CN115785277A - 一种抗IL4i1纳米抗体的制备及其应用 - Google Patents
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Abstract
本发明属于分子生物学技术领域，具体涉及一种抗IL4i1纳米抗体的制备及其应用。本发明提供的抗IL4i1纳米抗体具有独特的3个互补决定区CDR1、CDR2、CDR3，共筛选出9条抗IL4i1纳米抗体的序列。本发明还提供了编码上述纳米抗体或其VHH链的编码序列、相应的表达载体和能够表达该纳米抗体的宿主细胞，以及本发明抗IL4i1纳米抗体的生产方法。本发明提供的纳米抗体对IL4i1具有特异的识别和结合能力，具有特异性强、灵敏度高等优势；同时有效地降低了IL4i1抗体的开发和生产成本，缩减了抗体表达时间。
Description
技术领域
本发明属于分子生物学技术领域，具体涉及一种抗IL4i1纳米抗体的制备及其应用。
背景技术
白细胞介素-4诱导基因1 (IL4I1)是一种由抗原提呈细胞表达的L -苯丙氨酸氧化酶。在体外，IL4I1抑制T细胞增殖和细胞因子的产生，促进naïve CD4+ T细胞分化为调节性T细胞，有一部分原因依靠其产生H2O2和从T细胞微环境中消耗苯丙氨酸的能力。IL4I1在大多数人类肿瘤类型的肿瘤床中都能强烈地检测到，在某些情况下，如某些B细胞淋巴瘤，它是由肿瘤细胞本身表达的。局部产生IL4I1通过抑制抗肿瘤CD8+ T细胞的增殖和功能，改变肿瘤微环境中免疫细胞向免疫抑制人群的平衡，促进小鼠模型中肿瘤的生长。此外，IL4I1在细胞中表达的位置和密度与患者变差的临床参数相关，并倾向于导致黑色素瘤患者更短的生存期。研究者还发现，IL4I1蛋白或mRNA的存在与肾癌、神经胶质瘤、结肠癌和乳腺癌预后质量有关。这些数据都表明，旨在抑制肿瘤患者IL4I1表达或降低其活性的治疗策略可能会提高肿瘤患者的免疫控制。而且这种策略似乎不伤害宿主，因为有研究表明KO小鼠中IL4I1的缺失似乎不会导致特定的病理或过早死亡。因此，靶向IL4I1可能会是一种新的肿瘤免疫治疗策略。在Cell杂志发表的一项研究中，来自德国癌症研究中心（DKFZ）和柏林健康研究所（BIH）的科学家们发现，肿瘤中大量产生的一种代谢酶IL4I1（Interleukin-4-Induced-1）能促进肿瘤细胞的扩散并抑制免疫系统。他们的研究表明IL4I1是一种代谢免疫检查点，IL4i1抑制剂在将来有可能会为癌症治疗带来新的机遇。
纳米抗体有来源于成年骆驼体内HCAbs的最小的功能性抗原结合片段，具有高度稳定性和与抗原结合的高亲合力，能与蛋白裂隙和酶活性位点相互作用，使之作用类似抑制剂。因此，纳米抗体可以为从肽模拟药物设计小分子酶抑制物提供新的思路。由于仅有重链，纳米抗体的制造较单克隆抗体容易。纳米抗体的独特性质，如处于极端温度和pH环境中的稳定性，可以低成本制造大产量。
因此，纳米抗体在疾病的治疗和诊断中具有很大的价值，在肿瘤的抗体靶向诊断和治疗中也具有很大的发展前景。相对于常规的四链抗体的scFv而言，纳米抗体在亲和力方面与其对应的scFv相当，但在可溶性、稳定性、对聚集的抗性、可重折叠性、表达产率以及DNA操作、文库构建和3-D结构测定的容易性方面超越scFv。
噬菌体展示技术是一种将多肽或蛋白质展示在噬菌体的表面，从而将所需性质的多肽或蛋白质进行体外筛选的技术。将编码目的蛋白的基因融合到噬菌体衣壳蛋白的基因之中，就能使目的蛋白展示在噬菌体的表面，从而将基因型和表型联系起来。随后，在体外进行几轮亲和筛选（如生物淘选）得到一些噬菌体克隆，再将这些噬菌体克隆扩增，进行更多轮的筛选，便可进一步得到特异性较强的目的蛋白。
目前最广泛使用的单细胞分析技术是高通量单细胞RNA测序（Single-cell RNAsequencing , scRNA-seq）。自动化的高通量单细胞RNA测序技术可以提供一种无偏见和高效的方法对单细胞转录组进行高通量检测，以获得微环境的单细胞转录组图谱。基于液滴的高通量单细胞RNA测序技术出现，可以将单次检测细胞数量提高到上万个，真正实现了高通量检测。因此，高通量的单细胞RNA测序技术的实现依赖于微流控自动化技术和高通量测序技术的应用，前者实现高通量的单细胞分离而后者实现自动化的DNA测序。
噬菌体展示技术、生物淘洗、高通量测序等都是进行纳米抗体生产的常用技术，其中噬菌体展示技术由于需要噬菌体的包装及淘选，此技术均是在原核表达系统中进行，淘选需要经过至少3轮，所以耗时较长，且原核表达的抗体相对于真核表达系统对蛋白的修饰与折叠仍有一定的不足，一定程度上影响了抗体的亲和力。高通量测序技术数据规模庞大，类型复杂，包括转录组，基因组，蛋白质组等，可重复性不强，数据储存与可视化也是待优化的问题。样品污染也是影响测序结果准确度的一大问题。
发明内容
本发明的目的就是提供一种能够充分发挥纳米抗体的优越性能，既具有优异的特异性抗原结合能力，又能克服传统纳米抗体筛选方法耗时长，原核表达不能加工修饰的缺点，通过微流控筛选的方法结合高通量表达的平台，筛选到抗IL4i1抗体，并进一步提供其在肿瘤治疗药物和诊断制剂制备中的应用。
针对上述目的，本发明采用的技术方案为：
一种抗IL4i1纳米抗体，所述纳米抗体包括框架区和互补决定区；所述互补决定区包括CDR1、CDR2、CDR3，氨基酸序列如下：所述互补决定区CDR1序列为SEQ ID NO.1，所述互补决定区CDR2序列为SEQ ID NO.2，所述互补决定区CDR3序列为SEQ ID NO.3；或者所述互补决定区CDR1为SEQ ID NO.4，所述互补决定区CDR2为SEQ ID NO.5，所述互补决定区CDR3为SEQ ID NO.6；或者所述互补决定区CDR1为SEQ ID NO.7，所述互补决定区CDR2为SEQ IDNO.8，所述互补决定区CDR3为SEQ ID NO.9；或者所述互补决定区CDR1为SEQ ID NO.7，所述互补决定区CDR2为SEQ ID NO.10，所述互补决定区CDR3为SEQ ID NO.11；或者所述互补决定区CDR1为SEQ ID NO.12，所述互补决定区CDR2为SEQ ID NO.13，所述互补决定区CDR3为SEQ ID NO.14；或者所述互补决定区CDR1为SEQ ID NO.15，所述互补决定区CDR2为SEQ IDNO.16，所述互补决定区CDR3为SEQ ID NO.17；或者所述互补决定区CDR1为SEQ ID NO.18，所述互补决定区CDR2为SEQ ID NO.19，所述互补决定区CDR3为SEQ ID NO.20；或者所述互补决定区CDR1为SEQ ID NO.21，所述互补决定区CDR2为SEQ ID NO.22，所述互补决定区CDR3为SEQ ID NO.23；或者所述互补决定区CDR1为SEQ ID NO.24，所述互补决定区CDR2为SEQ ID NO.25，所述互补决定区CDR3为SEQ ID NO.26；具体序列信息如下表1所示。
表1 本发明IL4i1纳米抗体互补决定区具体序列
优选地，所述抗IL4i1纳米抗体具有选自以下任一种的氨基酸序列：ANb32-M384-4M-673对应的氨基酸序列如SEQ ID NO.27所示；ANb32-M384-4M-865对应的氨基酸序列如SEQ ID NO.28；ANb32-M384-4M-584对应的氨基酸序列如SEQ ID NO.29所示；ANb32-M384-4M-769对应的氨基酸序列如SEQ ID NO.30所示；ANb32-M384-4M-761对应的氨基酸序列如SEQ ID NO.31所示；ANb32-M384-4M-480对应的氨基酸序列如SEQ ID NO.32所示；ANb32-M384-4M-577对应的氨基酸序列如SEQ ID NO.33所示；ANb32-M384-4M-665对应的氨基酸序列如SEQ ID NO.34所示；ANb32-M384-4M-672对应的氨基酸序列如SEQ ID NO.35所示。
优选地，编码所述抗IL4i1纳米抗体的核苷酸序列分别如下：ANb32-M384-4M-673对应的核苷酸序列如SEQ ID NO.36所示；ANb32-M384-4M-865对应的核苷酸序列如SEQ IDNO.37所示；ANb32-M384-4M-584对应的核苷酸序列如SEQ ID NO.38所示；ANb32-M384-4M-769对应的核苷酸序列如SEQ ID NO.39所示；ANb32-M384-4M-761对应的核苷酸序列如SEQID NO.40所示；ANb32-M384-4M-480对应的核苷酸序列如SEQ ID NO.41所示；ANb32-M384-4M-577对应的核苷酸序列如SEQ ID NO.42所示；ANb32-M384-4M-665对应的核苷酸序列如SEQ ID NO.43所示；ANb32-M384-4M-672对应的核苷酸序列如SEQ ID NO.44所示。
本发明还提供了一种分子表达载体，所述载体包含所述SEQ ID NO.36~SEQ IDNO.44的核苷酸序列中的一种。
本发明还提供了一种含有所述表达载体的宿主细胞，所述细胞为真核细胞，优选为哺乳动物细胞。
本发明还提供了一种所述纳米抗体的制备方法，制备过程如下：
S1、依据IL4i1的蛋白序列和基因序列信息，分析并设计免疫抗原，在其C端连接His-tag，获得经修饰的抗原；
S2、用步骤S1获得的抗原对羊驼进行免疫，通过液滴微流控技术筛选得到可以分泌目标抗体的B细胞；
S3、以步骤S2获得的B细胞为原材料，提取RNA，并反转录成cDNA，经PCR获得目的基因片段，然后将目的基因片段克隆至真核表达载体中，并转化入感受态细胞，构建IL4i1-VHH单克隆表达库；
S4、以步骤S3所构建的IL4i1-VHH单克隆表达库为模板，提取质粒，并进一步经细胞转染，筛选阳性克隆，即得。
优选地，步骤S3所述真核表达载体为PcDNA3.4载体。
本发明还提供了一种所述纳米抗体在制备预防和治疗肿瘤药物中的应用。
本发明还提供了一种所述的纳米抗体在制备肿瘤检测试剂中的应用。
与现有技术相比，本发明提供的抗IL4i1纳米抗体具有如下优势：
本发明提供的抗IL4i1纳米，其氨基酸序列的排列方式为框架序列+CDR1+框架序列+CDR2+框架序列+CDR3的方式排列。
（1）本发明结合使用细胞微流控技术，能够比较直观的获得抗体序列，在较短时间内获得了大量的IL4i1纳米抗体基因，实现了纳米抗体的大规模生产，有利于IL4i1的进一步深入研究利用；
（2）本发明利用高通量哺乳动物细胞表达系统对IL4i1纳米抗体表达，有效地降低了IL4i1抗体的开发和生产成本，缩减了抗体表达时间，同时增加了通量，提高了效率。
附图说明
图1为ELISA检测结果图；
图2为目的片段克隆部分结果图；
图3为单克隆ELISA检测结果图；
图4为阳性克隆筛选结果图。
具体实施方式
下面结合具体实施例对本发明作进一步解释，但是应当注意的是，以下实施例仅用以解释本发明，而不能用来限制本发明，所有与本发明相同或相近的技术方案均在本发明的保护范围之内。本实施例中未注明具体技术或条件者，按照本领域常规技术方法和仪器说明书内容进行操作；所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
本发明首先构建IL4i1抗原对羊驼进行四次免疫后，获得羊驼PBMC细胞；其次通过微流控技术对PBMC细胞进行筛选分离；对单个细胞进行RNA的捕获，通过巢氏PCR的方法获得抗体基因片段，构建真核表达载体；然后通过哺乳动物细胞高通量表达系统诱导抗体高通量表达；最后通过ELISA与酶活抑制检测（利用Amplex® Red Catalase Assay Kit试剂盒进行），及测序结果分析获得了具有高的灵敏度和酶活抑制的抗体。
实施例一种抗IL4i1纳米抗体
所述抗IL4i1纳米抗体共9种，分别为ANb32-M384-4M-673、ANb32-M384-4M-865、ANb32-M384-4M-584、ANb32-M384-4M-769、ANb32-M384-4M-761、ANb32-M384-4M-480、ANb32-M384-4M-577、ANb32-M384-4M-665、ANb32-M384-4M-672。
所述抗IL4i1纳米抗体的制备过程如下：
S1、依据IL4i1的蛋白序列和基因序列信息，分析并设计可有效诱导羊驼产生针对human IL4i1的特异性抗体的抗原，在其C端连接His-tag，获得经修饰的抗原；
S2、用步骤S1获得的经修饰的抗原与弗氏佐剂的混合液对羊驼进行四次免疫，通过液滴微流控技术筛选得到可以分泌VHH有结合活性的B细胞；
用200μg、human IL4i1/His蛋白与200μL弗氏完全佐剂的乳化混合物对羊驼进行初免，在第21天、42天、63天用human IL4i1/His蛋白（即步骤S1获得的经修饰的抗原）与200μL弗氏不完全佐剂加强免疫3次，每次免疫1周后，采血检测Anti-IL4i1/His血清滴度；第4次免疫1周后，采血50mL用于微流控平台进行单细胞分选。
Anti-IL4i1/His血清效价通过ELISA检测，用浓度为2μg/mL的IL4i1/His蛋白包被酶标板，每孔加入2倍梯度稀释的血清100μL（对照为免疫前羊驼血清），37℃孵育1.5h，洗涤2次，每孔加入1：10000稀释的辣根过氧化物酶标记的Goat anti-Alpaca IgG(H+L)二抗，37℃孵育1h，洗涤5次后，加100μL TMB底物，37℃孵育10min，50μL 0.1M的H2SO4中止反应，测定OD 450nm。结果如图1所示，ELISA检测血清效价为在OD450是空白对照的2倍的稀释数值，4免后的抗血清效价为102400。由此可见，该抗原可诱导羊驼产生特异性针对IL4i1蛋白的高滴度抗血清。
分选细胞：第4次免疫1周后，采血50mL用于微流控平台进行有功能的抗体分泌细胞分选，液滴微流控技术可以将细胞与抗原包裹在一个个皮升级别的单分散油滴中，可以达到每秒数千个单分散液滴的生成速度，获取单细胞每个微液滴都是独立的，实现了细胞与细胞之间的环境独立，互不交叉污染；在油滴内通过荧光标记的羊驼二抗去识别IL4i1纳米抗体，同时如果细胞分泌的VHH抗体能够识别荧光标记的抗原，就能形成FRET信号而被分选出来，筛选得到可以分泌VHH有结合活性的Ｂ细胞。
S3、以步骤S2获得的B细胞为原材料，提取RNA，并反转录成cDNA，经PCR获得目的基因片段，然后将目的基因片段克隆至真核表达载体中，并转化入感受态细胞，构建IL4i1-VHH单克隆表达库；
以步骤S2筛选得到的B细胞为模板，利用Trizol法提取RNA，并利用oligo(dT)反转为cDNA，通过引物扩增，以及分子克隆等技术获得目的片段，目的片段的扩增体系如下表2所示，扩增程序如下表3所示。目的片段克隆结果如图2（部分结果）；将羊驼的VHH基因克隆至真核表达载体pCDNA3.4中，转化至感受态细胞Top 10中，得到VHH单克隆表达库。为了进一步鉴定IL4i1-VHH单克隆表达库是否构建成功，在涂的单克隆菌板中，挑选10个克隆进行测序，测序结果显示，阳性克隆率和序列多样性为100％；比对结果显示，差异序列大多在CDR结合区。经检测，该构建得到一个IL4i1-VHH单克隆表达库。
上游引物DFL-（03-16）的序列信息分别如下：
DFL-03：AGATCTACACATGGCCCAGGTGC
DFL-04：GAGATCTACACATGGCCCAGKTGCAGC
DFL-05：TACACATGGCCGAGGTGCAG
DFL-07：ATCTACACATGGCCCAGTCGCA
DFL-08：CTACACATGGCCCAGCCGCAS
DFL-09：AGATCTACACATGGCCCAGCCGCAS
DFL-10：ATCTACACATGGCCCAGTTG
DFL-11：ATCTACACATGGCCCAGGTGCA
DFL-12：GAGATCTACACATGGCCGAGGTGCAGY
DFL-13：CGAGATCTACACATGGCCGAGGTRS
DFL-14：GATCTACACATGGCCGCGGTA
DFL-15：GATCTACACATGGCCGAGTTGCAAC
DFL-16：CGAGATCTACACATGGCCAGYTKGGTG
下游引物DFL-06的序列信息为：CATACGAGAT ACTAGTTGAGGAGACR
其中，Y代表C或T碱基，K代表G或T碱基，R代表A或G碱基，S代表C或G碱基。
表2 目的片段扩增体系
表3 扩增程序
S4、以步骤S3所构建的IL4i1-VHH单克隆表达库为模板，提取质粒，并进一步经细胞转染，筛选阳性克隆，即得。
3.4 细胞转染与筛选：
将质粒转染至哺乳动物细胞HEK-293中，将细胞悬液分别加入对应的96孔细胞培养板中，每孔约5×104个细胞，37℃、5% CO2细胞培养箱内培养，连续培养72h。
转染后的细胞上清与IL4i1蛋白进行结合检测。即binding ELISA，具体检测过程为：抗原IL4i1包被2μg/mL 4℃过夜，细胞上清每孔100μL，25℃孵育1h，加入二抗25℃孵育1h，显色终止读数即可。单克隆ELISA结果显示，十板96孔细胞板共961个单克隆得到377个阳性克隆（如图3所示，部分结果展示），并将这些序列进行测序比对剔除重复序列。为了进一步验证文库中结合IL4i1-VHH蛋白的阳性抗体，将细胞上清进行酶活抑制实验，结果显示，377个ELISA阳性克隆中有9个抑制活性较好的阳性抗体（如图4所示，部分结果展示）。
最后，需要说明的是，上述实施例仅例示性说明本发明的原理、性能及功效，而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下，对上述实施例进行修饰或改变。因此，举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变，仍应由本发明的权利要求所涵盖。
Claims (9)
1.一种抗IL4i1纳米抗体，其特征在于，所述纳米抗体包括框架区和互补决定区；所述互补决定区包括CDR1、CDR2、CDR3，氨基酸序列如下：所述互补决定区CDR1序列为SEQ IDNO.1，所述互补决定区CDR2序列为SEQ ID NO.2，所述互补决定区CDR3序列为SEQ ID NO.3；或者所述互补决定区CDR1为SEQ ID NO.4，所述互补决定区CDR2为SEQ ID NO.5，所述互补决定区CDR3为SEQ ID NO.6；或者所述互补决定区CDR1为SEQ ID NO.7，所述互补决定区CDR2为SEQ ID NO.8，所述互补决定区CDR3为SEQ ID NO.9；或者所述互补决定区CDR1为SEQID NO.7，所述互补决定区CDR2为SEQ ID NO.10，所述互补决定区CDR3为SEQ ID NO.11；或者所述互补决定区CDR1为SEQ ID NO.12，所述互补决定区CDR2为SEQ ID NO.13，所述互补决定区CDR3为SEQ ID NO.14；或者所述互补决定区CDR1为SEQ ID NO.15，所述互补决定区CDR2为SEQ ID NO.16，所述互补决定区CDR3为SEQ ID NO.17；或者所述互补决定区CDR1为SEQ ID NO.18，所述互补决定区CDR2为SEQ ID NO.19，所述互补决定区CDR3为SEQ IDNO.20；或者所述互补决定区CDR1为SEQ ID NO.21，所述互补决定区CDR2为SEQ ID NO.22，所述互补决定区CDR3为SEQ ID NO.23；或者所述互补决定区CDR1为SEQ ID NO.24，所述互补决定区CDR2为SEQ ID NO.25，所述互补决定区CDR3为SEQ ID NO.26。
2.如权利要求1所述的抗IL4i1纳米抗体，其特征在于，所述抗IL4i1纳米抗体具有选自以下任一种的氨基酸序列：SEQ ID NO.27、SEQ ID NO.28、SEQ ID NO.29、SEQ ID NO.30、SEQ ID NO.31、SEQ ID NO.32、SEQ ID NO.33、SEQ ID NO.34、SEQ ID NO.35。
3.如权利要求2所述的抗IL4i1纳米抗体，其特征在于，编码所述抗IL4i1纳米抗体氨基酸序列的核苷酸序列为下列序列之一：SEQ ID NO.36、SEQ ID NO.37、SEQ ID NO.38、SEQID NO.39、SEQ ID NO.40、SEQ ID NO.41、SEQ ID NO.42、SEQ ID NO.43、SEQ ID NO.44。
4.一种分子表达载体，其特征在于，所述载体包含权利要求3所述SEQ ID NO.36~SEQID NO.44的核苷酸序列中的一种。
5.一种含有权利要求4所述表达载体的宿主细胞，其特征在于，所述细胞为真核细胞。
6.一种如权利要求1-5任一项所述抗IL4i1纳米抗体的制备方法，其特征在于，制备过程如下：
S1、依据IL4i1的蛋白序列和基因序列信息，分析并设计免疫抗原，在其C端连接His-tag，获得经修饰的抗原；
S2、用步骤S1获得的抗原对羊驼进行免疫，通过液滴微流控技术筛选得到可以分泌目标抗体的Ｂ细胞；
S3、以步骤S2获得的B细胞为原材料，提取RNA，并反转录成cDNA，经PCR获得目的基因片段，然后将目的基因片段克隆至真核表达载体中，并转化入感受态细胞，构建IL4i1-VHH单克隆表达库；
S4、以步骤S3所构建的IL4i1-VHH单克隆表达库为模板，提取质粒，并进一步经细胞转染，筛选阳性克隆，即得。
7.如权利要求6所述的制备方法，其特征在于，步骤S3所述真核表达载体为PcDNA3.4载体。
8.一种权利要求1所述抗IL4i1纳米抗体在制备预防和治疗肿瘤药物中的应用。
9.一种权利要求1所述的抗IL4i1纳米抗体在制备肿瘤检测试剂中的应用。
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