CN116023492A - 一种阻断pd1与pdl1结合的抗人pdl1纳米抗体的制备方法及其编码序列 - Google Patents
一种阻断pd1与pdl1结合的抗人pdl1纳米抗体的制备方法及其编码序列 Download PDFInfo
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Abstract
本发明公开了一种抗人PDL1纳米抗体以及其VHH链,包括框架区FR和互补决定区CDR区,框架区选自FR1‑FR4的氨基酸序列,CDR区选自CDR1‑CDR3氨基酸序列。同时还公开了编码该纳米抗体的基因序列和表达该纳米抗体的宿主细胞。该PDL1纳米抗体分子量小,特异性强。该纳米抗体能在大肠杆菌内可溶性表达蛋白,制备方法简单,成本低廉,在对人PDL1检测方面以及在抗肿瘤药物中具有良好的应用前景。
Description
技术领域
本发明属于生物医学或者生物制药技术领域,涉及一种针对阻断PD1与PDL1结合的抗人PDL1纳米抗体的制备方法及其编码序列。
背景技术
目前肿瘤的免疫治疗方法是肿瘤治疗方法中较为有前景的方法之一。免疫治疗的主要靶点是针对免疫检查点分子。T细胞介导的细胞免疫在肿瘤细胞的识别和杀伤方面都起着重要作用,由于癌细胞表达的蛋白质突变以及异常加工而产生的新抗原,这些特异性抗原可以被T细胞表面受体所识别,从而激发免疫系统的作用,消除癌细胞。但是T细胞表面受体在识别特异性抗原的过程有一系列的免疫检查点分子调节。在正常体内,免疫检查点是免疫系统维持自我耐受和阻止自身免疫的抑制途径。但是癌细胞能利用免疫检查点的机制,通过靶向作用免疫检查点,从而逃避 T细胞调节的免疫反应。当身体受到肿瘤攻击时,免疫检查点被激活,从而抑制自身免疫,促进肿瘤细胞生长和逃逸。
目前对免疫检查点抑制剂药物的研发应用在肿瘤的治疗领域取得了较好的结果,免疫检查点抑制剂的研究引起了越来越多的关注。特别是针对程序性死亡配体1(PDL1,B7-H1,CD274)和程序性死亡受体-1(PD1,CD279)的药物,对多种类型肿瘤的治疗均有显著效果。PD-1是在活化T细胞表面诱导的一种共刺激分子。是CD4+和CD8+T细胞表达的CD28免疫球蛋白家族的成员,与B7家族中的配体(PD-L1和PD-L2)相互作用,PD-L1比PD-L2表达广泛,PD-L1 (B7-H1,CD274)在肿瘤细胞、以及包括树突状细胞、巨噬细胞、B细胞和活化T细胞等抗原呈递细胞(APCs)上均表达,因此,PDL1常被用作抗癌药物设计的靶点。在正常细胞中,PD-1与其配体的结合限制了T细胞的活性,从而防止过度刺激和维持对自身抗原的免疫耐受。但是肿瘤细胞通过上调表达PDL1蛋白,PD-1/PD-L1相互作用,减弱内源性抗肿瘤免疫反应,抑制T淋巴细胞增殖、细胞因子释放和细胞毒性,导致肿瘤特异性T细胞的耗竭和凋亡。因此,阻断PD-1/PD-L1相互作,能增强T细胞活性,从而达到肿瘤免疫治疗的效果。用阻断PD-1/PDL1通路的抗体进行免疫治疗,已显示出令人印象深刻的临床结果:肿瘤持续消退,患者生存率提高。目前FDA通过的靶向PD1的单克隆抗体有Nivolumab和Pembrolizumab,靶向PDL1的单克隆抗体有Atezolizumab、avelumab、durvalumab。
目前针对PDL1的抗体大多数为单克隆抗体,但是传统的单克隆抗体的制备过程较复杂,生产成本较高。除此之外,单克隆抗体的分子量较大,存在组织穿透能力较差的问题,不能在肿瘤组织处发挥最大的治疗效果。纳米抗体,是目前最小的可以结合抗原的功能性片段,只有单克隆抗体大小的1/10。由于其不同于单抗的独特结构,所以不会造成红细胞和血小板发生凝聚,则在治疗过程中不会产生出血和血小板减少等副作用。除此之外,纳米抗体还具有可溶性好,稳定性较高,在原核表达体系中也能表达,这就克服了传统抗体制备周期长,制造成本较高的问题,是一种非常有应用前景的新型抗体分子。
目前针对PDL1靶点的纳米抗体还未有临床应用,所以本领域迫切需要开发新的有效针对PDL1靶点的特异性纳米抗体。
发明内容
发明目的:本发明所要解决的技术问题是提供一种可以阻断PD1与其配体PDL1结合的抗PDL1纳米抗体,同时提供该纳米抗体的编码序列及纳米抗体的制备方法。
技术方案:为实现上述目的,本发明的第一部分,一种抗人PDL1纳米抗体的VHH链,包括框架区(FR区)和抗原互补决定区(CDR区)。所述框架区选自以下的FR1-FR4的氨基酸序列:
SEQ ID No .1所示的FR1,SEQ ID No .2 所示的FR2,SEQ ID No .3 所示的FR3 ,SEQ ID No .4 所示的FR4;
或SEQ ID No .5所示的FR1,SEQ ID No .6 所示的FR2,SEQ ID No .7 所示的FR3, SEQ ID No .8 所示的FR4;
或SEQ ID No .9所示的FR1,SEQ ID No .10所示的FR2 ,SEQ ID No .11所示的FR3 , SEQ ID No .12 所示的FR4;
或SEQ ID No .13所示的FR1,SEQ ID No .14所示的FR2,SEQ ID No .15 所示的FR3,SEQ ID No .16 所示的FR4;
或SEQ ID No .17所示的FR1,SEQ ID No .18 所示的FR2,SEQ ID No .19 所示的FR3,SEQ ID No .20 所示的FR4;
所述的抗原互补决定区选自以下的CDR1-CDR3的氨基酸序列:
SEQ ID No .21所示的CDR1,SEQ ID No .22所示的CDR2,SEQ ID No .23所示的CDR3;
或SEQ ID No .24所示的CDR1,SEQ ID No .25所示的CDR2,SEQ ID No .26所示的CDR3;
或 SEQ ID No .27所示的CDR1,SEQ ID No .28所示的CDR2,SEQ ID No .29所示的CDR3;
或SEQ ID No .30所示的CDR1,SEQ ID No .31所示的CDR2,SEQ ID No .32所示的CDR3;
或SEQ ID No .33所示的CDR1,SEQ ID No 34所示的CDR2,SEQ ID No .35所示的CDR3;
优选地,它具有SEQ ID No .36,SEQ ID No .37,SEQ ID No .38,SEQ ID No .39,SEQ ID No .40所示的氨基酸序列。
本发明的第二部分,一种抗人PDL1纳米抗体(Nb1,Nb2,Nb3,Nb4,Nb5),它是针对人PDL1分子表位的纳米抗体,包括具有SEQ ID No .36,SEQ ID No .37,SEQ ID No .38,SEQID No .39,SEQ ID No .40所示的氨基酸序列的VHH链。
本发明的第三部分,提供了一种DNA分子,它编码选自下组的蛋白质:权利要求1或2所示的抗人PDL1纳米抗体的VHH链,或权利3所示的抗人PDL1纳米抗体。
优选地,所述的DNA分子,它具有选自下组的DNA序列:SEQ ID No .41,SEQ ID No.42,SEQ ID No .43,SEQ ID No .44,SEQ ID No .45。
本发明的第四部分,提供了一种表达载体,其特征在于,它含SEQ ID No .41,SEQID No .42,SEQ ID No .43,SEQ ID No .44,SEQ ID No .45所示的核苷酸序列。
本发明的第五部分,提供了一种宿主细胞,其转化了所述的重组表达载体或者基因组上整合了所述的编码所述的抗PDL1纳米抗体的核苷酸序列的宿主细胞及其后代细胞。
所述的宿主细胞及其后代细胞指细菌细胞、真菌细胞、动物细胞或者植物细胞及这些宿主细胞的后代。
本发明的第六部分,本发明提供一种制备抗PDL1抗体的方法,其包括:
(1)利用人PDL1真核蛋白免疫骆驼,制备噬菌体展示文库。
(2)用人PDL1蛋白亲和筛选噬菌体展示文库;(3)PE-ELISA鉴定阳性克隆;(4)抗PDL1纳米抗体的表达和纯化;(5)抗PDL1纳米抗体功能的鉴定。
本发明的第七部分,提供了本发明所述的人PDL1纳米抗体用于检测人PDL1的用途。
所述抗PDL1纳米抗体可用于制备检测样品中PDL1的检测试剂盒或诊断试剂盒。
所述的抗PDL1纳米抗体在治疗癌症药物中的应用。
所述的抗PDL1纳米抗体可作为免疫检查点抑制剂,作为癌症治疗药物单独使用,也可以联合其他抗癌药物。
与现有技术相比较,本发明的技术方案有以下优点:
本发明利用真核人PDL1真核蛋白免疫新疆双峰驼,分离骆驼外周血淋巴细胞扩增VHH基因序列,构建纳米抗体噬菌体展示文库。利用固相亲和淘洗的方法筛选文库,最终获得抗人PDL1特异性纳米抗体基因序列。
本发明未将筛选得到的阳性克隆序列亚克隆至其他表达载体上,而是直接利用pMECS载体和TG1宿主菌直接表达抗体蛋白,这就大大缩短抗体获得的时间和成本。
本发明构建的噬菌体展示文库库容较大,筛选得到的纳米抗体的多样性较高。
与常规抗体相比较,本发明的抗PDL1纳米抗体分子量小(约18kDa),亲和力高,特异性较好。有开发作为免疫检查点抑制剂的潜力,为肿瘤治疗提供备选方案。
附图说明
图1是分离骆驼外周血中的淋巴细胞后提取总RNA(TRIZOL法);
图2是第一轮PCR扩增产物切胶回收750bp的DNA片段琼脂糖凝胶电泳图;
图3是第二轮PCR扩增产物切胶回收450bp左右的DNA片段琼脂糖凝胶电泳图;
图4是纳米抗体表达后SDS-PAGE电泳后考马斯亮蓝染色图,通过高低渗的方法获得蛋白,图片从左至右分别为泳道1菌体未诱导总蛋白、泳道2为通过高低渗获得的周质蛋白、泳道3为利用HIS亲和层析纯化的抗体蛋白、泳道5为蛋白超滤、泳道6为蛋白分子量Marker,
图5是Nb1,Nb2,Nb3,Nb4,Nb5五个抗PDL1纳米抗体纯化结果图;
图6是间接ELISA法检测抗PDL1纳米抗体的特异性结合结果图;
图7是其中一个抗PDL1纳米抗体亲和力测定曲线图;
具体实施方式
下面结合实施例和附图对本发明做进一步详细的说明,但这些具体实施例不应以任何方式被解释为限制本发明的应用范围。
本发明首先利用真核人PDL1蛋白免疫了一只雌性新疆双峰驼,在连续免疫了六次后,抽取骆驼外周血分离获得该骆驼的外周血淋巴细胞,构建了PDL1特异的单域重链抗体免疫文库。然后采用固相亲和淘选的方法利用真核人PDL1蛋白对免疫文库进行筛选获得针对人PDL1的单域重链抗体,从而获得在大肠杆菌中高效表达的纳米抗体菌株。
实施案例1:针对人PDL1的单域重链抗体免疫文库的构建:
(1)用真核人PDL1蛋白免疫新疆双峰驼,每两周免疫一次,共连续免疫了六次。(2)六次免疫结束后,利用间接ELISA检测血清效价。分离骆驼外周血中的淋巴细胞并提取总RNA(TRIZOL法)。(3)按照TAKARA公司的Primescript TmRT reagent kit with gDNAEraser试剂说明书将提取的总RNA,如图1所示。再将RNA反转录为cDNA,然后利用PCR方法扩增VHH链,共包括两轮PCR,第一轮PCR:
上游引物CALL01-Leader:5'-GTCCTGGCTGCTCTTCTACAAGG-3'
下游引物CALL02-CH2:5'-GGTACGTGCTGTTGAACTGTTCC-3'
第一轮PCR反应条件:95℃预变性5min,95℃,10s,56℃,30s,72℃,1min ,20个循环。95℃,10s,68℃,1min,72℃延伸10min。
将第一轮PCR扩增产物利用1.5%琼脂糖凝胶电泳分离,切胶回收大小在750bp处的DNA片段,如图2所示。
第二轮PCR以第一轮切胶回收产物,作为扩增所需的模板,进行第二轮PCR:
上游引物VHH-Back :5'-GATGTGCAGCTGCAGGAGTCTGGRGGAGG-3'
下游引物PMCF primer:5'-CTAGTGCGGCCGCTGAGGAGACGGTGACCTGGGT-3'
第二轮PCR反应条件:95℃预变性5min,95℃,30s,56℃,30s,72℃,40s,28个循环,72℃延伸10min。
将第二轮PCR产物利用2%琼脂糖凝胶电泳分离,切胶回收大小在450bp左右的DNA片段。如图3所示;(4)将噬菌粒pMECS和第二轮PCR回收产物VHH分别使用限制性内切酶(购买于TAKARA)QuickCut™ Not I 、QuickCut™ PstI双酶切,经琼脂糖凝胶电泳回收,经核酸定量后,利用T4连接酶将载体与基因以质量比3:1连接,16℃过夜连接。(5)纯化并浓缩连接产物,将连接产物电转化入感受态TG1细胞中,构建PDL1纳米抗体噬菌体展示文库并计算文库库容,库容大小为4×108,收集文库分装保存于-80℃中。
实施案例2:抗PDL1纳米抗体的筛选、阳性克隆的的鉴定和测序
1.大容量PDL1噬菌体纳米抗体文库扩增
在10mL的2×YT液体培养基(含2 % 葡萄糖,100μg/mL 氨苄青霉素)接种文库至OD600为0.1,37 ℃,200 r/min培养至OD600达0 .5,按感染复数20∶1加入辅助噬菌体M13K07,37℃,静置感染30min后,37℃,100r/min 培养30 min。将培养物离心,用50 mL的2×YT(含100 μg/mL 氨苄青霉素和50 μg/mL卡那霉素)重悬沉淀,30℃,220 r/min过夜培养后,4℃,8000rpm离心取上清,加入5×PEG/NaCl溶液冰上放置2h,12000rpm离心30min,重悬沉淀于磷酸缓冲液(PBS,0 .01 M,pH 7 .4),即得到抗PDL1单域重链抗体免疫文库,取10 μL测定扩增文库的滴度。
2.抗PDL1纳米抗体的筛选
采用固相亲和淘洗的方法,将真核人PDL1蛋白用包被缓冲液稀释至1-100ug/mL,包被至高亲和力ELISA板上,4℃包被过夜。第二天,用PBS洗板三遍,每孔加入200uL含3%BSA的PBS,37℃,封闭2 h。用PBS稀释噬菌体的滴度至1×1012CFU(后面每轮滴度降低),将稀释后的噬菌粒与3%的脱脂奶粉等体积混合,在室温低速旋转混合30min,以中和与BSA结合的噬菌粒。PBS洗板5次,每孔加入100uL预孵育的噬菌粒,37℃孵育2小时。吸出孔中未结合的噬菌粒,加入PBST洗10-20遍(逐轮增加洗涤次数),再用PBS洗三遍后加入100ul的PH=2 .2的甘氨酸-盐酸洗脱缓冲液,37℃震荡10分钟。轻轻吹打孔将吸附的噬菌体洗下来并转移至新EP管中,加入适量中和液使溶液的pH值在7.0-7.4之间。取10uL测定滴度,其余的扩增后用于下一轮淘选。共经过三轮亲和筛选。
3.PE-ELISA筛选PDL1特异性单个阳性克隆
在96孔深孔板中加入200uL 2×YT-AG液体培养基(葡萄糖含量为2%,氨苄为100ug/mL),挑取第三轮筛选后输出平板上的单克隆至各孔中,37℃恒温箱静置过夜培养。第二天在96孔深孔板中加入500ul 2×YT-A(氨苄为100ug/mL),接种20uL/孔前夜培养菌,37℃ 220rpm培养 2h后,每孔加入IPTG至终浓度为1mM,30℃ 200rpm 过夜培养。96孔深孔板离心弃上清,每孔加入200uLTES高渗缓冲液重悬细胞,4℃振荡2h,再每孔加入300μLTES/4低渗液,4℃振荡2h,离心取上清。包被两块96孔ELISA板,一个为人PDL1抗原包被板,另一个是阴性对照BSA包被板,4℃过夜。次日,0 .05%PBST洗板2次,用3%脱脂牛奶室温封闭2h。吸去封闭液,用0 .05%PBST洗板2次。在抗原包被孔以及阴性对照孔中分别加入100uL相对应的深孔板上清,37℃孵育2h。PBST洗板,每孔加入100uL 1:3000稀释比例的抗HA鼠单克隆抗体,37℃孵育1h。用0 .05%PBST洗板3次。每孔加入100uL羊抗鼠-HRP抗体,37℃孵育1h;吸出抗体稀释液,洗板后加入TMB显色,酶标仪测定OD 450nm。吸光值大于1,且阳性OD450/阴性OD450>2.5认定为阳性克隆,将阳性克隆全部测序。
阳性克隆测序得到DNA序列,通过Vbase软件将DNA翻译为氨基酸序列,根据国际免疫遗传学信息系统(IMGT)对蛋白序列的框架区(FR区)和抗体识别区域(CDR区)进行区分,把CDR1、CDR2、CDR3序列均相同的克隆视为同一抗体株,将CDR序列不同的克隆视为不同抗体株,最终得到5个不同的纳米抗体序列。
实施案例3:PDL1纳米抗体的表达纯化
1.PDL1纳米抗体的表达
本专利将测序鉴定后的5个阳性克隆,未亚克隆至其他表达载体上,而是利用原载体pMECS,原宿主菌TG1进行蛋白表达,克隆命名为TG1-pMECS-VHH。将原始菌种划线至2YT-AG固体培养基板上,正放10min后,倒置过夜培养。第二天挑取固体板上的单克隆至含100ug/mL氨苄抗生素的10mL 2YT-AG液体培养基中,37℃,220rpm震荡培养8h,作为细菌种子液。
将细菌种子液按1%接种至2YT-A液体培养基中,37℃,220rpm震荡培养至OD600=0.8,加入IPTG至终浓度为1mM,30℃,150rpm过夜培养。第二天菌液离心弃上清,用1×PBS洗涤菌体两遍后,菌体沉淀-80℃反复冻溶两次。解冻后,加入适量TES重悬细胞,4℃,200rpm振荡4h。再加入两倍TES体积的TES/4,4℃,200rpm振荡过夜。4℃,12000rpm离心30min,取上清。
2.PDL1纳米抗体的纯化
经镍柱离子亲和层析方法纯化抗体蛋白,在得到的纳米抗体溶液中加入PBS缓冲液,4℃,4000rpm离心超滤至所需体积。利用12%SDS-PAGE蛋白电泳检测PDL1纳米抗体的分子量大小及纯度:如图4所示为其中一种纳米抗体从蛋白诱导至纯化超滤的蛋白表达情况,SDS-PAGE电泳结果显示在18kDa附近出现明显条带,与预期相符;超滤后样品泳道条带较单一,说明抗体纯度较高。
实施案例4:PDL1纳米抗体的理化性质检测
1.间接ELISA检测PDL1纳米抗体的特异性
本实施案例中使用的人PD1、人PDL1、人SRIPa蛋白以及人CD47,人CD47-Fc,CD47-HIS蛋白均购买于北京义翘神州生物科技公司。
使用包被缓冲液将人PD1蛋白、人PDL1蛋白、人SRIPa蛋白、人CD47蛋白、人CD47-Fc蛋白,CD47-HIS蛋白浓度分别稀释至1ug/mL,每孔100uL包被至96孔ELISA板中,4℃包被过夜。次日,0 .05%PBST洗板2次,用5%脱脂牛奶37℃封闭2h。洗板3次后,各抗原孔中加入100uL一定浓度(1ug/mL)的抗PDL1纳米抗体(Nb1,Nb2,Nb3,Nb4,Nb5),37℃孵育2h。洗板3次后每孔加入100uL二抗(Mouse anti-HA tag antibody,购自北京义翘神州生物科技公司)反应,37℃孵育1h。洗板3次后加入三抗(耦联HRP的Goat anti-mouse IgG mAb,购自康为世纪),37℃孵育1h。洗板5次后加入TMB显色,终止后酶标仪测定OD 450nm。
吸光值反应出抗PDL1纳米抗体与人PDL1,人PD1,人SRIPa、人CD47、人CD47-Fc,CD47-HIS的抗原的结合能力。如图5所示,,结果显示抗PDL1纳米抗体(Nb1,Nb2,Nb3,Nb4,Nb5)与抗原PDL1的结合值较高,但是与其他对照抗原基本不结合,说明制备的抗PDL1纳米抗体的特异性结合能力较强。
2.抗PDL1纳米抗体的亲和常数测定
采用非竞争性ELISA方法测定PDL1纳米抗体的亲和常数。
分别配置浓度为1ug/mL,0.5ug/mL,0.25ug/mL,0.125ug/mL的4种不同抗原浓度的人PDL1蛋白溶液,每孔100uL包被至96孔ELISA板中,于4℃包被过夜。次日0 .05%PBST洗板2次,用5%脱脂牛奶37℃封闭2h。将抗PDL1纳米抗体以倍比稀释的方法将抗体浓度从4ug/mL向后稀释12个浓度梯度,洗板2次后将样品加入到96孔酶标板内,100μL/孔,37℃孵育2h。洗板3次后每孔加入100uL二抗(Mouse anti-HA tag antibody,购自北京义翘神州生物科技公司)反应,37℃孵育1h。洗板3次后加入三抗(耦联HRP的Goat anti-mouse IgG mAb,购自康为世纪),37℃孵育1h。洗板5次后加入TMB显色,终止后酶标仪测定OD 450nm。拟合“S”型曲线,得出4个半数吸光值所对应的PDL1纳米抗体浓度(EC50),结果如图6,7所示。
纳米抗体为单价抗体,按公式KA=(n-1)/(nAb '-Ab)计算亲和常数(式中Ab和Ab'分别表示当抗原浓度为Ag和Ag '时,产生半数吸光值的抗体浓度(mol/L),n=Ag/Ag '),得6个KA值,计算平均值及标准差。结果如表1所示,以其中一个抗PDL1(Nb2)纳米抗体的亲和常数测定为例,计算得出亲和常数为(0.93304)×106L·mol-1 ,说明制备的抗PDL1纳米抗体具有较高的亲和力。
序列表
<110> 新疆优迈生物技术有限公司
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<213> 人工序列(Artificial Sequence)
<400> 5
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ile Ala Ser
20 25
<210> 6
<211> 17
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Asp Arg Met Ala Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly
1 5 10 15
Val
<210> 7
<211> 38
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Ile
1 5 10 15
Gly Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Glu Pro Glu Asp
20 25 30
Thr Ala Thr Tyr Tyr Cys
35
<210> 8
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210> 9
<211> 25
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Glu Val Ser
20 25
<210> 10
<211> 17
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Leu Gly Trp Phe Arg Gln Thr Pro Gly Lys Glu Arg Glu Gly Val Ala
1 5 10 15
Gly
<210> 11
<211> 38
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Thr Tyr Ala Asp Ser Val Lys Gly Arg Phe Ala Leu Ser Arg Asp Asn
1 5 10 15
Gly Gln Asn Thr Val Tyr Leu Glu Met Ser Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 12
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210> 13
<211> 25
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ile Ala Ser
20 25
<210> 14
<211> 17
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Asp Arg Met Val Trp Phe Arg Pro Val Pro Glu Lys Glu Arg Glu Gly
1 5 10 15
Val
<210> 15
<211> 38
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Ile
1 5 10 15
Gly Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Thr Tyr Tyr Cys
35
<210> 16
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 16
Trp Gly Gln Gly Thr His Val Thr Val Ser Ser
1 5 10
<210> 17
<211> 25
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 17
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Thr Ala Ser
20 25
<210> 18
<211> 17
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 18
Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Ala Val Gly
1 5 10 15
Cys
<210> 19
<211> 38
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 19
Val Tyr Ala Val Pro Val Glu Gly Arg Phe Thr Met Ser Arg Glu Asn
1 5 10 15
Arg Gln Asn Thr Val Tyr Leu Gln Met Asp Ser Leu Lys Ala Glu Asp
20 25 30
Thr Ala Val Tyr Tyr Cys
35
<210> 20
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 20
Trp Gly Gln Gly Thr Gln Val Thr Val Ser
1 5 10
<210> 21
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 21
Gly Asp Thr Ser Ser Leu Asn Ile
1 5
<210> 22
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 22
Ile Tyr Thr Asp Ala Gly Thr Pro Thr Tyr
1 5 10
<210> 23
<211> 19
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 23
Ala Ala Gly Thr Met Pro Val Arg Ala Ala Thr Leu Arg Pro Ala Arg
1 5 10 15
Tyr Ile Tyr
<210> 24
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 24
Gly Val Asp Tyr Asn Ser Arg Thr
1 5
<210> 25
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 25
Ala Ala Ile Tyr Thr Tyr Asn Asn Arg Thr
1 5 10
<210> 26
<211> 18
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 26
Ala Arg Asp Trp Arg Phe Ser Ser Gly Met Leu Asp Lys Asn Met Tyr
1 5 10 15
Lys Tyr
<210> 27
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 27
Gly Asp Thr Ser Thr Leu Asn Ile
1 5
<210> 28
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 28
Ile Tyr Phe Ala Ala Gly Thr Pro Thr Tyr
1 5 10
<210> 29
<211> 19
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 29
Ala Ala Gly Thr Met Pro Val Arg Ala Ala Thr Leu Arg Pro Ala Arg
1 5 10 15
Tyr Thr Tyr
<210> 30
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 30
Gly Asp Asp Tyr Asn Leu Lys Thr
1 5
<210> 31
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 31
Ala Ser Ile Tyr Thr Tyr Asn Asn Thr Thr
1 5 10
<210> 32
<211> 18
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 32
Ala Arg Asp Trp Ser Phe Ser Ser Gly Met Leu His Lys Asn Met Tyr
1 5 10 15
Lys Tyr
<210> 33
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 33
Gly Tyr Thr Ser Gly Ile Leu Tyr
1 5
<210> 34
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 34
Ile Tyr Pro Ser Gly Arg Ser Thr
1 5
<210> 35
<211> 16
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 35
Gly Ala Arg Pro Gln Ser Cys Gly Ala Pro Val Ser Thr Val Asp Tyr
1 5 10 15
<210> 36
<211> 128
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 36
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Ala Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Glu Val Ser Gly Asp Thr Ser Ser Leu Asn
20 25 30
Ile Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Gly Ile Tyr Thr Asp Ala Gly Thr Pro Thr Tyr Thr Tyr Ala Asp
50 55 60
Ser Val Lys Gly Arg Phe Ala Phe Ser Arg Asp Asn Gly Gln Asn Thr
65 70 75 80
Val Tyr Leu Glu Met Ser Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr
85 90 95
Tyr Cys Ala Ala Gly Thr Met Pro Val Arg Ala Ala Thr Leu Arg Pro
100 105 110
Ala Arg Tyr Ile Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 37
<211> 127
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 37
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ile Ala Ser Gly Val Asp Tyr Asn Ser Arg
20 25 30
Thr Asp Arg Met Ala Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu
35 40 45
Gly Val Ala Ala Ile Tyr Thr Tyr Asn Asn Arg Thr Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Ile Gly Lys Asn Thr
65 70 75 80
Val Tyr Leu Gln Met Asn Ser Leu Glu Pro Glu Asp Thr Ala Thr Tyr
85 90 95
Tyr Cys Ala Arg Asp Trp Arg Phe Ser Ser Gly Met Leu Asp Lys Asn
100 105 110
Met Tyr Lys Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 38
<211> 127
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 38
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ile Ala Ser Gly Asp Asp Tyr Asn Leu Lys
20 25 30
Thr Asp Arg Met Val Trp Phe Arg Pro Val Pro Glu Lys Glu Arg Glu
35 40 45
Gly Val Ala Ser Ile Tyr Thr Tyr Asn Asn Thr Thr Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Ile Gly Lys Asn Thr
65 70 75 80
Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Thr Tyr
85 90 95
Tyr Cys Ala Arg Asp Trp Ser Phe Ser Ser Gly Met Leu His Lys Asn
100 105 110
Met Tyr Lys Tyr Trp Gly Gln Gly Thr His Val Thr Val Ser Ser
115 120 125
<210> 39
<211> 123
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 39
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Thr Ala Ser Gly Tyr Thr Ser Gly Ile Leu
20 25 30
Tyr Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Ala Val
35 40 45
Gly Cys Ile Tyr Pro Ser Gly Arg Ser Thr Val Tyr Ala Val Pro Val
50 55 60
Glu Gly Arg Phe Thr Met Ser Arg Glu Asn Arg Gln Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Gly Ala Arg Pro Gln Ser Cys Gly Ala Pro Val Ser Thr Val Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 40
<211> 128
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 40
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Glu Val Ser Gly Asp Thr Ser Thr Leu Asn
20 25 30
Ile Leu Gly Trp Phe Arg Gln Thr Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Gly Ile Tyr Phe Ala Ala Gly Thr Pro Thr Tyr Thr Tyr Ala Asp
50 55 60
Ser Val Lys Gly Arg Phe Ala Leu Ser Arg Asp Asn Gly Gln Asn Thr
65 70 75 80
Val Tyr Leu Glu Met Ser Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr
85 90 95
Tyr Cys Ala Ala Gly Thr Met Pro Val Arg Ala Ala Thr Leu Arg Pro
100 105 110
Ala Arg Tyr Thr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 41
<211> 385
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
caggtgcagc tgcaggagtc tggaggaggc tcggcgcagg ctggagggtc tctgaggctc 60
tcctgtgaag tctctggaga cacctccagt ctgaacatcc tgggttggtt ccgccaggct 120
ccagggaagg agcgcgaggg ggtggcaggc atttatactg acgctggtac cccaacctat 180
acctatgccg actccgtgaa gggccgattc gccttctccc gtgacaacgg ccagaacacg 240
gtgtatcttg aaatgagcag cctgaagcct gaggacactg ccatgtacta ctgcgcggcg 300
ggtacgatgc ccgttcgggc ggccactctt cgccccgctc gttatattta ttggggccag 360
gggacccagg tcaccgtctc ctcag 385
<210> 42
<211> 382
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
caggtgcagc tgcaggagtc tggaggaggc tcggtgcagg ctggagggtc tctgagactc 60
tcctgtatag cttctggagt tgattacaat agtaggaccg atcgcatggc ttggttccgc 120
caggttccag ggaaggagcg cgagggagtg gcagccattt atacttataa taatagaaca 180
tattatgccg actccgtgaa gggccgattc accatctccc aagacattgg caagaatacg 240
gtatatctgc agatgaatag cctggaacct gaggacactg ccacatacta ctgtgcgcga 300
gattggcgtt tctcgagtgg gatgttggat aaaaatatgt ataagtactg gggccagggg 360
acccaggtca ccgtctcctc ag 382
<210> 43
<211> 215
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
aggtgcagct gcaggagtct gggggaggct cggtgcagcc tggagggtct ctgaggctct 60
cctgtgaagt ctctggagac acctccactc tgaacatcct gggttggttc cgccagactc 120
cagggaagga gcgcgagggg gtggcaggca tttattttgc cgctggtacc ccaacctata 180
cctatgccga ctccgtgaag ggccgattcg ccctc 215
<210> 44
<211> 381
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 44
caggtgcagc tgcaggagtc tgggggaggc tcggtgcagg ctggagggtc tctgagactc 60
tcctgtatag cttctggaga tgattacaat cttaagaccg atcgcatggt ttggttccgt 120
ccggttccag agaaggagcg cgagggagtg gcatccattt atacttataa caatacaaca 180
tattatgccg actccgtgaa gggccgattc accatctccc aagacattgg caagaatacg 240
gtatatctgc aaatgaatag cctgaaacct gaggacactg ccacatacta ctgtgcgcga 300
gattggagtt tctcgagtgg gatgttgcat aaaaatatgt ataagtactg gggccagggg 360
acccacgtca ccgtctcctc a 381
<210> 45
<211> 370
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 45
caggtgcagc tgcaggagtc tggaggaggc tcggtgcagg ctggagggtc tctgaaactc 60
tcctgtacag cctctggata caccagcggt atcttgtaca tggcctggtt ccgccaggct 120
ccggggaagg agcgcgaggc ggtcggctgc atttatccca gtggacgtag cacagtctat 180
gccgtccccg tggagggccg attcaccatg tccagggaga accgccagaa cacggtgtat 240
ctgcagatgg acagcctgaa agctgaggac actgccgtgt actattgtgg ggcaaggccg 300
cagtcctgtg gagcccctgt ttctacggtt gactactggg gccaggggac ccaggtcacc 360
gtctcctcag 370
Claims (7)
1.一种人PDL1纳米抗体的VHH链,包括框架区FR区和抗原互补决定区CDR区,其特征在于,所述框架区FR区来自其FR1-FR4的氨基酸序列:
SEQ ID No .1所示的FR1,SEQ ID No .2 所示的FR2,SEQ ID No .3 所示的FR3 , SEQID No .4 所示的FR4;
或SEQ ID No .5所示的FR1,SEQ ID No .6 所示的FR2,SEQ ID No .7 所示的FR3 ,SEQ ID No .8 所示的FR4;
或SEQ ID No .9所示的FR1,SEQ ID No .10所示的FR2 ,SEQ ID No .11所示的FR3 ,SEQ ID No .12 所示的FR4;
或SEQ ID No .13所示的FR1,SEQ ID No .14所示的FR2,SEQ ID No .15 所示的FR3,SEQ ID No .16 所示的FR4;
或SEQ ID No .17所示的FR1,SEQ ID No .18 所示的FR2,SEQ ID No .19 所示的FR3,SEQ ID No .20 所示的FR4;
所述的抗原互补决定区CDR区选自以下的CDR1-CDR3氨基酸序列:
SEQ ID No .21所示的CDR1,SEQ ID No .22所示的CDR2,SEQ ID No .23所示的CDR3;
或SEQ ID No .24所示的CDR1,SEQ ID No .25所示的CDR2,SEQ ID No .26所示的CDR3;
或 SEQ ID No .27所示的CDR1,SEQ ID No .28所示的CDR2,SEQ ID No .29所示的CDR3;
或SEQ ID No .30所示的CDR1,SEQ ID No .31所示的CDR2,SEQ ID No .32所示的CDR3;
或SEQ ID No .33所示的CDR1,SEQ ID No 34所示的CDR2,SEQ ID No .35所示的CDR3;
根据以上所述的人PDL1纳米抗体VHH链,其特征在于,它具有SEQ ID No .36,SEQ IDNo .37,SEQ ID No .38,SEQ ID No .39,SEQ ID No .40所示的氨基酸序列。
2.一种人PDL1纳米抗体,其特征在于,它是针对人PDL1分子表位的纳米抗体,包括具有SEQ ID No .36,SEQ ID No .37,SEQ ID No .38,SEQ ID No .39,SEQ ID No .40所示的氨基酸序列的VHH链。
3.一种DNA分子,其特征在于,它编码选自下组的蛋白质:权利要求1或2所示的人PDL1纳米抗体的VHH链,或权利要求2所示的人PDL1纳米抗体。
4.根据权利要求3所述的DNA分子,其特征在于,它具有选自以下组的DNA序列:SEQ IDNo .41,SEQ ID No .42,SEQ ID No .43,SEQ ID No .44,SEQ ID No .45。
5.一种表达载体,其特征在于,它含SEQ ID No .41,SEQ ID No .42,SEQ ID No .43,SEQ ID No .44,SEQ ID No .45所示的核苷酸序列。
6.一种宿主细胞,其特征在于,它表达针对人PDL1的纳米抗体。
7.一种制备抗PDL1纳米抗体的方法,其包括以下步骤:
(1)人PDL1真核蛋白免疫新疆双峰驼,分离外周血淋巴细胞,构建噬菌体展示文库;
(2)利用亲和筛选的方法,通过真核人PDL1蛋白筛选噬菌体展示文库;
(3)PE-ELISA的方法挑选结合PDL1阳性克隆并测序分析;
(4)PDL1重组纳米抗体的表达与纯化;
(5)抗PDL1纳米抗体的功能鉴定;
权利要求1-5所述的抗人PDL1纳米抗体的用途,其用于制备检测PDL1的诊断试剂盒,或在癌症治疗药物中的应用。
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