7)，当P146W(代表146位点由Pro突变为Trp)、G105I、V144W、A72I和S149C时，突变能均小于-1，对应的突变效果是稳定性提高，说明这五个位点的突变均能使scFv4C7的热稳定性提高。对这五个氨基酸进行同时突变，突变能最多可降低-9.95kcal/mol(表3)，将该突变体命名为MscFv4C7(SEQ ID NO:2)。
表2单链抗体scFv4D7的单点突变的突变能变化
注：前七位氨基酸突变能降低最为显著，从第八位开始仅选取部分结果展示。
表3单链抗体scFv4D7的多点突变的突变能变化
注：合计突变约5000次，分批突变，表中所示为含突变能降低最为显著的氨基酸突变的一批数据。
通过对五个氨基酸位点进行定点突变，由Discovery Studio 2019中的CalculateBinding Energies模块可计算得知突变后结合能没有明显降低(图8)，甚至部分结合能还有了较大提高，说明部分亲和力得到了改善。
将表达的scFv4C7及突变型抗体MscFv4C7通过蛋白稳定性分析仪Uncle测得突变前Tm值只有63.1℃(图5)，突变后Tm值为74.4℃(图6)，Tm值提高了11.3℃。由于氨基酸突变全部在抗体的框架区进行，均远离抗体-抗原结合区域，所以对单链抗体的亲和力影响非常小，通过提高热稳定性的同时没有降低单链抗体的亲和力。因此，本发明提供了一种既不降低单链抗体亲和力又能提高单链抗体热稳定性的方法，为抗体分子热稳定性的改造提供理论依据。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之做一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
序列表
<110> 中国农业大学
<120> 热稳定性抗体的制备方法
<130> KHP191115473.2
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<170> SIPOSequenceListing 1.0
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Asp Val Val Met Thr Gln Thr Pro Ile Thr Leu Ser Val Thr Ile Gly
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Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser
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Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser
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Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Arg Val Pro
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Asp Arg Phe Thr Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Lys Ile
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Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Trp Gln Gly
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Thr His Phe Pro Gln Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
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Gly Gly Gly Ser Asp Val Gln Leu Arg Glu Ser Gly Pro Asp Leu Val
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Thr Pro Ser Gln Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser
145 150 155 160
Ile Thr Ser Gly Tyr Ser Trp His Trp Asn Arg Gln Phe Pro Gly Asn
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Lys Leu Glu Trp Met Gly Tyr Ile His Tyr Ser Gly Ser Thr Asn Tyr
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Asn Pro Ser Leu Arg Gly Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys
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Asn Gln Phe Phe Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala
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Thr Tyr Tyr Cys Ala Arg Tyr Gly Gly Tyr Trp Gly Gln Gly Thr Ser
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Val Thr Val Ser Ser
245
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Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Trp Gln Gly Thr
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Claims (10)
1.热稳定性抗体的制备方法，其特征在于，包括以下步骤：
1)对于序列已知的抗体，利用生物学软件构建抗体的空间模型，并对模型进行评价；
2)确定关键氨基酸的位点并进行定点突变；
3)通过软件模拟计算评估突变前后抗体结合能的变化。
2.根据权利要求1所述的方法，其特征在于，所述抗体来自人、小鼠、大鼠、兔、猪、羊、骆驼、羊驼、牛或鲨鱼。
3.根据权利要求1或2所述的方法，其特征在于，所述抗体为IgG、Fab、scFv或Nanobody；和/或
突变位点为抗体的可变区和/或恒定区。
4.磺胺类药物单链抗体scFv4C7的突变体，其特征在于，所述突变体至少包含单链抗体scFv4C7的如下①～⑤突变之一：
①P146W突变；
②G105I突变；
③V144W突变；
④A72W突变；
⑤S149C突变。
5.根据权利要求4所述的突变体，其特征在于，所述突变体包含①～⑤突变。
6.编码权利要求4或5所述突变体的核酸分子。
7.含有权利要求6所述核酸分子的生物材料，所述生物材料为重组DNA、表达盒、转座子、质粒载体、噬菌体载体、病毒载体、工程菌或转基因细胞系。
8.磺胺类药物检测试剂或试剂盒，其特征在于，有效成分为权利要求4或5所述突变体。
9.权利要求4或5所述突变体的以下任一应用：
1)用于磺胺类药物检测；
2)用于制备磺胺类药物检测试剂或试剂盒。
10.根据权利要求9所述的应用，其特征在于，所述磺胺类药物包括氨苯磺胺、磺胺醋酰、磺胺胍、磺胺苯吡唑、酞磺胺噻唑、磺胺二甲异噁唑、磺胺噻唑、磺胺甲基异噁唑、磺胺二甲噁唑、磺胺甲二唑、磺胺硝苯、柳氮磺胺吡啶、磺胺吡啶、磺胺氯哒嗪、磺胺甲氧哒嗪、磺胺乙氧哒嗪、磺胺喹噁啉、磺胺氯吡嗪、磺胺多辛、磺胺苯酰、磺胺林、磺胺二甲基异嘧啶、磺胺间甲氧嘧啶、磺胺对甲氧嘧啶、磺胺嘧啶、磺胺二甲基嘧啶、磺胺间二甲氧嘧啶、磺胺甲基嘧啶、磺胺溴二甲嘧啶。
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