CN116082509A - 一种单域抗体及其制备方法和应用 - Google Patents
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Abstract
本发明公开了一种抗CTLA4单域抗体，所述的抗CTLA4单域抗体的可变区氨基酸序列如SEQ ID NO.1所示；所述的抗CTLA4单域抗体为鲨鱼源抗体。本发明还公开了一种编码所述的抗CTLA4单域抗体的基因，所述基因的核苷酸序列如SEQ ID NO.2所示。本发明还公开了一种重组质粒，所述的重组质粒含有SEQ ID NO.2所示的核苷酸序列。本发明还公开了一种重组表达菌，所述的重组表达菌含有所述的重组质粒。本发明制备的抗CTLA4单域抗体对CTLA4有良好的结合活性和抗肿瘤活性，因此，该单域抗体具有良好的应用前景。本发明制备的重组CTLA4单域抗体还具有重组单域抗体的优点。
Description
技术领域
本发明属于生物技术领域，具体涉及一种单域抗体及其制备方法和应用。
背景技术
CTLA4或CTLA-4(细胞毒性T淋巴细胞相关蛋白4)，也称为CD152(分化簇152)，是一种蛋白受体，其作为免疫检查点起作用并下调免疫应答。CTLA4在调节性T细胞中组成型表达，但在活化后仅在常规T细胞中上调-这种现象在癌症中特别显著。当与抗原呈递细胞表面上的CD80或CD86结合时，它起到“关闭”开关的作用。抗体药物应用存在很多问题，比如研发周期长，生产成本过高；难以大规模生产；稳定性差易降解，贮存成本高；容易被污染，维护成本费用高昂；并具有免疫原性等，限制了其在临床上的应用范围。
传统抗体分子(IgG)在结构上是由两条相同的重链和两条相同的轻链组成，此种结构在哺乳动物中均是非常保守的。抗体分子的轻链包含了1个VL区和1个CL区，而重链则拥有1个VH区和3个CH区(CH1、CH2和CH3)。VH区和VL区之间通过二硫键相互连接形成抗体的可变区(Fv)，是抗体识别抗原的最小单位，抗体可变区的序列差异决定了抗体能够特异地识别不同的抗原。而CL区和CH区则是相对保守的，被称为抗体的恒定区，其中CH区的CH2和CH3两个区域对于抗体招募免疫细胞发挥ADCC(抗体依赖的细胞介导的细胞毒性作用)和CDC(补体依赖的细胞毒性作用)功能有着重要的作用。
1995年，FlajnikMF等研究者首次从护士鲨(nurse shark)的血清中分离出一种类似免疫球蛋白重链的同源二聚物，这种同源二聚物被称为新抗原受体(Immunoglobulinnewantigenrecepter，IgNAR)，IgNAR在结构上和鱼类常有的IgW和IgM不同，其在结构上与HCABs相似，天然缺失Ig轻链结构，仅具有两条重链结构，每条重链由5个恒定区、1个铰链区和1个可变区组成。这种抗体被称为单域抗体(sdAb)，也称为域抗体。
尽管sdAb比其他形式的抗体(如Fab和scFv)小得多(通常为12-15KD)，但它具有抗原结合所需的所有元素。因此，sdAb具有许多优点，包括：1)高热稳定性和对变性剂(尿素)、蛋白酶和消化道低pH环境的耐受性；2)组织穿透力高，可穿越脑血屏障；3)更易溶于水；4)识别分子深处不能被其他形式的抗体结合的小表位；5)轻松跟踪活细胞/组织中的目标；6)高产量。sdAb的这些优点使其在生物化学研究和开发新的诊断和治疗方法方面具有巨大潜力。
发明内容
为了弥补现有技术的不足，本发明的目在于提供抗CTLA4单域抗体及其制备方法和应用。
因此，本发明一方面提供了一种抗CTLA4单域抗体，所述的抗CTLA4单域抗体的可变区氨基酸序列如SEQ ID NO.1所示。
优选地，本发明所述的抗CTLA4单域抗体为鲨鱼源抗体。
再一方面，本发明还提供了一种编码权利要求1所述的抗CTLA4单域抗体的基因，所述基因的核苷酸序列如SEQ ID NO.2所示。
再一方面，本发明还提供了一种重组质粒，所述的重组质粒含有权利要求3所述的核苷酸序列。
再一方面，本发明还提供了一种重组表达菌，所述的重组表达菌含有权利要求4所述的重组质粒。
再一方面，本发明还提供了一种所述的抗CTLA4单域抗体在制备CTLA4治疗性抗体药物中的应用。
再一方面，本发明还提供了一种所述的核苷酸序列在制备抗CTLA4单域抗体中的应用。
本发明制备的抗CTLA4单域抗体对CTLA4良好的结合活性和抗肿瘤活性。因此，该单域抗体具有良好的应用前景。
本发明制备的重组CTLA4单域抗体还具有重组单域抗体的优点。重组单域抗体的分子量小，因此组织穿透能力很强，可以进入致密组织，并且能够被快速清除；重组单域抗体稳定性高，高温下重组单域抗体的展开被证明是完全可逆的，不像传统的抗体片段，重组单域抗体在极端的pH值下也很稳定，并能够在胃液中存活。
附图说明
图1双酶切鉴定结果。其中M为marker，1和2为质粒酶切后结果。
图2菌落观察结果。
图3重组蛋白SDS-PAGE检测结果。其中M为marker，1是重组蛋白。
具体实施方式
以下结合具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。除非特别说明，以下实施例所用试剂和材料均为市购。
实施例1：抗CTLA4单域抗体可变区序列的确定
重组人CTLA4蛋白(Active)(ab167727)用鲨鱼生理盐水稀释至20μg/ml，取稀释好的抗原与佐剂1:1混合乳化。首次免疫时采用的是弗氏完全佐剂，后续免疫使用弗氏不完全佐剂。
取免疫后鲨鱼血清进行转录组测序分析鲨鱼体内抗体产生情况，具体工作委托第三方公司进行(如联川生物)。取免疫后鲨鱼的血清进行质谱检测(委托第三方公司完成)并提取免疫鲨鱼脾脏和淋巴细胞RNA进行RT-PCR获得仅含鲨鱼可变区的高通量数据库。将获得的转录组高通量测序结果与可变区的高通量数据库进行比对，筛选在两个数据库中均含有且含量较多的序列。将质谱结果与鲨鱼可变区的高通量数据库进行比对，筛选在两个数据库中均含有且含量较多的序列，结合两个筛选结果获得的可变区序列如SEQ ID NO.1所示。
可变区序列(SEQ ID NO.1)：EEVTPKSSAE NGHPGGQAAVLTCKTSGFHF PKDAMSWVRQVPGQGLGDKP YSNDYAPAIK DRFTASIDTS NNIFSVLKPD TAIYYDKPKC RYFSPSGPKD IWTPSVLKSGTQLTVQPRQK。
实施例2：抗CTLA4单域抗体在原核系统中的表达和制备
2.1密码子优化及合成
抗CTLA4单域抗体可变区基因密码子优化、基因合成及亚克隆至pET28a的Nco I和Xho I位点，并保存在DH5α菌株中，委托南京金斯瑞生物科技有限公司完成。其中密码子优化后的可变区的的核苷酸序列如SEQ ID NO.2所示(含6个His标签，共378bp)。
密码子优化后的可变区的的核苷酸序列(SEQ ID NO.2)：
gaagaagtta ccccgaaatc tagcgcagaa aacggtcatc caggtggtca ggctgctgtg
ctgacttgca aaaccagcgg ctttcacttc ccgaaagacg ctatgagctg ggttcgccag
gtaccgggtc agggcctggg cgacaaacct tacagcaacg actacgctcc ggcgatcaaa
gaccgtttca ccgcatccat cgacacctcc aacaacatct tttccgtact gaaaccggat
accgcgattt actatgataa accgaaatgc cgttacttct ccccgtccgg tccaaaagat
atttggaccc cgagcgttct gaaaagcggt actcagctga ctgtgcagcc gcgtcagaaa
caccaccacc accaccat.
2.2重组质粒鉴定
将合成的可变区基因命名为CTLA4-VNAR基因，重组质粒为pET28a-CTLA4-VNAR。具体操作为：将pET28a-CTLA4-VNAR质粒的菌液接种于卡那霉素抗性LB培养液中，提取质粒，用Nco I和Xho I双酶切对质粒进行鉴定。将鉴定为阳性的克隆菌送至尚亚生物科技有限公司进行基因测序。
用Nco I/Xho I双酶切对质粒进行鉴定，结果显示CTLA4-VNAR基因成功连入载体，凝胶电泳结果(图1)显示，Nco I/Xho I双酶切，条带大小约5231bp和378bp，质粒酶切正确。且测序结果经比对分析序列正确，为SEQ ID NO.2所示序列。
2.3菌株的构建与鉴定
2.3.1质粒转化与菌落形态观察将pET28a-CTLA4-VNAR质粒转化至BL21(DE3)感受态细胞中，在含卡那霉素的LB琼脂平板上进行涂布，36～37℃，恒温倒置培养16～20小时。观察菌落形态。
2.3.2菌种保存与菌体形态观察从2.3.1平板上挑取单菌落，接种至3ml含卡那霉素的LB培养基中，36～37℃，220～240r/min振荡培养至OD600nm值达0.4～0.6，收获部分菌液加入无菌甘油，甘油终浓度为25％，保存备用。在光学显微镜下进行菌体形态观察。
将pET28a-CTLA4-VNAR质粒转化至BL21(DE3)感受态细胞中，在含卡那霉素的LB琼脂平板上进行涂布，36～37℃，恒温倒置培养16～20小时观察，菌落为乳白色光滑圆形形态，形态大小均一，边缘整齐。见图2所示，为典型的大肠杆菌菌落形态，无其他杂菌生长。
2.4重组蛋白表达和纯化
取2.3.2菌液经37℃放大培养，然后降温至20℃，经IPTG诱导表达，收集菌体；将菌体进行重悬、破碎、离心收集上清，进行镍柱纯化，将收集的目的蛋白通过硫酸铵沉淀浓缩后复溶和换液，通过0.22μm低蛋白吸附滤膜过滤除菌后获得重组蛋白，定量分装(0.5ml/管)，置于-80℃冰箱保存备用。
2.5总蛋白含量测定
取2.4制备的重组蛋白，使用BCA检测试剂盒进行到蛋白含量检测，该重组蛋白含量为1.56mg/ml。
2.6重组蛋白纯度检测
取2.4制备的重组蛋白，使用SDS-PAGE进行纯度检测。经过跑胶和灰度计算，该重组蛋白的纯度为95％。具体见图3所示，分子量约为13.8kDa。
实施例3：抗CTLA4单域抗体的体外活性检测
使用双抗夹心ELISA进行体外活性检测验证，具体方法为：用碳酸钠-碳酸氢钠缓冲液(CBS，pH 9.6)将实施例2制备的重组蛋白稀释至1μg/ml浓度，每孔100μl铺板，4℃包板过夜。PBST洗3次后，每孔加入含2％BSA的PBST溶液37℃孵育2小时。PBST洗3次后，将实施例1中的CTLA4蛋白分别用含2％BSA的PBST溶液稀释至0.1μg/ml、1μg/ml，每孔加入100μl，37℃孵育1h。HRP Anti-CTLA4抗体[KT50](ab106490)作为二抗，使用PBST进行1:5000稀释，37℃孵育1h，PBST洗3次，拍干。每孔加入100μl TMB显色液，室温下避光反应10min，反应结束后每孔加入50μl 2M H2SO4，通过酶标仪测定样品450nm的吸光值。
检测结果见表1所示，该单域抗体能特异性的与CTLA4蛋白结果，说明该单域抗体具有良好的活性。
表1双抗体夹心法检测结果
实施例4：抗CTLA4单域抗体小鼠体内抗肿瘤活性评价
参照CN 110272490 B实施例10的方法，简述如下：采用MC38同系小鼠模型，使用人CTLA4基因敲入的C57BL/6小鼠评价抗体在小鼠体内的抗肿瘤活性。实验设计为，选择40只人CTLA4基因敲入的C57BL/6小鼠，分为4组，每组10只，使用Ipilimumab、同型抗体hIgG1和重组Anti-CTLA4抗体[CAL49](ab237712)作为对照，样品为抗CTLA4单域抗体。给药途径为腹腔注射，给药剂量为10mg/kg，在0、3、6、10天腹腔注射，第24天处死小鼠，测量肿瘤体积，小鼠体重，肿瘤重量及小鼠存活率，计算每组的中位生存期。
结果表明，本发明的抗体能够显著抑制肿瘤生长，抑制效果显著优于对照抗体(本发明单域抗体的中位生存期为22天，优于Ipilimumab的中位生存期18天，优于重组Anti-CTLA4抗体[CAL49](ab237712)的中位生存期17天，对照IgG的中位生存期为13天)。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
Claims (7)
1.一种抗CTLA4单域抗体，其特征在于，所述的抗CTLA4单域抗体的可变区氨基酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的抗CTLA4单域抗体，其特征在于，所述的抗CTLA4单域抗体为鲨鱼源抗体。
3.一种编码权利要求1所述的抗CTLA4单域抗体的基因，其特征在于，所述基因的核苷酸序列如SEQ ID NO.2所示。
4.一种重组质粒，其特征在于，所述的重组质粒含有权利要求3所述的核苷酸序列。
5.一种重组表达菌，其特征在于，所述的重组表达菌含有权利要求4所述的重组质粒。
6.一种如权利要求1所述的抗CTLA4单域抗体在制备CTLA4治疗性抗体药物中的应用。
7.一种如权利要求3所述的核苷酸序列在制备抗CTLA4单域抗体中的应用。
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