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CN115197968B - 抗原结合域自动优化的嵌合抗原受体修饰细胞文库的构建、筛选方法及其应用 - Google Patents

抗原结合域自动优化的嵌合抗原受体修饰细胞文库的构建、筛选方法及其应用 Download PDF

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CN115197968B
CN115197968B CN202110383729.1A CN202110383729A CN115197968B CN 115197968 B CN115197968 B CN 115197968B CN 202110383729 A CN202110383729 A CN 202110383729A CN 115197968 B CN115197968 B CN 115197968B
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牛志远
朱武凌
郭长江
支灵通
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Abstract

本发明公开了一种抗原结合域自动优化的嵌合抗原受体修饰细胞文库的构建、筛选方法及其应用,该方法通过在细胞中插入一段嵌合抗原受体序列,并通过顺式作用元件及反式作用因子在胞外抗原结合域不断诱导突变产生,建立一个可供后续筛选的抗体序列动态库;抗体序列动态库被受体2展示在细胞表面,形成嵌合受体库细胞文库;受体1能够识别靶标抗原上的标签,并与能够识别靶标抗原的受体2形成二聚体,从而启动筛选基因的表达,并同时启动抑制系统来维持抗原结合域的稳定。筛选嵌合抗原受体文库,将筛得的阳性细胞嵌合受体的抗原结合域进行扩增测序,即可得到抗原识别区的核酸序列。此方法可用于多种抗体制备,更为简便高效。

Description

抗原结合域自动优化的嵌合抗原受体修饰细胞文库的构建、 筛选方法及其应用
技术领域
本发明涉及抗原结合域自动优化的嵌合抗原受体修饰细胞文库的构建、筛选方法及其应 用,属于基因工程技术领域。
背景技术
目前抗体在科研领域,检测领域,诊断、医疗领域等都发挥着不可替代的作用,随着嵌 合抗原受体T细胞免疫疗法(CAR-T)及免疫检查点疗法如火如荼的发展,目前对抗体的制备也有了更大的需求以及更高的要求。,例如,嵌合抗原受体(CAR)胞外抗原结合域的单链 抗体(scFv)制备。CAR的胞外抗原结合域也多采用骆驼科动物和鲨鱼产生的天然缺乏轻链 的单域抗体(sdAb),其被称为仅重链抗体(HcAb),HcAb每个臂中的抗原结合片段具有单 个重链可变域(VHH),可在无需轻链的帮助下,对抗原具有高亲和力,VHH被称为最小的功能性抗原结合片段,分子量为大约15Kd,在CAR受体中被广泛应用。另外在对人的治疗 过程中经常需要人源化的抗体。
目前scFv及VHH大多通过传统的抗体制备方法获取,其主要步骤包括:1.抗原及佐剂 配制;2.动物免疫;3.抗血清采取;4.B淋巴细胞分离;5.杂交瘤制备/抗体序列扩增及建库;6.抗体筛选;7.抗体序列的获取;8.scFv及VHH序列的进一步制备。另外还需要将制 备的抗体进行人源化处理,涉及人源化小鼠的应用,或抗体之间的拼接,整个流程步骤繁琐,耗时费力,且成本高昂。
发明内容
本发明的目的是提供一种抗原结合域自动优化的嵌合抗原受体修饰细胞文库的构建方 法。
本发明的第二个目的是提供由上述构建方法制备得到的嵌合抗原受体修饰细胞文库。
本发明的第三个目的是提供一种抗原结合域自动优化的嵌合抗原受体修饰细胞文库的筛 选方法。
本发明的第四个目的是提供上述筛选方法在制备抗体中的应用。
本发明的第五个目的是提供由上述方法筛选得到的抗体。
为了实现上述目的,本发明采用以下技术方案:
第一方面,本发明提供了一种抗原结合域自动优化的嵌合抗原受体修饰细胞文库的构建 方法,将包含第一基因元件、第二基因元件、第三基因元件、第四基因元件以及第五基因元 件的一个或多个载体转染至细胞内,即得到嵌合抗原受体修饰细胞文库;
所述第一基因元件为编码受体1的核苷酸序列,所述受体1包括胞外域,跨膜域及胞内 域;所述胞外域可识别靶标抗原上所标记的生物素或6×His标签,所述胞内域包括烟草蚀纹 病毒蛋白酶TEVP;
所述第二基因元件为编码受体2的核苷酸序列,所述受体2包括胞外抗原结合域、跨膜 域以及胞内信号域;所述抗原结合域为抗体序列动态库,所述动态库是在第四基因元件的作 用下诱导产生一系列突变,引入序列多样性所得;所述胞内信号域包括tTA转录因子结构域 以及连接序列,所述连接序列连接tTA转录因子结构域和跨膜域,包含TEVP可识别切割的 序列;
所述第三基因元件为编码筛选基因的核苷酸序列,tTA转录因子可以启动所述筛选基因 的表达,使宿主产生荧光、获得某种抗性、或者获得在某种条件下的生长优势;
所述第四基因元件为编码一种或多种顺式调控元件和一种或多种反式作用因子的核苷酸 序列;所述顺式调控元件能够使DNA产生特殊结构的DNA序列,所述特殊结构是指DNA 结构中含有未发生碱基互补配对的DNA单链结构,所述的特殊结构包括R-loop,G-三联体 以及G-四联体;所述反式作用因子为Cas蛋白家族及Cas蛋白家族的变体、gRNA、尿嘧啶糖基化酶抑制剂、腺嘌呤脱氨酶、胞苷脱氨酶、RNA-DNA杂合链结合蛋白、感光蛋白、Nucleolin、DNA解旋酶、易错DNA聚合酶、重组酶、核酸内切酶、核酸外切酶、烷基腺嘌 呤糖基化酶中的一种或多种的组合;
所述第五基因元件为编码抑制系统的核苷酸序列,tTA转录因子可以启动所述抑制系统 的表达,进而敲减或敲除反式作用因子,使抗体序列动态库维持稳定;或所述第五基因元件 为编码光控制系统的核苷酸序列,所述光控制系统能够调控第四基因元件的表达:在光照条 件下启动第四基因元件的表达,开始构建抗体动态序列库;停止光照后,第四基因元件功能 被抑制,使抗体动态序列库维持稳定。
优选的,所述细胞是真核细胞或原核细胞,进一步优选的,所述细胞是人源或鼠源细胞, 更为优选的,所述细胞为293T。
优选的,所述抗体序列动态库包括传统抗体库、单链抗体库及纳米抗体库。
优选的,所述受体1的核苷酸序列如SEQ ID NO.1所示。
优选的,所述受体2是由Notch受体改造而来的synNotch受体,其核苷酸序列如SEQID NO.2或SEQ ID NO.3所示。
优选的,所述反式作用因子包括以下Cas蛋白家族成员及其部分结构域:Cas3-8s、Cas10s、 Cas11s、Cas9s、xCas9、Cas12s、Cas13s和Cas14s。
优选的,所述反式作用因子包括以下腺嘌呤脱氨酶及其部分结构域:ecTadA、mADA、 hADAR2和hADAT2。
优选的,所述反式作用因子包括以下胞苷脱氨酶及其部分结构域:AID(activation-induced cytidine deaminase)、APOBEC3G、APOBEC1和CDA1。
优选的,所述编码光控制系统的核苷酸序列包括以下感光蛋白及其部分结构域:BphS、 Cph1、PCB、PhyB-PIFs、PΦB、BphP1-PpsR2、BphP1-Q-PAS1、LOV2-Jα、PhyB、PIF6、Epdz和Photoreceptor Vivid(VVD)。
优选的,所述Cas蛋白家族是一类通过gRNA对基因组特定位点进行识别、导向的蛋白 家族。
优选的,所述光控制系统发挥对第四基因元件的调控作用需要光照的配合,所述光照包 括可见光、不可见光、蓝光、红光、远红外光及紫外光。
优选的,所述筛选基因包括GFP、mCherry、Luciferase、嘌呤霉素抗性基因、博来霉素 和新霉素抗性基因。
优选的,所述抑制系统是通过tTA转录因子启动负调控蛋白,阻遏蛋白,siRNA,CAS9/gRNA表达或通过反向转录方式,敲减或敲除反式作用因子,使抗体序列动态库维持稳定。
第二方面,本发明提供了由上述构建方法获得的抗原结合域自动优化的嵌合抗原受体修 饰细胞文库。
第三方面,本发明提供了对于上述抗原结合域自动优化的嵌合抗原受体修饰细胞文库的 筛选方法,包括以下步骤:
1)制备靶标抗原,所述靶标抗原上携带能够被嵌合受体2所识别的标签,所述标签包括生 物素或6×His;
2)将靶标抗原与嵌合抗原受体修饰细胞文库共孵育,根据筛选基因的表达情况,筛选出阳性嵌合抗原受体表达细胞;
3)对阳性细胞的嵌合抗原受体胞外抗原结合域进行扩增测序,得到能够与靶标抗原特异性 结合的抗体核苷酸序列;
优选的,在得到能够与靶标抗原能够特异性识结合的抗体核苷酸序列后,采用基因工程 的方法获得针对靶标抗原的抗体。
优选的,所述靶标抗原表达于细胞表面、包被于载体表面或为可溶性蛋白。
优选的,所述载体包括磁性微球,非磁性微球,石墨烯,琼脂糖微球,纳米颗粒,硅基 磁珠,GMA磁珠和聚苯乙烯磁性微球。
第四方面,本发明提供上述筛选方法在制备针对某一特定靶标抗原的抗体中的应用。
第五方面,本发明提供由上述方法筛选得到的抗体。
本发明以GFP-biotin为靶标抗原,采用不同方式构建嵌合受体修饰细胞文库并进行筛选, 得到GFP纳米抗体,所述抗GFP纳米抗体的氨基酸序列包含SEQ ID NO.4-13中的任一者, 或者SEQ ID NO.4-13中CDR1,CDR2,CDR3区域的任意组合。
本发明的有益效果在于:
本发明提供的嵌合抗原受体修饰细胞文库的构建方法构建得到的文库是动态库,能够在 筛选同时进行抗原结合域的自动优化,克服了现有技术中静态库序列多样性有限的缺陷;且 整个过程可以选用人源抗体作为框架进行优化,直接实现抗体的人源化。该方法构建的动态库可重复利用,根据本发明提供的嵌合抗原受体修饰细胞文库的筛选方法,能够获得针对多 种抗原的相应的抗原结合域序列。与传统的抗原结合域序列/抗体序列获取方法相比较,效率 极大提高,成本大大降低。
附图说明
图1是本发明技术方案示意图;
图2是gRNA引导碱基编辑器示意图;
图3是G-四联体引导碱基编辑器示意图;
图4是优化的gRNA引导碱基编辑器示意图;
图5是载体1结构示意图;
图6是载体2结构示意图;
图7是载体3结构示意图;
图8是载体4结构示意图;
图9是载体5结构示意图;
图10是dCas9融合的腺苷脱氨酶结构示意图;
图11是dCas9融合的胞苷脱氨酶结构示意图;
图12是dCas9-Vivid-Mcm7结构示意图;
图13是Vivid融合的胞苷脱氨酶和腺苷脱氨酶结构示意图;
图14是ELISA检测所得VHH序列识别eGFP结果图;
图中,a,根据DNA的G-四联体引导碱基编辑器定位建库并对筛选到的纳米抗体进行鉴 定;b,根据gRNA引导碱基编辑器定位建库并对筛选到的纳米抗体进行鉴定;c,根据gRNA 引导改进型碱基编辑器定位建库并对筛选到的纳米抗体进行鉴定。
图15是PCR扩增所有抗体重链结果图;
图16是大量PCR扩增所有抗体重链结果图;
图17是PCR扩增纳米抗体结果图;
图18是流式细胞术分别验证受体1和受体2的表达;
图19是细胞ELISA和免疫荧光验证受体1和受体2的表达;
图中,a,二聚化激活双受体系统在细胞中表达结构模式及检测方法示意图;b,细胞ELISA 检测链霉亲和素(受体1)表达情况;c,GFP与表达二聚化激活双受体系统的细胞共孵育15 min荧光显微镜下观察受体2表达情况。
图20是用生物素标记的GFP蛋白筛选纳米抗体;
图21是纳米抗体特异性鉴定结果。
图中,a,用未标记的GFP蛋白做流式,检测筛选到的5个阳性细胞克隆;b,ELISA 检测原核表达纯化后的5个纳米抗体对GFP蛋白的特异性,用抗CD19的纳米抗体做阴性 对照;c,用携带VHH5基因的阳性细胞与未标记的GFP蛋白共孵育30min,荧光显微镜 下观察GFP蛋白与细胞的结合状况。
具体实施方式
下面结合具体实施方式对本发明作进一步描述,但本发明的保护范围并不仅限于此;若 未特别指明,实施例中所用的设备和试剂均常规市售可得。
本发明构建了表达受体1和受体2的载体,受体1能够识别biotin标签,受体2的胞外 抗原结合域能够展示出纳米抗体库,当带有biotin标签的靶标抗原与嵌合抗原受体修饰细胞 文库共孵育时,靶标抗原能够诱导受体1及受体2产生二聚体,二聚化后,受体1胞内域的 烟草蚀纹病毒蛋白酶(TEV Proteinase,TEVP)识别并切割释放受体2胞内信号传导域的tTA 转录因子结构域,tTA转录因子可以启动筛选基因以及抑制系统的表达(如图1所示)。
而后取骆驼外周血单核细胞,获取天然纳米抗体序列,将天然纳米抗体序列库取代受体 2胞外的纳米抗体序列区,运用载体包装慢病毒并转染293T细胞,并用嘌呤霉素筛选转染阳 性的细胞,从而在293T细胞上通过受体2展示纳米抗体库,获得嵌合抗原受体修饰细胞文库。
通过原核表达纯化获得GFP蛋白,制备生物素标记的GFP蛋白(GFP-biotin)作为靶标 抗原,对上述修饰细胞文库进行筛选,证明由受体1与受体2二聚化激活的嵌合受体修饰细 胞文库构建成功并能够筛选得到GFP蛋白的纳米抗体。
进一步的,本发明在底盘细胞导入第四基因元件,即通过导入一种或多种顺式调控元件 和一种或多种反式作用因子,在抗原结合域不断诱导突变产生,引入序列多样性,从而建立 纳米抗体序列动态库,构建动态嵌合抗原受体修饰细胞文库并对其进行筛选,获得GFP蛋白 的纳米抗体,证明了本发明提供的嵌合抗原受体修饰细胞文库的构建及筛选方法的可行性。
实施例1:载体构建
插入一系列基因元件的载体能够在293T细胞表达受体1及受体2,受体1能够识别biotin 标签,受体2的胞外抗原结合域能够展示出纳米抗体库,当带有biotin标签的靶标抗原与嵌 合抗原受体修饰细胞文库共孵育时,靶标抗原能够诱导受体1及受体2产生二聚化,二聚化 后,受体1胞内域的烟草蚀纹病毒蛋白酶(TEV Proteinase,TEVP)识别并切割释放受体2 胞内信号传导域的tTA转录因子结构域,tTA转录因子可以启动筛选基因以及抑制系统的表 达(如图1所示)。
优选地,所述载体1(如图5所示),载体1的关键DNA序列如SEQ ID NO.14所示(SEQID NO.14序列为图5中cPPT/CTS元件和EF-1αcore promoter元件中间的序列,该段序列连接至任意具有类似结构的慢病毒载体上均可)。所述载体2(如图6)的关键DNA序列如SEQ IDNO.15所示(SEQ ID NO.15序列为图6中cPPT/CTS元件和EF-1αcore promoter元件中 间的序列,该段序列连接至任意具有类似结构的慢病毒载体上均可)。载体交由基因合成公司进行全基因合成。
实施例2:纳米抗体库构建
从天津漠尚客购买骆驼血200ml,进行骆驼血单核细胞分离实验,最终分离得到2.03×109个单核细胞,用Trizol重悬之后分装于10个离心管(2ml Trizol/离心管)中冻于-80℃冰箱。
将其中一个离心管中的细胞提取RNA,总共提取到95.8μg(A260/A280=2.0),用5×All In One RT MasterMix(Cat#G490,abm)全部反转录成cDNA冻存于-20℃冰箱。
1)第一次PCR扩增所有抗体重链
实验材料:
Hieff CanaceR Gold High-Fidelity DNA Polymerase高保真DNA聚合酶,产品编号 10148ES10;
扩增引物:
CALL001:5′-GTCCTGGCTGCTCTTCTACAAGG-3′;
CALL002:5′-GGTACGTGCTGTTGAACTGTTCC-3′;
反应体系:
反应程序:
反应结果:
PCR扩增结果如图15,由图可知,cDNA加5μl共0.5μg效果最好。
重新PCR扩增:cDNA加5μl重新进行一次PCR扩增,共扩增8管。
PCR扩增产物回收:
切割700bp目的条带,并分别用SanPrep柱式DNA凝胶回收试剂盒(REF B518131-0100, OLD#SK8132,LOT E710KA8969),回收目的条带DNA(如图16),8管PCR产物回收得到的总体积为240μl,产物浓度42ng/μl,纯度(OD260/280=1.81)。
2)第二次PCR扩增纳米抗体(用回收得到的8管PCR产物用巢式引物再次扩增)
实验材料:
Hieff CanaceR Gold High-Fidelity DNA Polymerase高保真DNA聚合酶,产品编号10148ES10。
引物如下表:Xba:ⅠTCTAGA,EcoR:ⅠGAATTC
反应体系:
反应程序:同前
扩增结果:如图17所示
反应体系:
反应程序:同前
3)PCR产物纯化
实验材料:DNA产物纯化试剂盒(D1300);
实验结果:纯化后产物浓度150ng/μl,总体积共240μl;纯度A260/A280=1.84
4)酶切载体1或酶切载体2,酶切PCR产物及酶连接。实验材料:
XmaI(NEB,R0180S),(NEB,R3136S),T4 DNA ligase(Takara,Cat#2011A, Lot#AIF2071A),SanPrep柱式PCR产物纯化试剂盒(REF#B518141-0100, LOT#B206KA5188)。
实验步骤:
a)酶切质粒载体
b)酶切PCR产物
c)酶切完成之后纯化酶切产物
用SanPrep柱式PCR产物纯化试剂盒纯化酶切产物。
载体1得到纯化产物60μL 150ng/μL A260/A280=1.86
VHH PCR产物40μL 100ng/μL A260/A280=1.85
d)酶连接
16℃连接过夜。
e)连接产物纯化
用SanPrep柱式PCR产物纯化试剂盒纯化连接产物,用100μL ddH2O缓冲液洗脱。
实施例3:大肠杆菌感受态细胞的制备
本研究使用电转化法将重组质粒转入大肠杆菌菌株Stbl3中。电转化法大肠杆菌的感受态 细胞制备方法如下(注:需要在酒精灯前操作):
1)将大肠杆菌Stbl3菌液使用接种环在不含抗生素的LB固体培养基平板上划线(超净工作 台中操作),将平板倒置放于37℃培养24h后获得直径为1mm左右的单菌落;
2)挑取一个Stbl3单菌落到3ml不含抗生素的LB液体培养基中,置于37℃恒温摇床,以 220rpm振荡过夜培养;
3)将3ml过夜培养菌液转移到250ml LB液体培养基(不含抗生素)中,置于37℃恒温摇 床,以220rpm振荡培养约3-5h,当菌液的OD值达到0.4-0.5时停止培养;
4)将250ml菌液分装在2个85ml无菌离心管中(分2次),置于低温离心机中,以4℃,4000rpm离心10min,倒去上清液,将离心管置于冰盒中;
注意:以下操作需要在低温下进行(冰浴操作)。
5)使用30ml无菌ddH2O(4℃冰箱预冷)重新充分悬浮细菌沉淀,置于低温离心机中,以 4℃,4000rpm离心10min,倒去上清液,将离心管置于冰盒中;
6)重复上一步骤1次;
7)使用30ml 10%甘油(4℃冰箱预冷)重新充分悬浮细菌沉淀,置于低温离心机中,以4℃, 4000rpm离心10min,倒去上清液,将离心管置于冰盒中;
8)重复上一步骤1次;
9)使用0.5ml 10%甘油(4℃冰箱预冷)重悬沉淀,用移液器轻轻吹打以打散小的菌块,将 菌液分装到1.5ml无菌离心管中,每管45μl,分装后置于冰盒中。
10)将分装好的离心管置于泡沫盒中,倒入适量液氮,冷冻1min后,保存于-20℃备用。
实施例4:重组载体的电转化与涂板
实验材料:美国Bio-Rad MicroPulser电穿孔仪,Bio-Rad电转杯(货号:165-2089)。
实验方法:将100μL纯化后连接产物+1mL电感受态细胞分20次进行电转(每次50μL), 其具体的操作为:
1)将电转杯(新、无菌)在-20℃冰箱预冷;
2)将点穿孔仪设置为“Bacteria"和"Time ms”,以自动模式进行实验;
3)将混匀的感受态细胞转移到预冷的无菌电转杯中,盖上盖子,并轻敲几下使菌液充分进入 电击凹槽中;
4)将电转杯放入点穿孔仪上,点击,正常的显示时间应在3-5ms之间;
5)电转完成后每个电转杯中加0.5mL预热的SOC复苏培养基,然后将20份细胞转移至同 一个50mL离心管中,37℃,170rpm孵育1h。
6)取100μL电转后的细胞用LB培养基10×梯度稀释(1:10,000,1:1,000,1:100),涂100μL 稀释后的细胞于9cm LB琼脂平板(100μg mL-1ampicillin,2%(wt/vol)glucose),37℃,过 夜。
7)将剩余的细胞分成4份,涂板于245mm的方形LB培养皿中(100μg ml-1ampicillinand 2% (wt/vol)glucose),37℃,过夜。
实施例5:计算电转效率收集文库
1)计数9cm LB琼脂平板上的菌落数目,通过菌落数×稀释倍数算出文库大小,挑取20个单 菌落用vF和vR作为引物进行菌落PCR,分析PCR产物大小确保>75%的菌落含有正确大小 的DNA片段。
2)挑取100个单菌落用vF和vR作为引物测序,得出100个单菌落的文库大小,进而计算出 整个纳米抗体文库大小为2.15×107
3)每个皿中加4mL LB培养基,用无菌细胞刮收集细胞,并转移至50mL的离心管中,每个 培养皿再用2mL LB培养基润洗一次,收集细胞,并加入20%体积的甘油,测量OD600,并分出来20份150μL体积的细胞悬液,剩余的保留在大的离心管里,-80℃冻存。
4)取出其中一份菌液扩增提取质粒,提取步骤按照试剂盒说明书进行。
实施例6:慢病毒的制备及稳转株筛选
按照4:3:1的比例,将连接好的载体1或载体2质粒23.3μg及两个慢病毒包装质粒PSPAX2 17.5μg,PMD2.G 5.8μg加入1.8mL氯化钙中。再将混合液加入1.8mL BBS中,静 置20min后加入175cm2的293T细胞培养瓶中。分别收取48h、72h病毒上清,使用5×PEG 浓缩。将得到的病毒和聚凝胺一起加入长有293T的培养皿中进行转染。2天后,用6μg/mL 的嘌呤霉素进行筛选,每天换液保持嘌呤霉素浓度不变连续5天。
实施例7:稳转株检测
由于实施例6中筛选得到的转染载体1的293T稳转株会表达受体1和受体2,这两个受 体的胞外域分别带有Flag及HA标签,因此我们通过识别这两个标签的流式抗体,运用流式 细胞术分别验证了受体1和受体2的表达,如图18所示,两个受体成功表达。
由于实施例6中筛选得到的转染载体2的293T稳转株表达的受体1胞外域是可以识别生 物素的单域链霉亲和素,因此我们用生物素标记的抗体及HRP标记的链霉亲和素做细胞 ELISA检测到了受体1的表达(如图19b)。受体2胞外域有一个纳米抗体结构域,我们用FITC 标记的可以识别纳米抗体的山羊抗体(Goat Anti-Llama IgG H&L,Abcam,ab112785)同样 检测到了受体2的表达(如图19c)。
实施例8:GFP蛋白纯化及GFP-biotin配体制备
我们原核表达并纯化了GFP蛋白,步骤如下:
1)eGFP蛋白表达载体为pET-28a(+),宿主菌为E.coli BL21(DE3);
2)将大肠杆菌BL21(DE3)菌液使用接种环在含卡那霉素的LB固体培养基平板上划线(超净 工作台中操作),将平板倒置放于37℃培养24h后获得直径为1mm左右的单菌落;
3)挑取一个单菌落到3ml不含抗生素的LB液体培养基中,置于37℃恒温摇床,以220rpm 振荡过夜培养;
4)将3ml过夜培养菌液转移到200ml LB液体培养基(不含抗生素)中,置于37℃恒温摇 床,以220rpm振荡培养约3-5h,当菌液的OD值达到0.8时停止培养;
5)加0.5mM IPTG诱导蛋白表达(IPTG母液浓度为1M,200ml LB液体培养基,加入100 μl),置于16℃恒温摇床,以170rpm振荡培养16h;
6)4000g离心5min,弃上清,用30ml Buffer A清洗一次;
7)加25ml Buffer A混匀,插到冰上超声破碎,工作时间2s,间隙时间5s,工作次数40次。
8)10000rpm,4℃离心30min,用0.45μm滤器过滤上清;
9)用10倍体积的Buffer A冲洗镍柱,冲洗之后加上清;
10)上清流过之后加5倍体积含有10mM咪唑的Buffer A冲洗蛋白一次,再加5倍体积含有 20mM咪唑的Buffer A冲洗蛋白一次,再加5倍体积含有40mM咪唑的Buffer A冲洗蛋白一次,再加5倍体积含有250mM咪唑的Buffer A,收集洗脱液至不同的1.5ml离心管 中。注意:低浓度咪唑冲洗蛋白的时候用Quick StartTMBradford Protein Assay蛋白检测液 观察是否有蛋白被洗脱下来。
11)用10倍体积的Buffer A冲洗镍柱,再用10倍体积的ddH2O冲洗镍柱,最后将镍柱保持 于20%乙醇中备用。
12)SDS-PAGE胶检测各个管中蛋白含量及纯度。
13)合并目的管中蛋白液,用合适的超滤管浓缩蛋白液,期间加入3次蛋白保存液置换洗脱 液。
14)用细长枪头转移出蛋白液。
15)用Biotin超级生物素快速标记试剂盒(ARL0020S-30K-0.5mL,购买自福因德科技(武汉) 有限公司)制备生物素标记的GFP蛋白(GFP-biotin),Biotin标记步骤按照说明书进行。
实施例9:纳米抗体筛选与特异性鉴定
实验材料:测序引物5’-GAGGGCAGAGGAAGTCTGCT-3’;Blasticidin(索莱宝);
将生物素标记的GFP蛋白(GFP-biotin,0.1μg/ml)配体与转染后的293T细胞共孵育24h, 用blastcidin筛选阳性的细胞(5μg/ml),将阳性细胞挑单克隆扩增后对纳米抗体区测序即得 到GFP特异性纳米抗体。图20左侧是未用blastcidin筛选的293T细胞,以及用blastcidin筛 选后其中一个单克隆细胞(图20右侧)。
筛选共得到5个阳性细胞克隆,VHH1、VHH2、VHH3、VHH4和VHH5,通过用未标 记的GFP蛋白做流式检测,结果如图21a所示,VHH3与VHH5这2个细胞克隆染色结果呈 阳性。
经过测序得到了5个纳米抗体序列,通过原核表达纯化(原核表达纯化步骤,表达载体, 诱导表达条件如实施例8中所述)之后通过ELISA验证筛选得到的5个纳米抗体对GFP蛋 白的特异性,结果如图21b所示,与流式结果相一致,流式结果阳性的VHH3与VHH5这2 个细胞株对应的纳米抗体ELISA结果同样是阳性。
ELISA实验步骤如下:
1)在酶标板中每孔加入100μL含有GFP蛋白(1μg/mL)的PBS,轻轻摇晃1min使其均匀混合,封好平板,室温包被2h。
2)倒掉溶液,每孔加入400μL Wash Solution洗涤,洗涤4次,每次洗涤1min。最后一 次清洗后,倒干液体,并且用干净吸水纸将残液吸干。
3)每孔加入100μL含有VHH1-5蛋白(1μg/mL)的PBS,以及含有anti-CD19 VHH 蛋白(1μg/mL)的PBS(对照组),封好平板,室温包被2h。
4)每孔加入100μL HRP标记的抗His标签抗体工作液,封好平板,室温包被2h。
5)倒掉溶液,用400μL Wash Solution清洗;重复三次;最后一次清洗后,倒干液体,并 且用干净吸水纸将残液吸干。
6)每孔加入200μL Substrate Solution,室温避光包被20-30min。
每孔加入50μL Stop Solution,摇匀。在30min内检测吸光值,酶标仪检测波长设置为 450nm,参比波长设置为540或570nm。
最后,采用携带VHH5基因的阳性细胞与未标记的GFP蛋白共孵育30min,结果如图21c所示,荧光显微镜下观察发现GFP蛋白能够有效地与细胞结合。
之后,为了增加VHH5纳米抗体对GFP蛋白的亲和力,我们对VHH5基因的三个CDR 区进行了一系列人工突变,最终得到了包含VHH5基因在内的一系列对GFP有不同亲和力的 纳米抗体。其中VHH5纳米抗体包含SEQ ID NO.16-25中的任一者,或者SEQ ID NO.16-25 中CDR1,CDR2,CDR3区域的任意组合。
实施例10:根据DNA的G-四联体引导碱基编辑器定位建库并进行纳米抗体筛选鉴定
本实施例为在已知序列的纳米抗体CDRs区,基于DNA的G-四联体高级结构引导碱基 编辑器进行体外碱基编辑建库。
原理如图3所示,由于目前所有的碱基编辑器只能在单链DNA上发挥作用,除了可以 用dCas9及gRNA的解螺旋作用产生一条DNA单链之外,还可以在一条DNA单链上诱导高级结构的产生,如G-四联体,从而导致另外一条DNA单链的产生,最少~25bp的富含G碱 基的DNA序列即可诱导G-四联体的形成。同时设计核仁素Nucleolin连接的胞苷脱氨酶(促 进G或C碱基向任意碱基突变)以及核仁素Nucleolin连接的腺苷脱氨酶(促进A碱基向任意碱基突变),由于核仁素Nucleolin可以识别并结合G-四联体,导致两种碱基编辑器定位于CDRs区。通过睡美人转座子系统SB100X将Nucleolin-Vivid以及Vivid连接的两个碱基编辑器基因一同转入293T细胞,构建稳转株。通过优化Vivid与脱氨酶之间Linker序列的长度及蓝光照射时间,实现在VHH的CDRs区广泛诱导突变。
将载体2(参见实施例1)包装慢病毒并转入293T细胞中,通过嘌呤霉素筛选得到稳转 株。
构建载体3(如图7),载体3的关键DNA序列如SEQ ID NO.26所示(SEQ ID NO.26 序列为图7中cPPT/CTS元件和EF-1αcore promoter元件中间的序列,该段序列连接至任意具有类似结构的慢病毒载体上均可);
构建载体4(如图8),载体4的关键DNA序列如SEQ ID NO.27所示(SEQ ID NO.27 序列为图8中cPPT/CTS元件和EF-1αcore promoter元件中间的序列,该段序列连接至任意具有类似结构的慢病毒载体上均可);(载体交由基因合成公司进行全基因合成)
通过包装慢病毒并构建293T细胞稳转株,将载体3和载体4依次转入293T细胞。
载体转染完毕之后培养5天,之后提取细胞基因组DNA,用巢式PCR扩增建库后的纳米抗体片段,两对巢式PCR引物序列如下:F1:5’-CTAGAGCCACCATGGCCC-3’;R1: 5’-CCAGGATGTGGCACAGCA-3’;F2:5’-TCTAGATGGCCCTGCTGCTGCACG-3’;R2:5’-GAATTCAGGTGCCCTGGTTGTAGC-3’,扩增出片端之后通过酶切(XbaⅠ/EcoRⅠ)酶连接 (T4连接酶)将片段连接到pET-28a-c(+)载体上(Novagen,Cat.No.69864-3),通过电转, 涂板,挑单克隆测序等步骤计算细胞展示纳米抗体库的大小约为2.67×106(具体步骤见参考 实施例2-5)。
将GFP-biotin配体与转染后的293T细胞共孵育24h,用blastcidin筛选阳性的细胞,将 阳性细胞挑单克隆扩增后对纳米抗体区测序即得到GFP特异性纳米抗体。
将所得阳性细胞中的纳米抗体序列挑选3条,进行原核表达纯化(蛋白表达纯化方法见 实施例8),得到VHH抗体蛋白。
ELISA检测所得VHH序列是否能够识别eGFP,具体步骤为,1.用eGFP蛋白包被96孔板,包被后清洗3次;2.加入3种纯化的VHH抗体蛋白,同时用抗CD19的VHH抗体蛋白做阴性对照,包被后清洗3次;3.加入HRP标记Anti-llama IgG(H+L)二抗,包被后清洗3 次。4.加入TMB显色液,鉴定得到VHH抗体蛋白是否识别eGFP。结果如图14a,3个抗体 中的1个对eGFP有较好的识别。
之后,为了增加纳米抗体对GFP蛋白的亲和力,我们对筛选到的VHH3基因的三个CDR 区进行了一系列人工突变,最终得到了包含VHH3基因在内的一系列对GFP有不同亲和力的 纳米抗体。其中VHH5纳米抗体包含SEQ ID NO.4-13中的任一者,或者SEQ ID NO.4-13中CDR1,CDR2,CDR3区域的任意组合。
实施例11:根据gRNA引导碱基编辑器定位建库并进行纳米抗体筛选鉴定
本实施例为在已知序列的纳米抗体CDRs区,基于gRNA引导碱基编辑器进行体外碱基 编辑建库。
原理如图2所示,首先获取一段VHH序列,设计3个分别靶向VHH的CDR1、CDR2 及CDR3区域的gRNA,同时设计失活Cas9(dCas9)连接的Vivid光敏蛋白(dCas9-Vivid), 再分别设计两个高活性碱基编辑器,即Vivid融合的胞苷脱氨酶(促进G或C碱基向任意碱 基突变)以及Vivid融合的腺苷脱氨酶(促进A碱基向任意碱基突变),在蓝光照射的条件下Vivid光敏蛋白会形成同源二聚体,因此含有Vivid结构域的蛋白会随机形成二聚体,导致两 种碱基编辑器随机结合到dCas9-Vivid蛋白上,从而定位于CDRs区。通过睡美人转座子系统 SB100X将gRNAs和dCas9-Vivid以及两个碱基编辑器基因一同转入293T细胞,构建稳转株。 通过优化Vivid光敏蛋白与脱氨酶之间Linker序列的长度及蓝光照射时间,实现在VHH的 CDRs区广泛诱导突变,从而将单一的VHH序列多样化,实现纳米抗体的建库,并通过检测纳米抗体库的多样性确定Vivid与脱氨酶之间Linker序列的长度及蓝光照射时间。
将载体2(参见实施例1)包装慢病毒并转入293T细胞中,通过嘌呤霉素筛选得到稳转 株。
构建载体5(如图9),载体5的关键DNA序列如SEQ ID NO.28所示(SEQ ID NO.28 序列为图9中U6 promoter元件和EF-1αcore promoter元件中间的序列,该段序列连接至任意具有类似结构的pLL7系列慢病毒载体上均可);
构建慢病毒表达载体6(如图10),载体6的关键DNA序列如SEQ ID NO.29所示(该段序列连接至任意慢病毒表达载体的合适位置上均可),在另一优选例中载体6表达的氨基酸 序列与SEQ ID NO.29序列的同源性≥85%。
构建慢病毒表达载体7(如图11),载体7的关键DNA序列如SEQ ID NO.30所示(该段序列连接至任意慢病毒表达载体的合适位置上均可),(在另一优选例中载体7表达的氨基 酸序列与SEQ ID NO.30序列的同源性≥85%);
载体5,6,7交由基因合成公司进行全基因合成。
通过包装慢病毒并构建293T细胞稳转株,将载体5、载体6和载体7依次转入293T细胞。
纳米抗体库大小检测具体步骤参见实施例2-5,检测到的纳米库大小约为2.75×107
将GFP-biotin配体与转染后的293T细胞共孵育24h,用blastcidin筛选阳性的细胞,将 阳性细胞挑单克隆扩增后对纳米抗体区测序即得到GFP特异性纳米抗体。
将所得阳性细胞中的纳米抗体序列挑选5条,进行原核表达纯化(蛋白表达纯化方法见 实施例8),得到VHH抗体蛋白。
ELISA检测所得VHH序列是否能够识别eGFP。结果如图14b所示,5个抗体中的2个对eGFP有较好的识别。
之后,为了增加纳米抗体对GFP蛋白的亲和力,我们对筛选到的两个VHH基因的三个 CDR区进行了一系列人工突变,最终得到了包含这两个VHH基因在内的一系列对GFP有不同亲和力的纳米抗体。其中VHH纳米抗体包含SEQ ID NO.31-40中的任两者,或者SEQ IDNO.31-40中CDR1,CDR2,CDR3区域的任意组合。
实施例12:根据gRNA引导碱基编辑器定位建库并进行纳米抗体筛选鉴定
本实施例可以在未知序列的纳米抗体CDRs区,基于gRNA引导进行体外碱基编辑建库。
原理如图4所示,由于在已知序列的纳米抗体CDRs区进行突变建库,经过几轮突变之 后会出现由于gRNA识别区或G-四联体区域序列改变而导致碱基编辑器无法识别CDRs区, 从而无法实现持续性建库,另外在得到特异性纳米抗体之后也无法进行亲和力成熟突变,因 此我们在图1的方案基础上做了改进,设计3个分别靶向VHH的CDR1、CDR2及CDR3右侧临近区域的gRNA,同时设计包含失活Cas9(dCas9)引导结构域以及在dCas9蛋白N端 通过柔性Linker依次连接Vivid光敏蛋白及解旋酶Mcm7(N端更靠近CDR区),解旋酶Mcm7 会和293T细胞中表达的Mcm2-6组成有解旋酶活性的完整六聚体,并在ATP水解提供能量 驱动下打开CDRs区双链(3'-5'解旋酶活性)并将Vivid光敏蛋白引向CDRs区一侧,通过另 外再表达两个高活性碱基编辑器,即Vivid融合的胞苷脱氨酶(促进G或C碱基向任意碱基突变)以及Vivid融合的腺苷脱氨酶(促进A碱基向任意碱基突变),在蓝光照射的条件下 Vivid光敏蛋白会形成同源二聚体,导致两种碱基编辑器随机结合到dCas9-Vivid-Mcm7的 Vivid蛋白上,从而定位于CDRs区。通过睡美人转座子系统SB100X将(dCas9-Vivid-Mcm7) 以及两个碱基编辑器基因一同转入293T细胞,构建稳转株。通过优化dCas9-Vivid-Mcm7之 间Linker序列的长度、Vivid与脱氨酶之间Linker序列的长度及蓝光照射时间,实现在VHH 的CDRs区广泛诱导突变,并通过检测纳米抗体库的多样性确定优化方案。
将载体2包装慢病毒并转入293T细胞中,通过嘌呤霉素筛选得到稳转株。
构建载体8(如图12),载体8所用的是和载体1同样的慢病毒载体,,载体8可表达包含Mcm7(SEQ ID NO.41)、Vivid(SEQ ID NO.42)、dCAS9(序列与文献中常用的酿脓 链球菌种Streptococcus pyogenes中的失活CAS9即dCAS9一致或同源性≥85%)三个结构域的融合蛋白(在另一优选例中载体8表达的Mcm7及Vivid两个结构域的氨基酸序列与SEQ IDNO.41、SEQ ID NO.42序列的同源性≥85%),三个结构域之间用5×GGGGS Linker(SEQ IDNO.43)依次连接(在另一优选例中Linker的长度可在2×GGGGS至10×GGGGS之间进行优化)。
构建慢病毒表达载体9(如图13),载体9可表达包含Vivid、rAPOBEC1和ecTadA*三个结构域的融合蛋白,Vivid氨基酸序列如SEQ ID NO.42所示,ecTadA*氨基酸序列如SEQID NO.44所示(在另一优选例中ecTadA*表达的氨基酸序列与SEQ ID NO.44序列的同源性≥85%),rAPOBEC1氨基酸序列如SEQ ID NO.45所示(在另一优选例中rAPOBEC1表达的 氨基酸序列与SEQ ID NO.45序列的同源性≥85%;在另一优选例中rAPOBEC1可用胞苷脱氨酶AID及其突变体替换),通过包装慢病毒并构建293T细胞稳转株,将载体8和载体9依次 转入293T细胞。
最后在293T细胞中转入载体10(载体结构如图9),载体10表达3个gRNA将包含dCAS9 结构域的蛋白定位至3个不同的位置,载体10的关键DNA序列如SEQ ID NO.46所示(SEQ ID NO.46序列为图9中U6 promoter元件和EF-1αcore promoter元件中间的序列,该段序列 连接至任意具有类似结构的pLL7系列慢病毒载体上均可)。
蓝光照射293T细胞2天后(每开5s关10s,蓝光强度0.84W/m2)计算纳米抗体库大小,纳米抗体库大小检测具体步骤参见实施例2-5,检测到的纳米库大小约为6.75×107。将GFP-biotin配体与转染后的293T细胞共孵育24h,用blastcidin筛选阳性的细胞,将阳性细胞挑单克隆扩增后对纳米抗体区测序即得到GFP特异性纳米抗体。
将所得阳性细胞中的纳米抗体序列挑选5条,进行原核表达纯化(蛋白表达纯化方法见 实施例8),得到VHH抗体蛋白。
ELISA检测所得VHH序列是否能够识别eGFP。结果如图14c所示,5个抗体中的1个对eGFP有较好的识别。
之后,为了增加纳米抗体对GFP蛋白的亲和力,我们对筛选到的VHH基因的三个CDR区进行了一系列人工突变,最终得到了包含VHH基因在内的一系列对GFP有不同亲和力的纳米抗体。其中VHH纳米抗体包含SEQ ID NO.31-40中的任一者,或者SEQ ID NO.31-40 中CDR1,CDR2,CDR3区域的任意组合。
<110> 新乡医学院
<120> 抗原结合域自动优化的嵌合抗原受体修饰细胞文库的构建、筛选方法及其应用
<160> 46
<170> PatentIn version 3.5
<210> 1
<211> 1416
<212> DNA
<213> 人工序列
<221> 受体1序列
<400> 1
atggccctgc tactggccct cagcctgctg gttctctgga cttccccagc cccaactctg 60
agtggcacca atgattaccc atacgatgtt ccagattacg ctgcggaagc gggtatcacc 120
ggcacgtggt acaaccagca tggttctacc ttcaccgtta ccgcgggtgc ggacggtaac 180
ctgaccggtc agtacgaaaa ccgtgcgcag ggcactggtt gccagaactc tccgtacacc 240
ctgaccggtc gttacaacgg taccaaactg gaatggcgtg ttgaatggaa caactctacc 300
gaaaactgcc actctcgtac cgaatggcgt ggtcagtacc agggtggtgc ggaagcgcgt 360
atcaacaccc agtggaacct gacctacgaa ggtggttctg gtccggcgac cgaacagggt 420
caggacacct tcaccaaagt taaaatgtac ttcagccact tcgtgccggt cttcctgcca 480
gcgaagccca ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 540
cagcccctgt ccctgcgccc agaggcgagc cggccagcgg cggggggcgc agtgcacacg 600
agggggctgg acttcgccag cgatatctac atctgggcgc ccttggccgg gacttgtggg 660
gtccttctcc tgtcactggt tatcaccctt tactgcggag gcggggagag tttgtttaag 720
gggccaaggg actataaccc aatatcatcc actatttgcc acctcactaa cgagagcgat 780
ggacatacaa cctctctcta cgggataggc ttcggtcctt tcatcattac caataagcat 840
ctgtttcgcc gaaacaacgg tactcttctg gttcaatctc ttcatggtgt cttcaaggtg 900
aaaaacacca ctacgcttca gcaacacctg attgatggta gggatatgat aattatcaga 960
atgccgaaag atttcccacc ttttccacag aagctgaaat tcagggaacc gcagagagag 1020
gagaggattt gtttggtaac gaccaacttc cagacgaaga gtatgagttc tatggtgtcc 1080
gacactagct gcacgttccc ctcaagtgat gggatattct ggaaacactg gatacagact 1140
aaagacggac agtgtggaag cccattggtt tccacccgag atggttttat cgtgggtatc 1200
catagcgcct ctaatttcac aaacacgaac aactatttca cttcagtgcc caaaaacttt 1260
atggagctgc tcacaaacca agaagcgcag cagtgggtaa gcggttggag acttaacgct 1320
gactcagttc tctggggggg gcacaaagta ttcatggtaa agccagagga gccattccaa 1380
ccagtcaaag aagccacaca acttatgaac agctaa 1416
<210> 2
<211> 2394
<212> DNA
<213> 人工序列
<221> 受体2序列1
<400> 2
atggccctgc ccgtgaccgc cctgctgctg cccctggccc tgctgctgca cgccgccagg 60
cccgactaca aggacgacga cgacaagccc gggatggccc aggtgcagct ggtggagagc 120
ggcggcggcc tggtgcaggc cggcggcagc ctgaggctga gctgcgccgc cccggggcgg 180
gccgggggcg gggtcccggc ggggtggttc aggcaggccc ccggcaagga gagggagttc 240
gtggccgcgg ggcgcttatg gggagggtgg ggagggtggg gaaggtgggg aggaggcagg 300
ttcaccatca gcagggacaa cgacaagaac accgtgtacg tgcagatgaa cagcctgatc 360
cccgaggaca ccgccatcta ctactgcgcc gcccgcggag gggcgggcgc gggaggaagg 420
gggcgggagc ggggctgtgg cgactactgg ggccagggca cccaggtgac cgtgggatcc 480
ccctgcgtgg gcagcaaccc ctgctacaac cagggcacct gcgagcccac cagcgagaac 540
cccttctaca ggtgcctgtg ccccgccaag ttcaacggcc tgctgtgcca catcctggac 600
tacagcttca ccggcggcgc cggcagggac atcccccccc cccagatcga ggaggcctgc 660
gagctgcccg agtgccaggt ggacgccggc aacaaggtgt gcaacctgca gtgcaacaac 720
cacgcctgcg gctgggacgg cggcgactgc agcctgaact tcaacgaccc ctggaagaac 780
tgcacccaga gcctgcagtg ctggaagtac ttcagcgacg gccactgcga cagccagtgc 840
aacagcgccg gctgcctgtt cgacggcttc gactgccagc tgaccgaggg ccagtgcaac 900
cccctgtacg accagtactg caaggaccac ttcagcgacg gccactgcga ccagggctgc 960
aacagcgccg agtgcgagtg ggacggcctg gactgcgccg agcacgtgcc cgagaggctg 1020
gccgccggca ccctggtgct ggtggtgctg ctgccccccg accagctgag gaacaacagc 1080
ttccacttcc tgagggagct gagccacgtg ctgcacacca acgtggtgtt caagagggac 1140
gcccagggcc agcagatgat cttcccctac tacggccacg aggaggagct gaggaagcac 1200
cccatcaaga ggagcaccgt gggctgggcc accagcagcc tgctgcccgg caccagcggc 1260
ggcaggcaga ggagggagct ggaccccatg gacatcaggg gcagcatcgt gtacctggag 1320
atcgacaaca ggcagtgcgt gcagagcagc agccagtgct tccagagcgc caccgacgtg 1380
gccgccttcc tgggcgccct ggccagcctg ggcagcctga acatccccta caagatcgag 1440
gcccataaga gcgagcccgt ggagcccccc ctgcccagcc agctgcacct gatgtacgtg 1500
gccgccgccg ccttcgtgct gctgttcttc gtgctccttt tctttctgct gagcaggaag 1560
aggaggaggc agctgtgcat ccagaagctg ctcgggatcg agggaagggg aggaggcgag 1620
ttcgctagcg agaacctgta tttccagggc atgtctagac tggacaagag caaagtcata 1680
aactctgctc tggaattact caatgaagtc ggtatcgaag gcctgacgac aaggaaactc 1740
gctcaaaagc tgggagttga gcagcctacc ctgtactggc acgtgaagaa caagcgggcc 1800
ctgctcgatg ccctggcaat cgagatgctg gacaggcatc atacccactt ctgccccctg 1860
gaaggcgagt catggcaaga ctttctgcgg aacaacgcca agtcattccg ctgtgctctc 1920
ctctcacatc gcgacggggc taaagtgcat ctcggcaccc gcccaacaga gaaacagtac 1980
gaaaccctgg aaaatcagct cgcgttcctg tgtcagcaag gcttctccct ggagaacgca 2040
ctgtacgctc tgtccgccgt gggccacttt acactgggct gcgtattgga ggatcaggag 2100
catcaagtag caaaagagga aagagagaca cctaccaccg attctatgcc cccacttctg 2160
agacaagcaa ttgagctgtt cgaccatcag ggagccgaac ctgccttcct tttcggcctg 2220
gaactaatca tatgtggcct ggagaaacag ctaaagtgcg aaagcggcgg gccggccgac 2280
gcccttgacg attttgactt agacatgctc ccagccgatg cccttgacga ctttgacctt 2340
gatatgctgc ctgctgacgc tcttgacgat tttgaccttg acatgctccc aggg 2394
<210> 3
<211> 1584
<212> DNA
<213> 人工序列
<221> 受体2序列2
<400> 3
atggccctgc ccgtgaccgc cctgctgctg cccctggccc tgctgctgca cgccgccagg 60
ccctacccat acgatgttcc agattacgct cccgggatgg cccaggtgca gctggtggag 120
agcggcggcg gcctggtgca ggccggcggc agcctgaggc tgagctgcgc cgccagcggc 180
aggaccttca gcaactacgc catgggctgg ttcaggcagg cccccggcaa ggagagggag 240
ttcgtggccg ccatcagctg gaccggcgtg agcacctact acgccgacag cgtgaagggc 300
aggttcacca tcagcaggga caacgacaag aacaccgtgt acgtgcagat gaacagcctg 360
atccccgagg acaccgccat ctactactgc gccgccgtga gggccaggag cttcagcgac 420
acctacagca gggtgaacga gtacgactac tggggccagg gcacccaggt gaccgtggga 480
tccatgtact tcagccactt cgtgccggtc ttcctgccag cgaagcccac cacgacgcca 540
gcgccgcgac caccaacacc ggcgcccacc atcgcgtcgc agcccctgtc cctgcgccca 600
gaggcgagcc ggccagcggc ggggggcgca gtgcacacga gggggctgga cttcgccagc 660
gatatctaca tctgggcgcc cttggccggg acttgtgggg tccttctcct gtcactggtt 720
atcacccttt actgcaattc gagctcgaac aacaacaaca ataacaataa caacaacctc 780
gggatcgagg gaaggggagg aggcgagttc gctagcgaga acctgtattt ccagggcatg 840
tctagactgg acaagagcaa agtcataaac tctgctctgg aattactcaa tgaagtcggt 900
atcgaaggcc tgacgacaag gaaactcgct caaaagctgg gagttgagca gcctaccctg 960
tactggcacg tgaagaacaa gcgggccctg ctcgatgccc tggcaatcga gatgctggac 1020
aggcatcata cccacttctg ccccctggaa ggcgagtcat ggcaagactt tctgcggaac 1080
aacgccaagt cattccgctg tgctctcctc tcacatcgcg acggggctaa agtgcatctc 1140
ggcacccgcc caacagagaa acagtacgaa accctggaaa atcagctcgc gttcctgtgt 1200
cagcaaggct tctccctgga gaacgcactg tacgctctgt ccgccgtggg ccactttaca 1260
ctgggctgcg tattggagga tcaggagcat caagtagcaa aagaggaaag agagacacct 1320
accaccgatt ctatgccccc acttctgaga caagcaattg agctgttcga ccatcaggga 1380
gccgaacctg ccttcctttt cggcctggaa ctaatcatat gtggcctgga gaaacagcta 1440
aagtgcgaaa gcggcgggcc ggccgacgcc cttgacgatt ttgacttaga catgctccca 1500
gccgatgccc ttgacgactt tgaccttgat atgctgcctg ctgacgctct tgacgatttt 1560
gaccttgaca tgctcccagg gtaa 1584
<210> 4
<211> 127
<212> PRT
<213> 人工序列
<221> G4纳米抗体1
<400> 4
Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala
1 5 10 15
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Gly Arg Ala Gly Gly
20 25 30
Gly Val Pro Ala Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
35 40 45
Phe Val Ala Ala Gly Arg Leu Trp Arg Gly Trp Gly Gly Trp Arg Arg
50 55 60
Trp Gly Gly Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr
65 70 75 80
Val Tyr Val Gln Met Asn Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr
85 90 95
Tyr Cys Ala Thr Arg Gly Gly Ala Gly Ala Gly Gly Arg Gly Arg Glu
100 105 110
Arg Gly Cys Gly Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val
115 120 125
<210> 5
<211> 121
<212> PRT
<213> 人工序列
<221> G4纳米抗体2
<400> 5
Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala
1 5 10 15
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Gly Arg Ala Gly Gly
20 25 30
Gly Val Pro Ala Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
35 40 45
Phe Val Ala Ala Gly Arg Leu Trp Gly Arg Trp Gly Gly Gly Arg Phe
50 55 60
Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr Val Tyr Val Gln Met Asn
65 70 75 80
Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Ala Arg Gly
85 90 95
Gly Ala Gly Ala Gly Gly Arg Gly Arg Glu Arg Gly Cys Gly Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val
115 120
<210> 6
<211> 124
<212> PRT
<213> 人工序列
<221> G4纳米抗体3
<400> 6
Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala
1 5 10 15
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Gly Arg Ala Gly Gly
20 25 30
Gly Val Pro Ala Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
35 40 45
Phe Val Ala Ala Gly Arg Leu Trp Gly Gly Trp Gly Arg Trp Gly Gly
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr Val Tyr Val
65 70 75 80
Gln Met Asn Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95
Ala Arg Gly Gly Ala Gly Ala Gly Gly Arg Gly Arg Glu Arg Gly Cys
100 105 110
Gly Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val
115 120
<210> 7
<211> 127
<212> PRT
<213> 人工序列
<221> G4纳米抗体4
<400> 7
Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala
1 5 10 15
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Gly Arg Ala Gly Gly
20 25 30
Gly Val Pro Ala Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
35 40 45
Phe Val Ala Ala Ile Ser Trp Thr Gly Val Ser Thr Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr
65 70 75 80
Val Tyr Val Gln Met Asn Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr
85 90 95
Tyr Cys Ala Ala Val Arg Ala Arg Ser Phe Ser Asp Thr Tyr Ser Arg
100 105 110
Val Asn Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val
115 120 125
<210> 8
<211> 127
<212> PRT
<213> 人工序列
<221> G4纳米抗体5
<400> 8
Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala
1 5 10 15
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Gly Arg Ala Gly Gly
20 25 30
Gly Val Pro Ala Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
35 40 45
Phe Val Ala Ala Ile Ser Trp Thr Gly Val Ser Thr Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr
65 70 75 80
Val Tyr Val Gln Met Asn Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr
85 90 95
Tyr Cys Ala Ala Val Arg Ala Arg Ser Phe Ser Asp Thr Tyr Ser Arg
100 105 110
Val Asn Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val
115 120 125
<210> 9
<211> 127
<212> PRT
<213> 人工序列
<221> G4纳米抗体6
<400> 9
Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala
1 5 10 15
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser
20 25 30
Thr Ser Ala Met Ala Trp Phe Arg Gln Ala Pro Gly Arg Glu Arg Glu
35 40 45
Phe Val Ala Ala Ile Thr Trp Thr Val Gly Asn Thr Ile Leu Gly Asp
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Arg Ala Lys Asn Thr
65 70 75 80
Val Asp Leu Gln Met Asp Asn Leu Glu Pro Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ser Ala Arg Ser Arg Gly Tyr Val Leu Ser Val Leu Arg Ser
100 105 110
Val Asp Ser Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val
115 120 125
<210> 10
<211> 124
<212> PRT
<213> 人工序列
<221> G4纳米抗体7
<400> 10
Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Arg Leu Val Gln Ala
1 5 10 15
Gly Asp Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser
20 25 30
Thr Ser Ala Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
35 40 45
Phe Val Ala Ala Gly Arg Leu Trp Gly Gly Trp Gly Arg Trp Gly Gly
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr Val Tyr Val
65 70 75 80
Gln Met Asn Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95
Ala Arg Gly Gly Ala Gly Ala Gly Gly Arg Gly Arg Glu Arg Gly Cys
100 105 110
Gly Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val
115 120
<210> 11
<211> 121
<212> PRT
<213> 人工序列
<221> G4纳米抗体8
<400> 11
Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala
1 5 10 15
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser
20 25 30
Asn Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
35 40 45
Phe Val Ala Ala Gly Arg Leu Trp Gly Arg Trp Gly Gly Gly Arg Phe
50 55 60
Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr Val Tyr Val Gln Met Asn
65 70 75 80
Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Ala Arg Gly
85 90 95
Gly Ala Gly Ala Gly Gly Arg Gly Arg Glu Arg Gly Cys Gly Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val
115 120
<210> 12
<211> 127
<212> PRT
<213> 人工序列
<221> G4纳米抗体9
<400> 12
Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Arg Leu Val Gln Ala
1 5 10 15
Gly Asp Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser
20 25 30
Thr Ser Ala Met Ala Trp Phe Arg Gln Ala Pro Gly Arg Glu Arg Glu
35 40 45
Phe Val Ala Ala Ile Thr Trp Thr Val Gly Asn Thr Ile Leu Gly Asp
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr
65 70 75 80
Val Tyr Val Gln Met Asn Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr
85 90 95
Tyr Cys Ala Ala Arg Gly Gly Ala Gly Ala Gly Gly Arg Gly Arg Glu
100 105 110
Arg Gly Cys Gly Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val
115 120 125
<210> 13
<211> 121
<212> PRT
<213> 人工序列
<221> G4纳米抗体10
<400> 13
Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala
1 5 10 15
Gly Gly Ser Leu Arg Leu Ser Tyr Ala Ala Pro Gly Arg Ala Gly Gly
20 25 30
Gly Val Pro Ala Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
35 40 45
Phe Val Ala Ala Gly Arg Leu Trp Gly Arg Trp Gly Gly Gly Arg Phe
50 55 60
Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr Val Tyr Val Gln Met Asn
65 70 75 80
Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Ala Arg Gly
85 90 95
Gly Ala Gly Ala Gly Gly Arg Gly Arg Glu Arg Gly Cys Gly Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val
115 120
<210> 14
<211> 4946
<212> DNA
<213> 人工序列
<221> 载体1关键序列
<400> 14
tccctatcag tgatagagaa aagtgaaagt cgagtttacc actccctatc agtgatagag 60
aaaagtgaaa gtcgagttta ccactcccta tcagtgatag agaaaagtga aagtcgagtt 120
taccactccc tatcagtgat agagaaaagt gaaagtcgag tttaccactc cctatcagtg 180
atagagaaaa gtgaaagtcg agtttaccac tccctatcag tgatagagaa aagtgaaagt 240
cgagtttacc actccctatc agtgatagag aactagttag gcgtgtacgg tgggaggcct 300
atataagcag agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt 360
ttgacctcca tagaagacac cgggaccgat ccagcctctc gacattcgtt ggatcgccgc 420
tagcgccacc atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat 480
gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga 540
gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg 600
ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta 660
cgtgaagcac cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa 720
ttgggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc 780
cctgcaggac ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga 840
cggccccgta atgcagtgtc gtaccatggg ctgggaggcc tccactgagc ggatgtaccc 900
cgaggacggc gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca 960
ctacgacgct gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc 1020
ctacaacgtc gacatcaagt tggacatcct ttcccacaac gaggactaca ccatcgtgga 1080
acagtacgaa cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagga 1140
gggcagagga agtctgctaa catgcggtga cgtcgaggag aatcctggcc caatggccaa 1200
gcctttgtct caagaagaat ccaccctcat tgaaagagca acggctacaa tcaacagcat 1260
ccccatctct gaagactaca gcgtcgccag cgcagctctc tctagcgacg gccgcatctt 1320
cactggtgtc aatgtatatc attttactgg gggaccttgt gcagaactcg tggtgctggg 1380
cactgctgct gctgcggcag ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa 1440
caggggcatc ttgagcccct gcggacggtg ccgacaggtg cttctcgatc tgcatcctgg 1500
gatcaaagcc atagtgaagg acagtgatgg acagccgacg gcagttggga ttcgtgaatt 1560
gctgccctct ggttatgtgt gggagggctg atacgtatta gtcatcgcta ttaccatggt 1620
gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc 1680
aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt 1740
tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg 1800
ggaggtttat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc 1860
acgctgtttt gacctccata gaagattcta gagccaccat ggccctgcta ctggccctca 1920
gcctgctggt tctctggact tccccagccc caactctgag tggcaccaat gatgactaca 1980
aagacgatga cgacaaggcg gaagcgggta tcaccggcac gtggtacaac cagcatggtt 2040
ctaccttcac cgttaccgcg ggtgcggacg gtaacctgac cggtcagtac gaaaaccgtg 2100
cgcagggcac tggttgccag aactctccgt acaccctgac cggtcgttac aacggtacca 2160
aactggaatg gcgtgttgaa tggaacaact ctaccgaaaa ctgccactct cgtaccgaat 2220
ggcgtggtca gtaccagggt ggtgcggaag cgcgtatcaa cacccagtgg aacctgacct 2280
acgaaggtgg ttctggtccg gcgaccgaac agggtcagga caccttcacc aaagttaaaa 2340
tgtacttcag ccacttcgtg ccggtcttcc tgccagcgaa gcccaccacg acgccagcgc 2400
cgcgaccacc aacaccggcg cccaccatcg cgtcgcagcc cctgtccctg cgcccagagg 2460
cgagccggcc agcggcgggg ggcgcagtgc acacgagggg gctggacttc gccagcgata 2520
tctacatctg ggcgcccttg gccgggactt gtggggtcct tctcctgtca ctggttatca 2580
ccctttactg cggaggcggg gagagtttgt ttaaggggcc aagggactat aacccaatat 2640
catccactat ttgccacctc actaacgaga gcgatggaca tacaacctct ctctacggga 2700
taggcttcgg tcctttcatc attaccaata agcatctgtt tcgccgaaac aacggtactc 2760
ttctggttca atctcttcat ggtgtcttca aggtgaaaaa caccactacg cttcagcaac 2820
acctgattga tggtagggat atgataatta tcagaatgcc gaaagatttc ccaccttttc 2880
cacagaagct gaaattcagg gaaccgcaga gagaggagag gatttgtttg gtaacgacca 2940
acttccagac gaagagtatg agttctatgg tgtccgacac tagctgcacg ttcccctcaa 3000
gtgatgggat attctggaaa cactggatac agactaaaga cggacagtgt ggaagcccat 3060
tggtttccac ccgagatggt tttatcgtgg gtatccatag cgcctctaat ttcacaaaca 3120
cgaacaacta tttcacttca gtgcccaaaa actttatgga gctgctcaca aaccaagaag 3180
cgcagcagtg ggtaagcggt tggagactta acgctgactc agttctctgg ggggggcaca 3240
aagtattcat ggtaaagcca gaggagccat tccaaccagt caaagaagcc acacaactta 3300
tgaacagcga gggcagagga agtctgctaa catgcggtga cgtcgaggag aatcctggcc 3360
caatggccct gcccgtgacc gccctgctgc tgcccctggc cctgctgctg cacgccgcca 3420
ggccctaccc atacgatgtt ccagattacg ctcccgggat ggcccaggtg cagctggtgg 3480
agagcggcgg cggcctggtg caggccggcg gcagcctgag gctgagctgc gccgccagcg 3540
gcaggacctt cagcaactac gccatgggct ggttcaggca ggcccccggc aaggagaggg 3600
agttcgtggc cgccatcagc tggaccggcg tgagcaccta ctacgccgac agcgtgaagg 3660
gcaggttcac catcagcagg gacaacgaca agaacaccgt gtacgtgcag atgaacagcc 3720
tgatccccga ggacaccgcc atctactact gcgccgccgt gagggccagg agcttcagcg 3780
acacctacag cagggtgaac gagtacgact actggggcca gggcacccag gtgaccgtgg 3840
gatccatgta cttcagccac ttcgtgccgg tcttcctgcc agcgaagccc accacgacgc 3900
cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg tccctgcgcc 3960
cagaggcgag ccggccagcg gcggggggcg cagtgcacac gagggggctg gacttcgcca 4020
gcgatatcta catctgggcg cccttggccg ggacttgtgg ggtccttctc ctgtcactgg 4080
ttatcaccct ttactgcaat tcgagctcga acaacaacaa caataacaat aacaacaacc 4140
tcgggatcga gggaagggga ggaggcgagt tcgctagcga gaacctgtat ttccagggca 4200
tgtctagact ggacaagagc aaagtcataa actctgctct ggaattactc aatgaagtcg 4260
gtatcgaagg cctgacgaca aggaaactcg ctcaaaagct gggagttgag cagcctaccc 4320
tgtactggca cgtgaagaac aagcgggccc tgctcgatgc cctggcaatc gagatgctgg 4380
acaggcatca tacccacttc tgccccctgg aaggcgagtc atggcaagac tttctgcgga 4440
acaacgccaa gtcattccgc tgtgctctcc tctcacatcg cgacggggct aaagtgcatc 4500
tcggcacccg cccaacagag aaacagtacg aaaccctgga aaatcagctc gcgttcctgt 4560
gtcagcaagg cttctccctg gagaacgcac tgtacgctct gtccgccgtg ggccacttta 4620
cactgggctg cgtattggag gatcaggagc atcaagtagc aaaagaggaa agagagacac 4680
ctaccaccga ttctatgccc ccacttctga gacaagcaat tgagctgttc gaccatcagg 4740
gagccgaacc tgccttcctt ttcggcctgg aactaatcat atgtggcctg gagaaacagc 4800
taaagtgcga aagcggcggg ccggccgacg cccttgacga ttttgactta gacatgctcc 4860
cagccgatgc ccttgacgac tttgaccttg atatgctgcc tgctgacgct cttgacgatt 4920
ttgaccttga catgctccca gggtaa 4946
<210> 15
<211> 5762
<212> DNA
<213> 人工序列
<221> 载体2关键序列
<400> 15
tccctatcag tgatagagaa aagtgaaagt cgagtttacc actccctatc agtgatagag 60
aaaagtgaaa gtcgagttta ccactcccta tcagtgatag agaaaagtga aagtcgagtt 120
taccactccc tatcagtgat agagaaaagt gaaagtcgag tttaccactc cctatcagtg 180
atagagaaaa gtgaaagtcg agtttaccac tccctatcag tgatagagaa aagtgaaagt 240
cgagtttacc actccctatc agtgatagag aactagttag gcgtgtacgg tgggaggcct 300
atataagcag agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt 360
ttgacctcca tagaagacac cgggaccgat ccagcctctc gacattcgtt ggatcgccgc 420
tagcgccacc atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat 480
gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga 540
gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg 600
ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta 660
cgtgaagcac cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa 720
ttgggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc 780
cctgcaggac ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga 840
cggccccgta atgcagtgtc gtaccatggg ctgggaggcc tccactgagc ggatgtaccc 900
cgaggacggc gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca 960
ctacgacgct gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc 1020
ctacaacgtc gacatcaagt tggacatcct ttcccacaac gaggactaca ccatcgtgga 1080
acagtacgaa cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagga 1140
gggcagagga agtctgctaa catgcggtga cgtcgaggag aatcctggcc caatggccaa 1200
gcctttgtct caagaagaat ccaccctcat tgaaagagca acggctacaa tcaacagcat 1260
ccccatctct gaagactaca gcgtcgccag cgcagctctc tctagcgacg gccgcatctt 1320
cactggtgtc aatgtatatc attttactgg gggaccttgt gcagaactcg tggtgctggg 1380
cactgctgct gctgcggcag ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa 1440
caggggcatc ttgagcccct gcggacggtg ccgacaggtg cttctcgatc tgcatcctgg 1500
gatcaaagcc atagtgaagg acagtgatgg acagccgacg gcagttggga ttcgtgaatt 1560
gctgccctct ggttatgtgt gggagggctg atacgtatta gtcatcgcta ttaccatggt 1620
gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc 1680
aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt 1740
tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg 1800
ggaggtttat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc 1860
acgctgtttt gacctccata gaagattcta gagccaccat ggccctgccc gtgaccgccc 1920
tgctgctgcc cctggccctg ctgctgcacg ccgccaggcc cgactacaag gacgacgacg 1980
acaagcccgg gatggcccag gtgcagctgg tggagagcgg cggcggcctg gtgcaggccg 2040
gcggcagcct gaggctgagc tgcgccgccc cggggcgggc cgggggcggg gtcccggcgg 2100
ggtggttcag gcaggccccc ggcaaggaga gggagttcgt ggccgcgggg cgcttatggg 2160
gagggtgggg agggtgggga aggtggggag gaggcaggtt caccatcagc agggacaacg 2220
acaagaacac cgtgtacgtg cagatgaaca gcctgatccc cgaggacacc gccatctact 2280
actgcgccgc ccgcggaggg gcgggcgcgg gaggaagggg gcgggagcgg ggctgtggcg 2340
actactgggg ccagggcacc caggtgaccg tgggatcccc ctgcgtgggc agcaacccct 2400
gctacaacca gggcacctgc gagcccacca gcgagaaccc cttctacagg tgcctgtgcc 2460
ccgccaagtt caacggcctg ctgtgccaca tcctggacta cagcttcacc ggcggcgccg 2520
gcagggacat cccccccccc cagatcgagg aggcctgcga gctgcccgag tgccaggtgg 2580
acgccggcaa caaggtgtgc aacctgcagt gcaacaacca cgcctgcggc tgggacggcg 2640
gcgactgcag cctgaacttc aacgacccct ggaagaactg cacccagagc ctgcagtgct 2700
ggaagtactt cagcgacggc cactgcgaca gccagtgcaa cagcgccggc tgcctgttcg 2760
acggcttcga ctgccagctg accgagggcc agtgcaaccc cctgtacgac cagtactgca 2820
aggaccactt cagcgacggc cactgcgacc agggctgcaa cagcgccgag tgcgagtggg 2880
acggcctgga ctgcgccgag cacgtgcccg agaggctggc cgccggcacc ctggtgctgg 2940
tggtgctgct gccccccgac cagctgagga acaacagctt ccacttcctg agggagctga 3000
gccacgtgct gcacaccaac gtggtgttca agagggacgc ccagggccag cagatgatct 3060
tcccctacta cggccacgag gaggagctga ggaagcaccc catcaagagg agcaccgtgg 3120
gctgggccac cagcagcctg ctgcccggca ccagcggcgg caggcagagg agggagctgg 3180
accccatgga catcaggggc agcatcgtgt acctggagat cgacaacagg cagtgcgtgc 3240
agagcagcag ccagtgcttc cagagcgcca ccgacgtggc cgccttcctg ggcgccctgg 3300
ccagcctggg cagcctgaac atcccctaca agatcgaggc ccataagagc gagcccgtgg 3360
agccccccct gcccagccag ctgcacctga tgtacgtggc cgccgccgcc ttcgtgctgc 3420
tgttcttcgt gctccttttc tttctgctga gcaggaagag gaggaggcag ctgtgcatcc 3480
agaagctgct cgggatcgag ggaaggggag gaggcgagtt cgctagcgag aacctgtatt 3540
tccagggcat gtctagactg gacaagagca aagtcataaa ctctgctctg gaattactca 3600
atgaagtcgg tatcgaaggc ctgacgacaa ggaaactcgc tcaaaagctg ggagttgagc 3660
agcctaccct gtactggcac gtgaagaaca agcgggccct gctcgatgcc ctggcaatcg 3720
agatgctgga caggcatcat acccacttct gccccctgga aggcgagtca tggcaagact 3780
ttctgcggaa caacgccaag tcattccgct gtgctctcct ctcacatcgc gacggggcta 3840
aagtgcatct cggcacccgc ccaacagaga aacagtacga aaccctggaa aatcagctcg 3900
cgttcctgtg tcagcaaggc ttctccctgg agaacgcact gtacgctctg tccgccgtgg 3960
gccactttac actgggctgc gtattggagg atcaggagca tcaagtagca aaagaggaaa 4020
gagagacacc taccaccgat tctatgcccc cacttctgag acaagcaatt gagctgttcg 4080
accatcaggg agccgaacct gccttccttt tcggcctgga actaatcata tgtggcctgg 4140
agaaacagct aaagtgcgaa agcggcgggc cggccgacgc ccttgacgat tttgacttag 4200
acatgctccc agccgatgcc cttgacgact ttgaccttga tatgctgcct gctgacgctc 4260
ttgacgattt tgaccttgac atgctcccag gggagggcag aggaagtctg ctaacatgcg 4320
gtgacgtcga ggagaatcct ggcccaatgg ccctgctact ggccctcagc ctgctggttc 4380
tctggacttc cccagcccca actctgagtg gcaccaatga ttacccatac gatgttccag 4440
attacgctgc ggaagcgggt atcaccggca cgtggtacaa ccagcatggt tctaccttca 4500
ccgttaccgc gggtgcggac ggtaacctga ccggtcagta cgaaaaccgt gcgcagggca 4560
ctggttgcca gaactctccg tacaccctga ccggtcgtta caacggtacc aaactggaat 4620
ggcgtgttga atggaacaac tctaccgaaa actgccactc tcgtaccgaa tggcgtggtc 4680
agtaccaggg tggtgcggaa gcgcgtatca acacccagtg gaacctgacc tacgaaggtg 4740
gttctggtcc ggcgaccgaa cagggtcagg acaccttcac caaagttaaa atgtacttca 4800
gccacttcgt gccggtcttc ctgccagcga agcccaccac gacgccagcg ccgcgaccac 4860
caacaccggc gcccaccatc gcgtcgcagc ccctgtccct gcgcccagag gcgagccggc 4920
cagcggcggg gggcgcagtg cacacgaggg ggctggactt cgccagcgat atctacatct 4980
gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc accctttact 5040
gcggaggcgg ggagagtttg tttaaggggc caagggacta taacccaata tcatccacta 5100
tttgccacct cactaacgag agcgatggac atacaacctc tctctacggg ataggcttcg 5160
gtcctttcat cattaccaat aagcatctgt ttcgccgaaa caacggtact cttctggttc 5220
aatctcttca tggtgtcttc aaggtgaaaa acaccactac gcttcagcaa cacctgattg 5280
atggtaggga tatgataatt atcagaatgc cgaaagattt cccacctttt ccacagaagc 5340
tgaaattcag ggaaccgcag agagaggaga ggatttgttt ggtaacgacc aacttccaga 5400
cgaagagtat gagttctatg gtgtccgaca ctagctgcac gttcccctca agtgatggga 5460
tattctggaa acactggata cagactaaag acggacagtg tggaagccca ttggtttcca 5520
cccgagatgg ttttatcgtg ggtatccata gcgcctctaa tttcacaaac acgaacaact 5580
atttcacttc agtgcccaaa aactttatgg agctgctcac aaaccaagaa gcgcagcagt 5640
gggtaagcgg ttggagactt aacgctgact cagttctctg gggggggcac aaagtattca 5700
tggtaaagcc agaggagcca ttccaaccag tcaaagaagc cacacaactt atgaacagct 5760
aa 5762
<210> 16
<211> 124
<212> PRT
<213> 人工序列
<221> 天然纳米抗体1
<400> 16
Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Ser Ala Ile Ser Ile Asp Gly Ser His Thr Thr Tyr Thr Ala Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Thr Leu Arg Leu Gln Tyr Gly Leu Ala Tyr Asp Leu Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 17
<211> 123
<212> PRT
<213> 人工序列
<221> 天然纳米抗体2
<400> 17
Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Tyr Phe Val Ile Pro Asp Ala Gln
20 25 30
His Gly Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Ser Asn Lys Asp Thr Gly Cys Tyr His Val Ala Arg Leu Pro Met Glu
50 55 60
Gln Phe Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Asn Thr Arg Glu Ile His Gly Lys Met Gln Trp Leu Phe Ser Val
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 18
<211> 123
<212> PRT
<213> 人工序列
<221> 天然纳米抗体3
<400> 18
Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Pro Phe Ser Leu Gly Cys Gln Glu
20 25 30
Arg Ala Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Ser Thr Glu Asn Lys His Ala Pro Leu Val Gln Tyr Asp Trp Ile Gly
50 55 60
Met Cys Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Val Met Asn Arg Ala Ile Glu Asp Gly Phe Lys Cys Ser Trp Gln
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 19
<211> 123
<212> PRT
<213> 人工序列
<221> 天然纳米抗体4
<400> 19
Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Trp Thr Gly Pro Asp Arg Glu Val
20 25 30
Met Phe Lys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Ser Thr Gln Ile Met Asp Arg Leu Phe Lys Val Pro Asn Ala Ser Glu
50 55 60
Gly Trp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Trp Cys Ala Arg Val His Gly Asp Pro Asn Phe Tyr Met Ile Glu
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 20
<211> 123
<212> PRT
<213> 人工序列
<221> 天然纳米抗体5
<400> 20
Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Cys Tyr Gly Ile His Val Trp Asn
20 25 30
Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Ser Asn Met Gln Pro Leu Ser Ala Ile Arg Glu Phe Trp Tyr Cys Val
50 55 60
His Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Val Cys Phe Pro Thr Asn Arg Tyr Glu His Met Asp Leu Gln Ile
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 21
<211> 123
<212> PRT
<213> 人工序列
<221> 天然纳米抗体6
<400> 21
Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Tyr Phe Asn Glu Trp His Gln Pro
20 25 30
Thr Gly Lys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Ser Cys Glu Ala Trp Pro Ile Ser Asp Thr Asn Met Phe Tyr Gln Leu
50 55 60
His Val Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Phe Ala Pro Gln Glu Ser Leu Asp Gly Ile Asn Arg Trp Tyr Lys
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 22
<211> 123
<212> PRT
<213> 人工序列
<221> 天然纳米抗体7
<400> 22
Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala His Ile Phe Pro Thr Gly Trp Asn
20 25 30
Cys Lys Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Ser Glu Met Thr Ile Tyr Ala Gly Val Trp His Asp Cys Pro Gln Lys
50 55 60
Asn Phe Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Gln Met Cys Asn Arg Val Pro Phe Glu Ala Thr Gly Asp Leu Trp
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 23
<211> 123
<212> PRT
<213> 人工序列
<221> 天然纳米抗体8
<400> 23
Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Lys Leu Phe Val Asn Gly Cys Tyr
20 25 30
Ser His Glu Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Ser Ala Cys Ser Gly Val Ile Pro Asp Met His Thr Gln Glu Leu Trp
50 55 60
Tyr Asn Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Cys Arg Ser Phe Asn Ile Gln Gly Met Asp Thr Tyr Val Trp His
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 24
<211> 123
<212> PRT
<213> 人工序列
<221> 天然纳米抗体9
<400> 24
Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Met Val Tyr Pro Ala Thr Glu Ile
20 25 30
His Trp Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Ser Tyr Asp Ala Glu Met Trp Lys Gln Thr Asn Arg Val Gly His Cys
50 55 60
Ser Phe Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Tyr Val Ala Gly Gln Leu Asn Asp Cys Pro Glu Met Lys Phe His
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 25
<211> 123
<212> PRT
<213> 人工序列
<221> 天然纳米抗体10
<400> 25
Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Phe Thr Trp Glu Arg Gly Ile Pro
20 25 30
Ser Ala Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Ser Glu Lys Asn Pro Thr Ser Phe Cys Leu Tyr Ile Asp Ala Gly Met
50 55 60
Trp Arg Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Arg Lys Ala Cys Phe Ser Met Gln Val Pro Ile Asp His Glu
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 26
<211> 3378
<212> DNA
<213> 人工序列
<221> 载体3
<400> 26
gactacaaag acgatgacga caagtccgaa gtcgagtttt cccatgagta ctggatgaga 60
cacgcattga ctctcgcaaa gagggcttgg gatgaacgcg aggtgcccgt gggggcagta 120
ctcgtgcata acaatcgcgt aatcggcgaa ggttggaata ggccgatcgg acgccacgac 180
cccactgcac atgcggaaat catggccctt cgacagggag ggcttgtgat gcagaattat 240
cgacttatcg atgcgacgct gtacgtcacg cttgaacctt gcgtaatgtg cgcgggagct 300
atgattcact cccgcattgg acgagttgta ttcggtgccc gcgacgccaa gacgggtgcc 360
gcaggttcac tgatggacgt gctgcatcac ccaggcatga accaccgggt agaaatcaca 420
gaaggcatat tggcggacga atgtgcggcg ctgttgtccg acttttttcg catgcggagg 480
caggagatca aggcccagaa aaaagcacaa tcctctactg actctggtgg ttcttctggt 540
ggttctagcg gcagcgagac tcccgggacc tcagagtccg ccacacccga aagttctggt 600
ggttcttctg gtggttcttc cgaagtcgag ttttcccatg agtactggat gagacacgca 660
ttgactctcg caaagagggc tcgagatgaa cgcgaggtgc ccgtgggggc agtactcgtg 720
ctcaacaatc gcgtaatcgg cgaaggttgg aatagggcaa tcggactcca cgaccccact 780
gcacatgcgg aaatcatggc ccttcgacag ggagggcttg tgatgcagaa ttatcgactt 840
atcgatgcga cgctgtacgt cacgtttgaa ccttgcgtaa tgtgcgcggg agctatgatt 900
cactcccgca ttggacgagt tgtattcggt gttcgcaacg ccaagacggg tgccgcaggt 960
tcactgatgg acgtgctgca ttacccaggc atgaaccacc gggtagaaat cacagaaggc 1020
atattggcgg acgaatgtgc ggcgctgttg tgttactttt ttcgcatgcc caggcaggtc 1080
tttaacgccc agaaaaaagc acaatcctct actgactctg gtggttcttc tggtggttct 1140
agcggcagcg agactcccgg gacctcagag tccgccacac ccgaaagttc tggtggttct 1200
tctggtggtt ctatggtgaa gctcgcgaag gcaggtaaaa atcaaggtga ccccaagaaa 1260
atggctcctc ctccaaagga ggtagaagaa gatagtgaag atgaggaaat gtcagaagat 1320
gaagaagatg atagcagtgg agaagaggtc gtcatacctc agaagaaagg caagaaggct 1380
gctgcaacct cagcaaagaa ggtggtcgtt tccccaacaa aaaaggttgc agttgccaca 1440
ccagccaaga aagcagctgt cactccaggc aaaaaggcag cagcaacacc tgccaagaag 1500
acagttacac cagccaaagc agttaccaca cctggcaaga agggagccac accaggcaaa 1560
gcattggtag caactcctgg taagaagggt gctgccatcc cagccaaggg ggcaaagaat 1620
ggcaagaatg ccaagaagga agacagtgat gaagaggagg atgatgacag tgaggaggat 1680
gaggaggatg acgaggacga ggatgaggat gaagatgaaa ttgaaccagc agcgatgaaa 1740
gcagcagctg ctgcccctgc ctcagaggat gaggacgatg aggatgacga agatgatgag 1800
gatgacgatg acgatgagga agatgactct gaagaagaag ctatggagac tacaccagcc 1860
aaaggaaaga aagctgcaaa agttgttcct gtgaaagcca agaacgtggc tgaggatgaa 1920
gatgaagaag aggatgatga ggacgaggat gacgacgacg acgaagatga tgaagatgat 1980
gatgatgaag atgatgagga ggaggaagaa gaggaggagg aagagcctgt caaagaagca 2040
cctggaaaac gaaagaagga aatggccaaa cagaaagcag ctcctgaagc caagaaacag 2100
aaagtggaag gcacagaacc gactacggct ttcaatctct ttgttggaaa cctaaacttt 2160
aacaaatctg ctcctgaatt aaaaactggt atcagcgatg tttttgctaa aaatgatctt 2220
gctgttgtgg atgtcagaat tggtatgact aggaaatttg gttatgtgga ttttgaatct 2280
gctgaagacc tggagaaagc gttggaactc actggtttga aagtctttgg caatgaaatt 2340
aaactagaga aaccaaaagg aaaagacagt aagaaagagc gagatgcgag aacacttttg 2400
gctaaaaatc tcccttacaa agtcactcag gatgaattga aagaagtgtt tgaagatgct 2460
gcggagatca gattagtcag caaggatggg aaaagtaaag ggattgctta tattgaattt 2520
aagacagaag ctgatgcaga gaaaaccttt gaagaaaagc agggaacaga gatcgatggg 2580
cgatctattt ccctgtacta tactggagag aaaggtcaaa atcaagacta tagaggtgga 2640
aagaatagca cttggagtgg tgaatcaaaa actctggttt taagcaacct ctcctacagt 2700
gcaacagaag aaactcttca ggaagtattt gagaaagcaa cttttatcaa agtaccccag 2760
aaccaaaatg gcaaatctaa agggtatgca tttatagagt ttgcttcatt cgaagacgct 2820
aaagaagctt taaattcctg taataaaagg gaaattgagg gcagagcaat caggctggag 2880
ttgcaaggac ccaggggatc acctaatgcc agaagccagc catccaaaac tctgtttgtc 2940
aaaggcctgt ctgaggatac cactgaagag acattaaagg agtcatttga cggctccgtt 3000
cgggcaagga tagttactga ccgggaaact gggtcctcca aagggtttgg ttttgtagac 3060
ttcaacagtg aggaggatgc caaagctgcc aaggaggcca tggaagacgg tgaaattgat 3120
ggaaataaag ttaccttgga ctgggccaaa cctaagggtg aaggtggctt cgggggtcgt 3180
ggtggaggca gaggcggctt tggaggacga ggtggtggta gaggaggccg aggaggattt 3240
ggtggcagag gccggggagg ctttggaggg cgaggaggct tccgaggagg cagaggagga 3300
ggaggtgacc acaagccaca aggaaagaag acgaagtttg aatctggtgg ttctcccaag 3360
aagaagagga aagtctag 3378
<210> 27
<211> 3183
<212> DNA
<213> 人工序列
<221> 载体4
<400> 27
gactacaaag acgatgacga caagagctca gagactggcc cagtggctgt ggaccccaca 60
ttgagacggc ggatcgagcc ccatgagttt gaggtattct tcgatccgag agagctccgc 120
aaggagacct gcctgcttta cgaaattaat tgggggggcc ggcactccat ttggcgacat 180
acatcacaga acactaacaa gcacgtcgaa gtcaacttca tcgagaagtt cacgacagaa 240
agatatttct gtccgaacac aaggtgcagc attacctggt ttctcagctg gagcccatgc 300
ggcgaatgta gtagggccat cactgaattc ctgtcaaggt atccccacgt cactctgttt 360
atttacatcg caaggctgta ccaccacgct gacccccgca atcgacaagg cctgcgggat 420
ttgatctctt caggtgtgac tatccaaatt atgactgagc aggagtcagg atactgctgg 480
agaaactttg tgaattatag cccgagtaat gaagcccact ggcctaggta tccccatctg 540
tgggtacgac tgtacgttct tgaactgtac tgcatcatac tgggcctgcc tccttgtctc 600
aacattctga gaaggaagca gccacagctg acattcttta ccatcgctct tcagtcttgt 660
cattaccagc gactgccccc acacattctc tgggccaccg ggttgaaaag cggcagcgag 720
actcccggga cctcagagtc cgccacaccc gaaagtatgg tgaagctcgc gaaggcaggt 780
aaaaatcaag gtgaccccaa gaaaatggct cctcctccaa aggaggtaga agaagatagt 840
gaagatgagg aaatgtcaga agatgaagaa gatgatagca gtggagaaga ggtcgtcata 900
cctcagaaga aaggcaagaa ggctgctgca acctcagcaa agaaggtggt cgtttcccca 960
acaaaaaagg ttgcagttgc cacaccagcc aagaaagcag ctgtcactcc aggcaaaaag 1020
gcagcagcaa cacctgccaa gaagacagtt acaccagcca aagcagttac cacacctggc 1080
aagaagggag ccacaccagg caaagcattg gtagcaactc ctggtaagaa gggtgctgcc 1140
atcccagcca agggggcaaa gaatggcaag aatgccaaga aggaagacag tgatgaagag 1200
gaggatgatg acagtgagga ggatgaggag gatgacgagg acgaggatga ggatgaagat 1260
gaaattgaac cagcagcgat gaaagcagca gctgctgccc ctgcctcaga ggatgaggac 1320
gatgaggatg acgaagatga tgaggatgac gatgacgatg aggaagatga ctctgaagaa 1380
gaagctatgg agactacacc agccaaagga aagaaagctg caaaagttgt tcctgtgaaa 1440
gccaagaacg tggctgagga tgaagatgaa gaagaggatg atgaggacga ggatgacgac 1500
gacgacgaag atgatgaaga tgatgatgat gaagatgatg aggaggagga agaagaggag 1560
gaggaagagc ctgtcaaaga agcacctgga aaacgaaaga aggaaatggc caaacagaaa 1620
gcagctcctg aagccaagaa acagaaagtg gaaggcacag aaccgactac ggctttcaat 1680
ctctttgttg gaaacctaaa ctttaacaaa tctgctcctg aattaaaaac tggtatcagc 1740
gatgtttttg ctaaaaatga tcttgctgtt gtggatgtca gaattggtat gactaggaaa 1800
tttggttatg tggattttga atctgctgaa gacctggaga aagcgttgga actcactggt 1860
ttgaaagtct ttggcaatga aattaaacta gagaaaccaa aaggaaaaga cagtaagaaa 1920
gagcgagatg cgagaacact tttggctaaa aatctccctt acaaagtcac tcaggatgaa 1980
ttgaaagaag tgtttgaaga tgctgcggag atcagattag tcagcaagga tgggaaaagt 2040
aaagggattg cttatattga atttaagaca gaagctgatg cagagaaaac ctttgaagaa 2100
aagcagggaa cagagatcga tgggcgatct atttccctgt actatactgg agagaaaggt 2160
caaaatcaag actatagagg tggaaagaat agcacttgga gtggtgaatc aaaaactctg 2220
gttttaagca acctctccta cagtgcaaca gaagaaactc ttcaggaagt atttgagaaa 2280
gcaactttta tcaaagtacc ccagaaccaa aatggcaaat ctaaagggta tgcatttata 2340
gagtttgctt cattcgaaga cgctaaagaa gctttaaatt cctgtaataa aagggaaatt 2400
gagggcagag caatcaggct ggagttgcaa ggacccaggg gatcacctaa tgccagaagc 2460
cagccatcca aaactctgtt tgtcaaaggc ctgtctgagg ataccactga agagacatta 2520
aaggagtcat ttgacggctc cgttcgggca aggatagtta ctgaccggga aactgggtcc 2580
tccaaagggt ttggttttgt agacttcaac agtgaggagg atgccaaagc tgccaaggag 2640
gccatggaag acggtgaaat tgatggaaat aaagttacct tggactgggc caaacctaag 2700
ggtgaaggtg gcttcggggg tcgtggtgga ggcagaggcg gctttggagg acgaggtggt 2760
ggtagaggag gccgaggagg atttggtggc agaggccggg gaggctttgg agggcgagga 2820
ggcttccgag gaggcagagg aggaggaggt gaccacaagc cacaaggaaa gaagacgaag 2880
tttgaatctg gtggttctac taatctgtca gatattattg aaaaggagac cggtaagcaa 2940
ctggttatcc aggaatccat cctcatgctc ccagaggagg tggaagaagt cattgggaac 3000
aagccggaaa gcgatatact cgtgcacacc gcctacgacg agagcaccga cgagaatgtc 3060
atgcttctga ctagcgacgc ccctgaatac aagccttggg ctctggtcat acaggatagc 3120
aacggtgaga acaagattaa gatgctctct ggtggttctc ccaagaagaa gaggaaagtc 3180
taa 3183
<210> 28
<211> 589
<212> DNA
<213> 人工序列
<221> 载体5
<400> 28
gaaacaccga acaaagcacc agtggtctag tggtagaata gtaccctgcc acggtacaga 60
cccgggttcg attcccggct ggtgcacccc gccgggaccc cgccccgttt tagagctaga 120
aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt 180
gcaacaaagc accagtggtc tagtggtaga atagtaccct gccacggtac agacccgggt 240
tcgattcccg gctggtgcag agggtgggga gggtggggag ttttagagct agaaatagca 300
agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcaacaa 360
agcaccagtg gtctagtggt agaatagtac cctgccacgg tacagacccg ggttcgattc 420
ccggctggtg cactcccgcg cccgcccctc cggttttaga gctagaaata gcaagttaaa 480
ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttggatcc 540
agcgcttagg tcttgaaagg agtgggaatt ggctccggtg cccgtcagt 589
<210> 29
<211> 5328
<212> PRT
<213> 人工序列
<221> 载体6
<400> 29
atgtccgaag tcgagttttc ccatgagtac tggatgagac acgcattgac tctcgcaaag 60
agggcttggg atgaacgcga ggtgcccgtg ggggcagtac tcgtgcataa caatcgcgta 120
atcggcgaag gttggaatag gccgatcgga cgccacgacc ccactgcaca tgcggaaatc 180
atggcccttc gacagggagg gcttgtgatg cagaattatc gacttatcga tgcgacgctg 240
tacgtcacgc ttgaaccttg cgtaatgtgc gcgggagcta tgattcactc ccgcattgga 300
cgagttgtat tcggtgcccg cgacgccaag acgggtgccg caggttcact gatggacgtg 360
ctgcatcacc caggcatgaa ccaccgggta gaaatcacag aaggcatatt ggcggacgaa 420
tgtgcggcgc tgttgtccga cttttttcgc atgcggaggc aggagatcaa ggcccagaaa 480
aaagcacaat cctctactga ctctggtggt tcttctggtg gttctagcgg cagcgagact 540
cccgggacct cagagtccgc cacacccgaa agttctggtg gttcttctgg tggttcttcc 600
gaagtcgagt tttcccatga gtactggatg agacacgcat tgactctcgc aaagagggct 660
cgagatgaac gcgaggtgcc cgtgggggca gtactcgtgc tcaacaatcg cgtaatcggc 720
gaaggttgga atagggcaat cggactccac gaccccactg cacatgcgga aatcatggcc 780
cttcgacagg gagggcttgt gatgcagaat tatcgactta tcgatgcgac gctgtacgtc 840
acgtttgaac cttgcgtaat gtgcgcggga gctatgattc actcccgcat tggacgagtt 900
gtattcggtg ttcgcaacgc caagacgggt gccgcaggtt cactgatgga cgtgctgcat 960
tacccaggca tgaaccaccg ggtagaaatc acagaaggca tattggcgga cgaatgtgcg 1020
gcgctgttgt gttacttttt tcgcatgccc aggcaggtct ttaacgccca gaaaaaagca 1080
caatcctcta ctgactctgg tggttcttct ggtggttcta gcggcagcga gactcccggg 1140
acctcagagt ccgccacacc cgaaagttct ggtggttctt ctggtggttc tgataaaaag 1200
tattctattg gtttagccat cggcactaat tccgttggat gggctgtcat aaccgatgaa 1260
tacaaagtac cttcaaagaa atttaaggtg ttggggaaca cagaccgtca ttcgattaaa 1320
aagaatctta tcggtgccct cctattcgat agtggcgaaa cggcagaggc gactcgcctg 1380
aaacgaaccg ctcggagaag gtatacacgt cgcaagaacc gaatatgtta cttacaagaa 1440
atttttagca atgagatggc caaagttgac gattctttct ttcaccgttt ggaagagtcc 1500
ttccttgtcg aagaggacaa gaaacatgaa cggcacccca tctttggaaa catagtagat 1560
gaggtggcat atcatgaaaa gtacccaacg atttatcacc tcagaaaaaa gctagttgac 1620
tcaactgata aagcggacct gaggttaatc tacttggctc ttgcccatat gataaagttc 1680
cgtgggcact ttctcattga gggtgatcta aatccggaca actcggatgt cgacaaactg 1740
ttcatccagt tagtacaaac ctataatcag ttgtttgaag agaaccctat aaatgcaagt 1800
ggcgtggatg cgaaggctat tcttagcgcc cgcctctcta aatcccgacg gctagaaaac 1860
ctgatcgcac aattacccgg agagaagaaa aatgggttgt tcggtaacct tatagcgctc 1920
tcactaggcc tgacaccaaa ttttaagtcg aacttcgact tagctgaaga tgccaaattg 1980
cagcttagta aggacacgta cgatgacgat ctcgacaatc tactggcaca aattggagat 2040
cagtatgcgg acttattttt ggctgccaaa aaccttagcg atgcaatcct cctatctgac 2100
atactgagag ttaatactga gattaccaag gcgccgttat ccgcttcaat gatcaaaagg 2160
tacgatgaac atcaccaaga cttgacactt ctcaaggccc tagtccgtca gcaactgcct 2220
gagaaatata aggaaatatt ctttgatcag tcgaaaaacg ggtacgcagg ttatattgac 2280
ggcggagcga gtcaagagga attctacaag tttatcaaac ccatattaga gaagatggat 2340
gggacggaag agttgcttgt aaaactcaat cgcgaagatc tactgcgaaa gcagcggact 2400
ttcgacaacg gtagcattcc acatcaaatc cacttaggcg aattgcatgc tatacttaga 2460
aggcaggagg atttttatcc gttcctcaaa gacaatcgtg aaaagattga gaaaatccta 2520
acctttcgca taccttacta tgtgggaccc ctggcccgag ggaactctcg gttcgcatgg 2580
atgacaagaa agtccgaaga aacgattact ccatggaatt ttgaggaagt tgtcgataaa 2640
ggtgcgtcag ctcaatcgtt catcgagagg atgaccaact ttgacaagaa tttaccgaac 2700
gaaaaagtat tgcctaagca cagtttactt tacgagtatt tcacagtgta caatgaactc 2760
acgaaagtta agtatgtcac tgagggcatg cgtaaacccg cctttctaag cggagaacag 2820
aagaaagcaa tagtagatct gttattcaag accaaccgca aagtgacagt taagcaattg 2880
aaagaggact actttaagaa aattgaatgc ttcgattctg tcgagatctc cggggtagaa 2940
gatcgattta atgcgtcact tggtacgtat catgacctcc taaagataat taaagataag 3000
gacttcctgg ataacgaaga gaatgaagat atcttagaag atatagtgtt gactcttacc 3060
ctctttgaag atcgggaaat gattgaggaa agactaaaaa catacgctca cctgttcgac 3120
gataaggtta tgaaacagtt aaagaggcgt cgctatacgg gctggggacg attgtcgcgg 3180
aaacttatca acgggataag agacaagcaa agtggtaaaa ctattctcga ttttctaaag 3240
agcgacggct tcgccaatag gaactttatg cagctgatcc atgatgactc tttaaccttc 3300
aaagaggata tacaaaaggc acaggtttcc ggacaagggg actcattgca cgaacatatt 3360
gcgaatcttg ctggttcgcc agccatcaaa aagggcatac tccagacagt caaagtagtg 3420
gatgagctag ttaaggtcat gggacgtcac aaaccggaaa acattgtaat cgagatggca 3480
cgcgaaaatc aaacgactca gaaggggcaa aaaaacagtc gagagcggat gaagagaata 3540
gaagagggta ttaaagaact gggcagccag atcttaaagg agcatcctgt ggaaaatacc 3600
caattgcaga acgagaaact ttacctctat tacctacaaa atggaaggga catgtatgtt 3660
gatcaggaac tggacataaa ccgtttatct gattacgacg tcgatcacat tgtaccccaa 3720
tcctttttga aggacgattc aatcgacaat aaagtgctta cacgctcgga taagaaccga 3780
gggaaaagtg acaatgttcc aagcgaggaa gtcgtaaaga aaatgaagaa ctattggcgg 3840
cagctcctaa atgcgaaact gataacgcaa agaaagttcg ataacttaac taaagctgag 3900
aggggtggct tgtctgaact tgacaaggcc ggatttatta aacgtcagct cgtggaaacc 3960
cgccaaatca caaagcatgt tgcacagata ctagattccc gaatgaatac gaaatacgac 4020
gagaacgata agctgattcg ggaagtcaaa gtaatcactt taaagtcaaa attggtgtcg 4080
gacttcagaa aggattttca attctataaa gttagggaga taaataacta ccaccatgcg 4140
cacgacgctt atcttaatgc cgtcgtaggg accgcactca ttaagaaata cccgaagcta 4200
gaaagtgagt ttgtgtatgg tgattacaaa gtttatgacg tccgtaagat gatcgcgaaa 4260
agcgaacagg agataggcaa ggctacagcc aaatacttct tttattctaa cattatgaat 4320
ttctttaaga cggaaatcac tctggcaaac ggagagatac gcaaacgacc tttaattgaa 4380
accaatgggg agacaggtga aatcgtatgg gataagggcc gggacttcgc gacggtgaga 4440
aaagttttgt ccatgcccca agtcaacata gtaaagaaaa ctgaggtgca gaccggaggg 4500
ttttcaaagg aatcgattct tccaaaaagg aatagtgata agctcatcgc tcgtaaaaag 4560
gactgggacc cgaaaaagta cggtggcttc gatagcccta cagttgccta ttctgtccta 4620
gtagtggcaa aagttgagaa gggaaaatcc aagaaactga agtcagtcaa agaattattg 4680
gggataacga ttatggagcg ctcgtctttt gaaaagaacc ccatcgactt ccttgaggcg 4740
aaaggttaca aggaagtaaa aaaggatctc ataattaaac taccaaagta tagtctgttt 4800
gagttagaaa atggccgaaa acggatgttg gctagcgccg gagagcttca aaaggggaac 4860
gaactcgcac taccgtctaa atacgtgaat ttcctgtatt tagcgtccca ttacgagaag 4920
ttgaaaggtt cacctgaaga taacgaacag aagcaacttt ttgttgagca gcacaaacat 4980
tatctcgacg aaatcataga gcaaatttcg gaattcagta agagagtcat cctagctgat 5040
gccaatctgg acaaagtatt aagcgcatac aacaagcaca gggataaacc catacgtgag 5100
caggcggaaa atattatcca tttgtttact cttaccaacc tcggcgctcc agccgcattc 5160
aagtattttg acacaacgat agatcgcaaa cgatacactt ctaccaagga ggtgctagac 5220
gcgacactga ttcaccaatc catcacggga ttatatgaaa ctcggataga tttgtcacag 5280
cttgggggtg actctggtgg ttctcccaag aagaagagga aagtctaa 5328
<210> 30
<211> 5130
<212> DNA
<213> 人工序列
<221> 载体7
<400> 30
atgagctcag agactggccc agtggctgtg gaccccacat tgagacggcg gatcgagccc 60
catgagtttg aggtattctt cgatccgaga gagctccgca aggagacctg cctgctttac 120
gaaattaatt gggggggccg gcactccatt tggcgacata catcacagaa cactaacaag 180
cacgtcgaag tcaacttcat cgagaagttc acgacagaaa gatatttctg tccgaacaca 240
aggtgcagca ttacctggtt tctcagctgg agcccatgcg gcgaatgtag tagggccatc 300
actgaattcc tgtcaaggta tccccacgtc actctgttta tttacatcgc aaggctgtac 360
caccacgctg acccccgcaa tcgacaaggc ctgcgggatt tgatctcttc aggtgtgact 420
atccaaatta tgactgagca ggagtcagga tactgctgga gaaactttgt gaattatagc 480
ccgagtaatg aagcccactg gcctaggtat ccccatctgt gggtacgact gtacgttctt 540
gaactgtact gcatcatact gggcctgcct ccttgtctca acattctgag aaggaagcag 600
ccacagctga cattctttac catcgctctt cagtcttgtc attaccagcg actgccccca 660
cacattctct gggccaccgg gttgaaaagc ggcagcgaga ctcccgggac ctcagagtcc 720
gccacacccg aaagtgataa aaagtattct attggtttag ccatcggcac taattccgtt 780
ggatgggctg tcataaccga tgaatacaaa gtaccttcaa agaaatttaa ggtgttgggg 840
aacacagacc gtcattcgat taaaaagaat cttatcggtg ccctcctatt cgatagtggc 900
gaaacggcag aggcgactcg cctgaaacga accgctcgga gaaggtatac acgtcgcaag 960
aaccgaatat gttacttaca agaaattttt agcaatgaga tggccaaagt tgacgattct 1020
ttctttcacc gtttggaaga gtccttcctt gtcgaagagg acaagaaaca tgaacggcac 1080
cccatctttg gaaacatagt agatgaggtg gcatatcatg aaaagtaccc aacgatttat 1140
cacctcagaa aaaagctagt tgactcaact gataaagcgg acctgaggtt aatctacttg 1200
gctcttgccc atatgataaa gttccgtggg cactttctca ttgagggtga tctaaatccg 1260
gacaactcgg atgtcgacaa actgttcatc cagttagtac aaacctataa tcagttgttt 1320
gaagagaacc ctataaatgc aagtggcgtg gatgcgaagg ctattcttag cgcccgcctc 1380
tctaaatccc gacggctaga aaacctgatc gcacaattac ccggagagaa gaaaaatggg 1440
ttgttcggta accttatagc gctctcacta ggcctgacac caaattttaa gtcgaacttc 1500
gacttagctg aagatgccaa attgcagctt agtaaggaca cgtacgatga cgatctcgac 1560
aatctactgg cacaaattgg agatcagtat gcggacttat ttttggctgc caaaaacctt 1620
agcgatgcaa tcctcctatc tgacatactg agagttaata ctgagattac caaggcgccg 1680
ttatccgctt caatgatcaa aaggtacgat gaacatcacc aagacttgac acttctcaag 1740
gccctagtcc gtcagcaact gcctgagaaa tataaggaaa tattctttga tcagtcgaaa 1800
aacgggtacg caggttatat tgacggcgga gcgagtcaag aggaattcta caagtttatc 1860
aaacccatat tagagaagat ggatgggacg gaagagttgc ttgtaaaact caatcgcgaa 1920
gatctactgc gaaagcagcg gactttcgac aacggtagca ttccacatca aatccactta 1980
ggcgaattgc atgctatact tagaaggcag gaggattttt atccgttcct caaagacaat 2040
cgtgaaaaga ttgagaaaat cctaaccttt cgcatacctt actatgtggg acccctggcc 2100
cgagggaact ctcggttcgc atggatgaca agaaagtccg aagaaacgat tactccatgg 2160
aattttgagg aagttgtcga taaaggtgcg tcagctcaat cgttcatcga gaggatgacc 2220
aactttgaca agaatttacc gaacgaaaaa gtattgccta agcacagttt actttacgag 2280
tatttcacag tgtacaatga actcacgaaa gttaagtatg tcactgaggg catgcgtaaa 2340
cccgcctttc taagcggaga acagaagaaa gcaatagtag atctgttatt caagaccaac 2400
cgcaaagtga cagttaagca attgaaagag gactacttta agaaaattga atgcttcgat 2460
tctgtcgaga tctccggggt agaagatcga tttaatgcgt cacttggtac gtatcatgac 2520
ctcctaaaga taattaaaga taaggacttc ctggataacg aagagaatga agatatctta 2580
gaagatatag tgttgactct taccctcttt gaagatcggg aaatgattga ggaaagacta 2640
aaaacatacg ctcacctgtt cgacgataag gttatgaaac agttaaagag gcgtcgctat 2700
acgggctggg gacgattgtc gcggaaactt atcaacggga taagagacaa gcaaagtggt 2760
aaaactattc tcgattttct aaagagcgac ggcttcgcca ataggaactt tatgcagctg 2820
atccatgatg actctttaac cttcaaagag gatatacaaa aggcacaggt ttccggacaa 2880
ggggactcat tgcacgaaca tattgcgaat cttgctggtt cgccagccat caaaaagggc 2940
atactccaga cagtcaaagt agtggatgag ctagttaagg tcatgggacg tcacaaaccg 3000
gaaaacattg taatcgagat ggcacgcgaa aatcaaacga ctcagaaggg gcaaaaaaac 3060
agtcgagagc ggatgaagag aatagaagag ggtattaaag aactgggcag ccagatctta 3120
aaggagcatc ctgtggaaaa tacccaattg cagaacgaga aactttacct ctattaccta 3180
caaaatggaa gggacatgta tgttgatcag gaactggaca taaaccgttt atctgattac 3240
gacgtcgatc acattgtacc ccaatccttt ttgaaggacg attcaatcga caataaagtg 3300
cttacacgct cggataagaa ccgagggaaa agtgacaatg ttccaagcga ggaagtcgta 3360
aagaaaatga agaactattg gcggcagctc ctaaatgcga aactgataac gcaaagaaag 3420
ttcgataact taactaaagc tgagaggggt ggcttgtctg aacttgacaa ggccggattt 3480
attaaacgtc agctcgtgga aacccgccaa atcacaaagc atgttgcaca gatactagat 3540
tcccgaatga atacgaaata cgacgagaac gataagctga ttcgggaagt caaagtaatc 3600
actttaaagt caaaattggt gtcggacttc agaaaggatt ttcaattcta taaagttagg 3660
gagataaata actaccacca tgcgcacgac gcttatctta atgccgtcgt agggaccgca 3720
ctcattaaga aatacccgaa gctagaaagt gagtttgtgt atggtgatta caaagtttat 3780
gacgtccgta agatgatcgc gaaaagcgaa caggagatag gcaaggctac agccaaatac 3840
ttcttttatt ctaacattat gaatttcttt aagacggaaa tcactctggc aaacggagag 3900
atacgcaaac gacctttaat tgaaaccaat ggggagacag gtgaaatcgt atgggataag 3960
ggccgggact tcgcgacggt gagaaaagtt ttgtccatgc cccaagtcaa catagtaaag 4020
aaaactgagg tgcagaccgg agggttttca aaggaatcga ttcttccaaa aaggaatagt 4080
gataagctca tcgctcgtaa aaaggactgg gacccgaaaa agtacggtgg cttcgatagc 4140
cctacagttg cctattctgt cctagtagtg gcaaaagttg agaagggaaa atccaagaaa 4200
ctgaagtcag tcaaagaatt attggggata acgattatgg agcgctcgtc ttttgaaaag 4260
aaccccatcg acttccttga ggcgaaaggt tacaaggaag taaaaaagga tctcataatt 4320
aaactaccaa agtatagtct gtttgagtta gaaaatggcc gaaaacggat gttggctagc 4380
gccggagagc ttcaaaaggg gaacgaactc gcactaccgt ctaaatacgt gaatttcctg 4440
tatttagcgt cccattacga gaagttgaaa ggttcacctg aagataacga acagaagcaa 4500
ctttttgttg agcagcacaa acattatctc gacgaaatca tagagcaaat ttcggaattc 4560
agtaagagag tcatcctagc tgatgccaat ctggacaaag tattaagcgc atacaacaag 4620
cacagggata aacccatacg tgagcaggcg gaaaatatta tccatttgtt tactcttacc 4680
aacctcggcg ctccagccgc attcaagtat tttgacacaa cgatagatcg caaacgatac 4740
acttctacca aggaggtgct agacgcgaca ctgattcacc aatccatcac gggattatat 4800
gaaactcgga tagatttgtc acagcttggg ggtgactctg gtggttctac taatctgtca 4860
gatattattg aaaaggagac cggtaagcaa ctggttatcc aggaatccat cctcatgctc 4920
ccagaggagg tggaagaagt cattgggaac aagccggaaa gcgatatact cgtgcacacc 4980
gcctacgacg agagcaccga cgagaatgtc atgcttctga ctagcgacgc ccctgaatac 5040
aagccttggg ctctggtcat acaggatagc aacggtgaga acaagattaa gatgctctct 5100
ggtggttctc ccaagaagaa gaggaaagtc 5130
<210> 31
<211> 127
<212> PRT
<213> 人工序列
<221> gRNA纳米抗体1
<400> 31
Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala
1 5 10 15
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Leu Thr Ala Trp Phe
20 25 30
Arg Gln Ile His Cys Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
35 40 45
Phe Val Ser Trp Val Pro Ala Lys Met Phe Cys Gln Ile Leu Thr Asp
50 55 60
Glu Arg Asn Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr
65 70 75 80
Val Tyr Val Gln Met Asn Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr
85 90 95
Tyr Cys Ala Leu Glu Phe His Arg Met Lys Asp Val Trp Ser Gly Pro
100 105 110
Tyr Ala Asn Gln Thr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val
115 120 125
<210> 32
<211> 127
<212> PRT
<213> 人工序列
<221> gRNA纳米抗体2
<400> 32
Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala
1 5 10 15
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Val Asn Leu Gln Gly
20 25 30
Ala Tyr Met Ile Cys Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
35 40 45
Phe Val Ile Ala Ser Gly Lys Val Glu Asp Tyr Met His Cys Trp Leu
50 55 60
Pro Asn Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr
65 70 75 80
Val Tyr Val Gln Met Asn Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr
85 90 95
Tyr Cys Ala Ile Gln Tyr His Trp Glu Asn Val Phe Thr Cys Pro Ser
100 105 110
Gly Arg Met Ala Leu Tyr Trp Gly Gln Gly Thr Gln Val Thr Val
115 120 125
<210> 33
<211> 127
<212> PRT
<213> 人工序列
<221> gRNA纳米抗体3
<400> 33
Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala
1 5 10 15
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Asp Ser Val Ile Pro
20 25 30
Glu Ala Asn Cys Met Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
35 40 45
Phe Val Met Ser Asn Gln Tyr Lys Phe Cys Asp His Ile Arg Gly Pro
50 55 60
Val Glu Ala Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr
65 70 75 80
Val Tyr Val Gln Met Asn Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr
85 90 95
Tyr Cys Ala Met Ala Glu Ile His Arg Thr Pro Val Leu Ser Tyr Lys
100 105 110
Phe Asn Cys Gln Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr Val
115 120 125
<210> 34
<211> 127
<212> PRT
<213> 人工序列
<221> gRNA纳米抗体4
<400> 34
Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala
1 5 10 15
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Pro Thr Cys Ser Gly
20 25 30
Ile Asn Tyr Val Glu Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
35 40 45
Phe Val Arg Gln Tyr Ala Pro Glu Val Asn Ser Gly Thr Phe Met Leu
50 55 60
Trp Ile Asp Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr
65 70 75 80
Val Tyr Val Gln Met Asn Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr
85 90 95
Tyr Cys Ala Gln Trp Ser Arg Ile Asn His Gly Phe Asp Leu Tyr Met
100 105 110
Ala Thr Lys Val Cys Tyr Trp Gly Gln Gly Thr Gln Val Thr Val
115 120 125
<210> 35
<211> 127
<212> PRT
<213> 人工序列
<221> gRNA纳米抗体5
<400> 35
Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala
1 5 10 15
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Asp Pro Ile Met Trp
20 25 30
Asn Gln Ala Tyr Arg Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
35 40 45
Phe Val Val Ser Phe Gln Pro Cys Tyr Leu His Glu Lys Ile Asn Trp
50 55 60
Thr Met Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr
65 70 75 80
Val Tyr Val Gln Met Asn Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr
85 90 95
Tyr Cys Ala Gln Met Ser Asp Arg Ala Tyr Pro His Ile Asn Phe Val
100 105 110
Leu Thr Trp Cys Glu Tyr Trp Gly Gln Gly Thr Gln Val Thr Val
115 120 125 <210> 36
<211> 127
<212> PRT
<213> 人工序列
<221> gRNA纳米抗体6
<400> 36
Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala
1 5 10 15
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Trp Arg Gln Pro Glu
20 25 30
Met Cys Tyr Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
35 40 45
Phe Val Gln Arg Phe Ile Gly Pro Asn Leu His Lys Ala Met Trp Glu
50 55 60
Thr Asp Tyr Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr
65 70 75 80
Val Tyr Val Gln Met Asn Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr
85 90 95
Tyr Cys Ala Trp Gly Met Pro Arg Phe Tyr Cys Asp Thr Ser Lys His
100 105 110
Val Gln Asn Leu Ala Tyr Trp Gly Gln Gly Thr Gln Val Thr Val
115 120 125
<210> 37
<211> 127
<212> PRT
<213> 人工序列
<221> gRNA纳米抗体7
<400> 37
Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala
1 5 10 15
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Val Ser Thr Cys Tyr
20 25 30
Lys Pro Trp Leu Phe Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
35 40 45
Phe Val Asp Phe His Thr Gln Gly Ala Leu Ser Glu Met Asn Pro Tyr
50 55 60
Cys Lys Ile Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr
65 70 75 80
Val Tyr Val Gln Met Asn Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr
85 90 95
Tyr Cys Ala Gln Tyr Met Glu Arg Val His Lys Cys Gly Ala Leu Thr
100 105 110
Ser Phe Trp Asn Ile Tyr Trp Gly Gln Gly Thr Gln Val Thr Val
115 120 125
<210> 38
<211> 127
<212> PRT
<213> 人工序列
<221> gRNA纳米抗体8
<400> 38
Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala
1 5 10 15
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Thr His Gly Leu Ser
20 25 30
Ile Asn Phe Met Cys Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
35 40 45
Phe Val Pro Met Tyr Ser Ala Lys Glu Ile Thr Asn Trp Cys Val Leu
50 55 60
Asp Arg Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr
65 70 75 80
Val Tyr Val Gln Met Asn Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr
85 90 95
Tyr Cys Ala Gln Glu Phe Thr Asp Arg Trp Ala Leu Cys Ser His Met
100 105 110
Pro Lys Ile Asn Tyr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val
115 120 125
<210> 39
<211> 127
<212> PRT
<213> 人工序列
<221> gRNA纳米抗体9
<400> 39
Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala
1 5 10 15
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Gly Arg Gln Glu His
20 25 30
Cys Asn Lys Ala Tyr Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
35 40 45
Phe Val Gly Glu Cys Ser Lys Asn Val Gln Asp Arg Tyr Pro Ala Leu
50 55 60
His Trp Ile Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr
65 70 75 80
Val Tyr Val Gln Met Asn Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr
85 90 95
Tyr Cys Ala Gln Phe Pro Thr Ala Lys Leu Met Gly His Glu Trp Tyr
100 105 110
Cys Arg Asn Ser Ile Tyr Trp Gly Gln Gly Thr Gln Val Thr Val
115 120 125
<210> 40
<211> 127
<212> PRT
<213> 人工序列
<221> gRNA纳米抗体10
<400> 40
Met Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala
1 5 10 15
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Arg Lys Thr Cys Tyr
20 25 30
Asn Pro Asp Phe His Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
35 40 45
Phe Val Pro Val Gln Arg Met Ala Asp Asn Lys Cys Phe Leu Ile Thr
50 55 60
Trp Tyr Gly Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Thr
65 70 75 80
Val Tyr Val Gln Met Asn Ser Leu Ile Pro Glu Asp Thr Ala Ile Tyr
85 90 95
Tyr Cys Ala Ile Arg Gln Pro Cys His Leu Tyr Thr Lys Val Phe Asn
100 105 110
Met Ser Glu Ala Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val
115 120 125
<210> 41
<211> 719
<212> PRT
<213> 人(Homo sapiens)
<221> Mcm7
<400> 41
Met Ala Leu Lys Asp Tyr Ala Leu Glu Lys Glu Lys Val Lys Lys Phe
1 5 10 15
Leu Gln Glu Phe Tyr Gln Asp Asp Glu Leu Gly Lys Lys Gln Phe Lys
20 25 30
Tyr Gly Asn Gln Leu Val Arg Leu Ala His Arg Glu Gln Val Ala Leu
35 40 45
Tyr Val Asp Leu Asp Asp Val Ala Glu Asp Asp Pro Glu Leu Val Asp
50 55 60
Ser Ile Cys Glu Asn Ala Arg Arg Tyr Ala Lys Leu Phe Ala Asp Ala
65 70 75 80
Val Gln Glu Leu Leu Pro Gln Tyr Lys Glu Arg Glu Val Val Asn Lys
85 90 95
Asp Val Leu Asp Val Tyr Ile Glu His Arg Leu Met Met Glu Gln Arg
100 105 110
Ser Arg Asp Pro Gly Met Val Arg Ser Pro Gln Asn Gln Tyr Pro Ala
115 120 125
Glu Leu Met Arg Arg Phe Glu Leu Tyr Phe Gln Gly Pro Ser Ser Asn
130 135 140
Lys Pro Arg Val Ile Arg Glu Val Arg Ala Asp Ser Val Gly Lys Leu
145 150 155 160
Val Thr Val Arg Gly Ile Val Thr Arg Val Ser Glu Val Lys Pro Lys
165 170 175
Met Val Val Ala Thr Tyr Thr Cys Asp Gln Cys Gly Ala Glu Thr Tyr
180 185 190
Gln Pro Ile Gln Ser Pro Thr Phe Met Pro Leu Ile Met Cys Pro Ser
195 200 205
Gln Glu Cys Gln Thr Asn Arg Ser Gly Gly Arg Leu Tyr Leu Gln Thr
210 215 220
Arg Gly Ser Arg Phe Ile Lys Phe Gln Glu Met Lys Met Gln Glu His
225 230 235 240
Ser Asp Gln Val Pro Val Gly Asn Ile Pro Arg Ser Ile Thr Val Leu
245 250 255
Val Glu Gly Glu Asn Thr Arg Ile Ala Gln Pro Gly Asp His Val Ser
260 265 270
Val Thr Gly Ile Phe Leu Pro Ile Leu Arg Thr Gly Phe Arg Gln Val
275 280 285
Val Gln Gly Leu Leu Ser Glu Thr Tyr Leu Glu Ala His Arg Ile Val
290 295 300
Lys Met Asn Lys Ser Glu Asp Asp Glu Ser Gly Ala Gly Glu Leu Thr
305 310 315 320
Arg Glu Glu Leu Arg Gln Ile Ala Glu Glu Asp Phe Tyr Glu Lys Leu
325 330 335
Ala Ala Ser Ile Ala Pro Glu Ile Tyr Gly His Glu Asp Val Lys Lys
340 345 350
Ala Leu Leu Leu Leu Leu Val Gly Gly Val Asp Gln Ser Pro Arg Gly
355 360 365
Met Lys Ile Arg Gly Asn Ile Asn Ile Cys Leu Met Gly Asp Pro Gly
370 375 380
Val Ala Lys Ser Gln Leu Leu Ser Tyr Ile Asp Arg Leu Ala Pro Arg
385 390 395 400
Ser Gln Tyr Thr Thr Gly Arg Gly Ser Ser Gly Val Gly Leu Thr Ala
405 410 415
Ala Val Leu Arg Asp Ser Val Ser Gly Glu Leu Thr Leu Glu Gly Gly
420 425 430
Ala Leu Val Leu Ala Asp Gln Gly Val Cys Cys Ile Asp Glu Phe Asp
435 440 445
Lys Met Ala Glu Ala Asp Arg Thr Ala Ile His Glu Val Met Glu Gln
450 455 460
Gln Thr Ile Ser Ile Ala Lys Ala Gly Ile Leu Thr Thr Leu Asn Ala
465 470 475 480
Arg Cys Ser Ile Leu Ala Ala Ala Asn Pro Ala Tyr Gly Arg Tyr Asn
485 490 495
Pro Arg Arg Ser Leu Glu Gln Asn Ile Gln Leu Pro Ala Ala Leu Leu
500 505 510
Ser Arg Phe Asp Leu Leu Trp Leu Ile Gln Asp Arg Pro Asp Arg Asp
515 520 525
Asn Asp Leu Arg Leu Ala Gln His Ile Thr Tyr Val His Gln His Ser
530 535 540
Arg Gln Pro Pro Ser Gln Phe Glu Pro Leu Asp Met Lys Leu Met Arg
545 550 555 560
Arg Tyr Ile Ala Met Cys Arg Glu Lys Gln Pro Met Val Pro Glu Ser
565 570 575
Leu Ala Asp Tyr Ile Thr Ala Ala Tyr Val Glu Met Arg Arg Glu Ala
580 585 590
Trp Ala Ser Lys Asp Ala Thr Tyr Thr Ser Ala Arg Thr Leu Leu Ala
595 600 605
Ile Leu Arg Leu Ser Thr Ala Leu Ala Arg Leu Arg Met Val Asp Val
610 615 620
Val Glu Lys Glu Asp Val Asn Glu Ala Ile Arg Leu Met Glu Met Ser
625 630 635 640
Lys Asp Ser Leu Leu Gly Asp Lys Gly Gln Thr Ala Arg Thr Gln Arg
645 650 655
Pro Ala Asp Val Ile Phe Ala Thr Val Arg Glu Leu Val Ser Gly Gly
660 665 670
Arg Ser Val Arg Phe Ser Glu Ala Glu Gln Arg Cys Val Ser Arg Gly
675 680 685
Phe Thr Pro Ala Gln Phe Gln Ala Ala Leu Asp Glu Tyr Glu Glu Leu
690 695 700
Asn Val Trp Gln Val Asn Ala Ser Arg Thr Arg Ile Thr Phe Val
705 710 715
SEQ ID NO. 76
<210> 42
<211> 150
<212> PRT
<213> 粗糙脉胞霉种(Neurospora crassa)
<221> Vivid
<400> 42
His Thr Leu Tyr Ala Pro Gly Gly Tyr Asp Ile Met Gly Tyr Leu Ile
1 5 10 15
Gln Ile Met Asn Arg Pro Asn Pro Gln Val Glu Leu Gly Pro Val Asp
20 25 30
Thr Ser Cys Ala Leu Ile Leu Cys Asp Leu Lys Gln Lys Asp Thr Pro
35 40 45
Ile Val Tyr Ala Ser Glu Ala Phe Leu Tyr Met Thr Gly Tyr Ser Asn
50 55 60
Ala Glu Val Leu Gly Arg Asn Cys Arg Phe Leu Gln Ser Pro Asp Gly
65 70 75 80
Met Val Lys Pro Lys Ser Thr Arg Lys Tyr Val Asp Ser Asn Thr Ile
85 90 95
Asn Thr Met Arg Lys Ala Ile Asp Arg Asn Ala Glu Val Gln Val Glu
100 105 110
Val Val Asn Phe Lys Lys Asn Gly Gln Arg Phe Val Asn Phe Leu Thr
115 120 125
Met Ile Pro Val Arg Asp Glu Thr Gly Glu Tyr Arg Tyr Ser Met Gly
130 135 140
Phe Gln Cys Glu Thr Glu
145 150
<210> 43
<211> 25
<212> PRT
<213> 人工序列
<221> Linker
<400> 43
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser Gly Gly Gly Gly Ser
20 25
SEQ ID NO. 79
<210> 44
<211> 166
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<221> ecTadA*
<400> 44
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala Ile
35 40 45
Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Cys Tyr Phe Phe Arg Met Pro Arg Gln Val Phe Asn Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 45
<211> 228
<212> PRT
<213> 大鼠(rattus norvegicus)
<221> rAPOBEC1
<400> 45
Ser Ser Glu Thr Gly Pro Val Ala Val Asp Pro Thr Leu Arg Arg Arg
1 5 10 15
Ile Glu Pro His Glu Phe Glu Val Phe Phe Asp Pro Arg Glu Leu Arg
20 25 30
Lys Glu Thr Cys Leu Leu Tyr Glu Ile Asn Trp Gly Gly Arg His Ser
35 40 45
Ile Trp Arg His Thr Ser Gln Asn Thr Asn Lys His Val Glu Val Asn
50 55 60
Phe Ile Glu Lys Phe Thr Thr Glu Arg Tyr Phe Cys Pro Asn Thr Arg
65 70 75 80
Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Gly Glu Cys Ser
85 90 95
Arg Ala Ile Thr Glu Phe Leu Ser Arg Tyr Pro His Val Thr Leu Phe
100 105 110
Ile Tyr Ile Ala Arg Leu Tyr His His Ala Asp Pro Arg Asn Arg Gln
115 120 125
Gly Leu Arg Asp Leu Ile Ser Ser Gly Val Thr Ile Gln Ile Met Thr
130 135 140
Glu Gln Glu Ser Gly Tyr Cys Trp Arg Asn Phe Val Asn Tyr Ser Pro
145 150 155 160
Ser Asn Glu Ala His Trp Pro Arg Tyr Pro His Leu Trp Val Arg Leu
165 170 175
Tyr Val Leu Glu Leu Tyr Cys Ile Ile Leu Gly Leu Pro Pro Cys Leu
180 185 190
Asn Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe Thr Ile Ala
195 200 205
Leu Gln Ser Cys His Tyr Gln Arg Leu Pro Pro His Ile Leu Trp Ala
210 215 220
Thr Gly Leu Lys
225
<210> 46
<211> 589
<212> DNA
<213> 人工序列
<221> 载体10关键序列
<400> 46
gaaacaccga acaaagcacc agtggtctag tggtagaata gtaccctgcc acggtacaga 60
cccgggttcg attcccggct ggtgcagttc aggcaggccc ccggcagttt tagagctaga 120
aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt 180
gcaacaaagc accagtggtc tagtggtaga atagtaccct gccacggtac agacccgggt 240
tcgattcccg gctggtgcaa ggcaggttca ccatcagcag ttttagagct agaaatagca 300
agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcaacaa 360
agcaccagtg gtctagtggt agaatagtac cctgccacgg tacagacccg ggttcgattc 420
ccggctggtg cactactggg gccagggcac ccgttttaga gctagaaata gcaagttaaa 480
ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttggatcc 540
agcgcttagg tcttgaaagg agtgggaatt ggctccggtg cccgtcagt 589

Claims (17)

1.一种抗原结合域自动优化的嵌合抗原受体修饰细胞文库的构建方法,其特征在于,将包含第一基因元件、第二基因元件、第三基因元件、第四基因元件以及第五基因元件的载体转染至细胞内,所述载体为一个或多个,即得到嵌合抗原受体修饰细胞文库;
所述第一基因元件为编码受体1的核苷酸序列,所述受体1包括胞外域,跨膜域及胞内域;所述胞外域可识别靶标抗原上所标记的生物素或6×His标签,所述胞内域包括烟草蚀纹病毒蛋白酶TEVP,所述受体1的核苷酸序列如SEQ ID NO. 1所示;
所述第二基因元件为编码受体2的核苷酸序列,所述受体2包括胞外抗原结合域、跨膜域以及胞内信号域;所述抗原结合域为抗体序列动态库,所述动态库是在第四基因元件的作用下诱导产生一系列突变,引入序列多样性所得;所述胞内信号域包括tTA转录因子结构域以及连接序列,所述连接序列连接tTA转录因子结构域和跨膜域,包含TEVP可识别切割的序列,所述受体2是由Notch受体改造而来的synNotch受体,其核苷酸序列如SEQ ID NO. 2或SEQ ID NO. 3所示;
所述第三基因元件为编码筛选基因的核苷酸序列,tTA转录因子可以启动所述筛选基因的表达;
所述第四基因元件为编码顺式调控元件或反式作用因子的核苷酸序列;所述顺式调控元件能够使DNA产生DNA的单链结构;所述反式作用因子为gRNA、dCas9、感光蛋白、脱氨酶和感光蛋白与脱氨酶之间的Linker序列;
所述第五基因元件为编码抑制系统的核苷酸序列,tTA转录因子可以启动所述抑制系统的表达,进而敲减或敲除反式作用因子,使抗体序列动态库维持稳定;或所述第五基因元件为编码光控制系统的核苷酸序列,所述光控制系统能够调控第四基因元件的表达:在光照条件下启动第四基因元件发挥作用,开始构建抗体动态序列库;停止光照后,第四基因元件功能被抑制,使抗体动态序列库维持稳定。
2.根据权利要求1所述嵌合抗原受体修饰细胞文库的构建方法,其特征在于,所述顺式调控元件使DNA产生特殊结构的DNA单链结构;所述特殊结构的DNA单链结构包括G-四联体,所述反式作用因子为Nucleolin、感光蛋白、脱氨酶和感光蛋白与脱氨酶之间的Linker序列。
3.根据权利要求1所述嵌合抗原受体修饰细胞文库的构建方法,其特征在于,所述细胞为人源或鼠源细胞。
4.根据权利要求1所述嵌合抗原受体修饰细胞文库的构建方法,其特征在于,所述抗体序列动态库包括双链抗体库、单链抗体库及纳米抗体库。
5.根据权利要求1所述嵌合抗原受体修饰细胞文库的构建方法,其特征在于,所述反式作用因子包括以下Cas蛋白家族成员及其部分结构域:Cas3-8s、Cas10s、Cas11s、Cas9s、xCas9、Cas12s、Cas13s和Cas14s。
6.根据权利要求1所述嵌合抗原受体修饰细胞文库的构建方法,其特征在于,所述反式作用因子包括以下腺嘌呤脱氨酶及其部分结构域:ecTadA、mADA、hADAR2和hADAT2。
7.根据权利要求1所述嵌合抗原受体修饰细胞文库的构建方法,其特征在于,所述反式作用因子包括以下胞苷脱氨酶及其部分结构域:activation-induced cytidinedeaminase、APOBEC3G、APOBEC1和CDA1。
8.根据权利要求1所述嵌合抗原受体修饰细胞文库的构建方法,其特征在于,所述编码光控制系统的核苷酸序列包括以下感光蛋白及其部分结构域:BphS、Cph1、PCB、PhyB-PIFs、PΦB、BphP1-PpsR2、BphP1-Q-PAS1、LOV2-Jα、PhyB、PIF6、Epdz和Photoreceptor Vivid。
9.根据权利要求1所述嵌合抗原受体修饰细胞文库的构建方法,其特征在于,所述光控制系统发挥对第四基因元件的调控作用需要光照的配合,所述光照包括可见光、不可见光、蓝光、红光、远红外光及紫外光。
10.根据权利要求1所述嵌合抗原受体修饰细胞文库的构建方法,其特征在于,所述筛选基因包括GFP、mCherry、Luciferase、嘌呤霉素抗性基因、博来霉素和新霉素抗性基因。
11.根据权利要求1所述嵌合抗原受体修饰细胞文库的构建方法,其特征在于,所述抑制系统是通过tTA转录因子启动负调控蛋白,阻遏蛋白,siRNA,CAS9/gRNA表达或通过反向转录方式,敲减或敲除反式作用因子,使抗体序列动态库维持稳定。
12.一种抗原结合域自动优化的嵌合抗原受体修饰细胞文库,其特征在于,所述嵌合抗原受体修饰细胞文库由权利要求1-11任一项所述方法构建得到。
13.一种抗原结合域自动优化的嵌合抗原受体修饰细胞文库筛选抗体的方法,其特征在于,采用权利要求12所述的嵌合抗原受体修饰细胞文库进行筛选,包括以下步骤:
制备靶标抗原,所述靶标抗原上标记能够被受体1所识别的标签,所述标签包括生物素或6×His;
将靶标抗原与嵌合抗原受体修饰细胞文库共孵育,根据筛选基因的表达情况,筛选出阳性嵌合抗原受体表达细胞;
对阳性细胞的嵌合抗原受体胞外抗原结合域进行扩增测序,得到能够与靶标抗原特异性结合的抗体核苷酸序列。
14.根据权利要求13所述的嵌合抗原受体修饰细胞文库筛选抗体的方法,其特征在于,在得到能够与靶标抗原特异性结合的抗体核苷酸序列后,还包括以下步骤:采用基因工程的方法获得针对靶标抗原的抗体。
15.据权利要求13所述的嵌合抗原受体修饰细胞文库筛选抗体的方法,其特征在于,所述靶标抗原表达于细胞表面、包被于载体表面或为可溶性蛋白。
16.根据权利要求15所述的嵌合抗原受体修饰细胞文库筛选抗体的方法,其特征在于,所述载体包括磁性微球,非磁性微球,石墨烯,纳米颗粒;所述磁性微球包括硅基磁珠,GMA磁珠和聚苯乙烯磁性微球;所述非磁性微球包括琼脂糖微球。
17.如权利要求13-16任一项所述的嵌合抗原受体修饰细胞文库筛选抗体的方法在制备针对某一特定靶标抗原的抗体中的应用。
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CN102643852A (zh) * 2011-02-28 2012-08-22 华东理工大学 光可控的基因表达系统
CN109576292A (zh) * 2018-12-21 2019-04-05 深圳市爱思迪生物科技有限公司 一种抗体文库的构建方法及其应用
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CN102643852A (zh) * 2011-02-28 2012-08-22 华东理工大学 光可控的基因表达系统
CN109576292A (zh) * 2018-12-21 2019-04-05 深圳市爱思迪生物科技有限公司 一种抗体文库的构建方法及其应用
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