CN107663239A - 一种识别hla‑a2/nlvpmvatv的单域抗体 - Google Patents
一种识别hla‑a2/nlvpmvatv的单域抗体 Download PDFInfo
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- CN107663239A CN107663239A CN201611230994.1A CN201611230994A CN107663239A CN 107663239 A CN107663239 A CN 107663239A CN 201611230994 A CN201611230994 A CN 201611230994A CN 107663239 A CN107663239 A CN 107663239A
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Abstract
本发明公开一种识别HLA‑A2/NLVPMVATV的单域抗体,及起特异性识别作用的CDR1、CDR2和CDR3序列。本发明还公开基于这种抗体的融合蛋白及特异性多肽分子,以及在生物医学领域的应用。
Description
技术领域
本发明属于肿瘤治疗技术领域,特别涉及一种识别HLA-A2/NLVPMVATV的单域抗体。
背景技术
巨细胞病毒(cytomegalovirus,CMV)为双链DNA病毒,属疱疹病毒科,具有潜伏-活化的生物学特性。它可感染人、牛、马、猪等多种哺乳动物,其中感染人类的CMV称为人巨细胞病毒(human cytomegalovirus,HCMV),且只有HCMV感染人类。在发达国家HCMV人群感染率在50%以上,而在我国HCMV人群感染率则为70%~90%,儿童感染率较高,流行病学资料显示,大约有30%~70%美国学龄前儿童感染HCMV。先天性及围产期感染HCMV是出生缺陷的主要病毒病因,主要导致肝脏损伤,严重时可伴肝功能衰竭,并发凝血功能异常,并可引起胆道闭锁,甚至危及患儿生命。此外,HCMV感染还能导致神经***、泌尿生殖***、肺以及血液***等病变。
目前认为在正常的易感人群中,感染HCMV后将成为HCMV的终生携带者,呈现为隐性感染的状态。而携带者体内潜伏的HCMV在机体免疫功能处于正常状态时是不具备致病能力的,当携带者处于免疫抑制或者缺陷的时候,体内潜伏的HCMV将可能诱发某些肿瘤。
HCMV感染所致的脑发育畸形表现主要包括头畸形、颅内钙化、脑积水、脑萎缩、脑瘫等,多数在婴儿期即出现明显的症状,CT表现类型包括脑发育异常、脑软化、髓鞘发育迟缓、脑积水(包括梗阻性脑积水和外部性脑积水)和脑萎缩改变等。新生小鼠的感染率最高,未成熟的发育期大脑较成熟大脑更易被感染,且在发育期的小鼠对CMV的敏感性也随着年龄的增长而下降。实验中发现,刚出生即被感染的小鼠脑片中几乎所有细胞都被感染,出生7d的脑片中被感染的细胞有向室管膜下区及皮层下层集中的趋势,而生后14d、21d小鼠脑片被感染的细胞更多分布于室管膜下区,在皮层下层分布较少。虽然还不能解释这种现象,但从中可知,这种空间上的差异性能保护皮层及白质,延迟其遭受病毒侵袭的时间,降低HCMV对神经***的不可逆损伤。感染发生时细胞所处细胞周期的时相也与感染严重程度密切相关。这种细胞周期时相上的区别是由于细胞周期各时相上发挥作用的不同细胞周期蛋白与HCMV相互作用的结果。
迄今,HCMV感染尚无特效治疗药物。临床上抗病毒疗法中的药物为GCV、缬更昔洛韦、膦甲酸、西多福韦,还可以将更昔洛韦与丙种球蛋白联用治疗HCMV感染,加强疗效。GCV是目前临床上使用的最主要治疗药物,是一种无环鸟苷的衍生物。然而,GCV只能抑制病毒感染,不能彻底消灭病毒。
通过对动物模型和CMV感染者研究发现T细胞免疫应答在抑制CMV复制,阻止其致病方面具有决定性的作用,机体对CMV的免疫应答主要由pp65所介导。Pp65蛋白是病毒颗粒的主要成分,在病毒复制早期就可以观察到,它也是一种较强的抗原,可刺激机体免疫应答,有助于清除病毒。Pp65495-503(NLVPMVATV)是由HLA-A2提呈的T细胞主要识别肽段。以下HLA-A2/Pp65495-503(NLVPMVATV)均简写为HLA-A2/NLVPMVATV。基于以上研究,在肿瘤生物治疗方面的研究上,Pp65抗原又成为研究的热点。尤其作为基因修饰的TCR,CART,TCR样抗体的优势靶点。
TCR虽然既能识别胞外抗原也能识别通过抗原加工递呈至细胞表面的胞内抗原,但TCR通常需要从T细胞克隆中分离出特异性的αβTCR基因,难度较大,重复性不高。此外,转基因TCR还有TCR错配带来的潜在安全隐患,外源TCR的两条链可能会和内源TCR亚基发生错配,组合成新的TCR。这种错配的TCR会形成未知的特异性,可能会靶向正常组织,导致严重的移植物抗宿主反应。
如果能制备出识别MHC多肽复合物的抗体分子,并表达至细胞表面,就可以将T细胞免疫和成熟的抗体技术相结合,开发出一种兼具抗体和T细胞优势的新型T细胞修饰治疗技术。
这样新型的识别MHC多肽复合物的分子与CAR相比,因具有天然T细胞受体的功能,沿用生命进化过程中获得的自我异体识别机制,即可识别肿瘤胞外抗原,也可识别通过抗原加工形成的病毒抗原,因而具有更广泛的应用,具有得天独厚的优势。
此外,以抗体为基础的靶向治疗常用的抗体是单链抗体或抗体Fab段,与它们相比,单域抗体仅含有抗体重链的一个可变区,是最小的全功能性的抗原结合片段,免疫原性较弱;更易通过血管壁,有利于疾病治疗;由于没有Fc段,不能与非靶标细胞的Fc受体结合,能更集中到达病灶部位因此容易进行基因工程操作。
发明内容
本发明针对现有技术中存在的技术问题,提供一种识别HLA-A2/NLVPMVATV抗原复合物的单域抗体,筛选得到的单域抗体能特异性的结合NLVPMVATV与HLA-A2的复合物,可开发成***的抗体药物及其他有有益效果生物治疗产品。
为解决上述技术问题,本发明采用的技术方案是:
一种识别HLA-A2/NLVPMVATV的单域抗体,通过三轮生物淘选,从噬菌体单域文库中筛选得到亲和力较高的抗体序列,将得到的抗体克隆至原核/真核表达载体中,与人源FC融合表达,转染宿主细胞,获得双价抗体再进行体外亲和力的验证。
一种识别HLA-A2/NLVPMVATV的单域抗体,该单域抗体的氨基酸序列包含互补决定区CDR1为SEQ ID NO.1,CDR2为SEQ ID NO.2和CDR3为SEQ ID NO.3;或该单域抗体的氨基酸序列包含互补决定区CDR1为SEQ ID NO.4,CDR2为SEQ ID NO.5和CDR3为SEQ ID NO.6。
本发明所提供的单域抗体为单区抗体或重链变区或轻链变区。
本发明所提供的单域抗体为scFv或Fc融合蛋白或完整抗体的一部分。
本发明所提供的单域抗体,其氨基酸序列为SEQ ID NO.7和SEQ ID NO.8。
所述单域抗体包含所述互补决定区(CDR1、CDR2和CDR3)中的一个或者两个以上的氨基酸序列,且至少与一个氨基酸序列最低具有79%同源性。
所述单域抗体的氨基酸序列包含所述框架(FR,单域抗体除互补决定区CDR以外的氨基酸序列)中的一个或者两个以上的氨基酸序列,且至少与一个氨基酸序列最低具有90%同源性。
使用上述抗体的互补决定区的氨基酸序列与至少包含一个具治疗功能的多肽制备的融合蛋白;所述的融合蛋白可以与各具识别功能多肽的直接融合,也可以是通过接头肽连接的含1个或1个以上的识别功能多肽分子融合蛋白。
核酸分子可以为编码各互补决定区或单域抗体或融合蛋白氨基酸序列的核苷酸序列,通过遗传密码子可以随时获得相应核酸分子的具体序列。
本发明所提供的核苷酸序列或者至少部分序列可以通过合适的表达***进行表达以得到相应的蛋白质或多肽。所述表达***包括细菌,酵母菌,丝状真菌,动物细胞,昆虫细胞,植物细胞,或无细胞表达***。
本发明还提供一种载体,包含所述核酸序列,由于遗传密码子具有兼并性,该核酸序列可以根据不同的应用目的而不同。
融合蛋白也可以是单域抗体与一个或多个其他具治疗功能或识别功能的多肽融合制备的融合蛋白;融合方式可以直接融合,也可以通过接头肽连接。
核酸分子可以为编码各互补决定区或单域抗体或融合蛋白氨基酸序列的核苷酸序列,由于遗传密码子具有兼并性,该核酸序列可以根据不同的应用目的不同。该核酸分子可以在细菌,酵母菌,丝状真菌,哺乳动物细胞,昆虫细胞,植物细胞,或无细胞表达***等表达***中表达出蛋白质。
宿主细胞可以为表达互补决定区或单域抗体或融合蛋白氨基酸序列的核苷酸序列的细胞。
相对于现有技术,本发明的有益效果是:本发明中所筛选得到的单域抗体能特异性的结合巨细胞病毒抗原与HLA-A2的复合物,可开发成治疗巨细胞病毒的抗体药及其他有益产品,对于治疗巨细胞病毒引起的疾病具有重要意义。
附图说明
图1为本发明中三轮淘选后第一板ELISA检测及数据分析图,其中:
A是ELISA所有孔均包被抗原HLA-A2/NLVPMVATV;
B是数据分析图,纵坐标为各孔在650nm下的光吸收值,横坐标为96个孔,其中,1-8为A1、B1、C1、D1、E1、F1、G1、H1,9-16为A2、B2、C2、D2、E2、F2、G2、H2,依次类推,89-96为A12、B12、C12、D12、E12、F12、G12、H12。
图2为本发明中三轮淘选后第二板ELISA检测及数据分析,其中:
A是ELISA所有孔均包被抗原HLA-A2/NLVPMVATV;
B是数据分析图,纵坐标为各孔在650nm下的光吸收值,横坐标为96个孔,其中,1-8为A1、B1、C1、D1、E1、F1、G1、H1,9-16为A2、B2、C2、D2、E2、F2、G2、H2,依次类推,89-96为A12、B12、C12、D12、E12、F12、G12、H12。
图3为本发明中的两种不同单域抗体对不同抗原的特异性检测及数据分析图,其中:
A是ELISA酶标板A、B两行包被抗原HLA-A2/ITDQVPFSV,C、D两行包被抗原HLA-A2/NLVPMVATV,E、F两行包被抗原HLA-A2/RMFPNAPYL,G、H两行包被抗原HLA-A2/SLLMWITQC;Line 1所加一抗为的M4-F4,line 2为阳性对照;line 3为某一个对四种抗原有交叉反应的抗体MA;line 4为M4-G5;
B是数据分析图,纵坐标为650nm下的光吸收值,横坐标1,2,3,4为四种抗原HLA-A2/ITDQVPFSV,HLA-A2/NLVPMVATV,HLA-A2/RMFPNAPYL,HLA-A2/SLLMWITQC。a,b,c,d依次为M4-F4,M5对照1,MA对照2,M4-G5。
图4为本发明中的单域抗体在pcDNA3.1中融合FC的质粒图谱示意图;
单域抗体与FC之间引入通过BamH I酶切位点,单域抗体前使用Hind III限制性内切酶,FC后使用Xba I限制性酶切。单域抗体前有信号肽以及kozak序列。
图5为本发明中的pcDNA3.1表达的融合蛋白M4-G5-FC的SDS-PAGE;
Marker的条带从小到大依次为14,25,30,40,50,70,100,120,160KD。Line1为还原态M4-G5-FC,Line2为非还原态M4-G5-FC。
图6为发明中的融合蛋白M4-G5-FC对不同抗原的特异性检测及数据分析。
纵坐标为650nm下的光吸收值,横坐标1,2,3,4为四种抗原HLA-A2/ITDQVPFSV,HLA-A2/NLVPMVATV,HLA-A2/RMFPNAPYL,HLA-A2/SLLMWITQC。样品为M4-G5-FC,MB对照。
具体实施方式
为使本领域技术人员更好的理解本发明的技术方案,下面结合附图和具体实施例对本发明作详细说明。
实施例1筛选HLA-A2/NLVPMVATV复合物的单域抗体
1.1单域抗体噬菌体文库制备
1.1.1辅助噬菌体(BM13)的制备
将M13KE噬菌体(购自NEB#N0316S)的复制子用AlwnI(购自NEB)和AfeI(购自NEB)双酶切,同时人工合成基因片段也用AlwnI和AfeI(购自NEB)双酶切,然后用T4连接酶连接在一起。连接后转染TG1得到辅助噬菌体BM13。如此,原复制子中tctggtggtggttctggtggcggctctgagggtggtggctctgagggtggcggttctgagggtggcggctctgagggaggcggttccggtggtggctct序列被人工合成基因序列所取代,即在噬菌体GIII编码区域,添加了胰蛋白酶切割序列,一旦用作辅助噬菌体时,增加胰蛋白酶消化步骤,减少不含融合目的基因蛋白噬菌体的数目。
人工合成基因序列如下:
CCA GCC GGC CTT TCT GAG GGG TCG ACT ATA GAA GGA CGA GGG GCC CAC GAAGGA GGT GGG GTA CCC GGT TCC GAG GGT
1.1.2噬菌体文库构建
1.1.2.1载体构建
将pUC19(购自NEB)用HindIII(购自NEB)和NdeI(购自NEB)双酶切,加入基于DP47抗体序列的重链人工单域抗体序列。单域抗体表达框架中,单域抗体与GIII蛋白融合,中间加入Myc和VSV-G标签,用于纯化或鉴定,构建成噬菌体展示载体pBG3。
1.1.2.2 ssDNA模板制备
大肠杆菌菌株CJ236(购自NEB)缺乏功能性尿嘧啶脱氧核糖核苷三磷酸酶和尿嘧啶-N糖苷酶,可以产生尿嘧啶化单链DNA模板。将pBG3质粒转染进入CJ236并涂布在含Carbenicillin(50μg/ml)和chloramphenicol(15μg/ml)琼脂平板上,培养过夜。挑选平板上筛选出的单个菌落到3ml 2×TY肉汤培养基中(含上述同样浓度的双抗),在37℃,250rpm条件下培养过夜。次日取0.3ml过夜培养物加入30ml新鲜2×TY肉汤培养基中(Carbenicillin 50μg/ml),持续3-4小时培养,使OD600=0.4-0.6,加入辅助噬菌体(按细菌:噬菌体个数比为1:10的比例加入),在37℃,150rpm的条件下离心1小时,将细菌沉淀重悬于60ml 2×TY双抗培养基中,在25℃,250rpm的条件下培养22小时,离心弃沉淀。含噬菌体的上清液用5%PEG(PEG800和300mM NaCl调至浓度为5%)沉淀,然后重悬于PBS中,使用QIAprep Spin M13试剂盒(购自Qiagen)制备出ssDNA。
1.1.2.3文库制备
文库制备用KunKel方法制备CDR突变寡核苷酸链由金唯智公司合成按下表合成:
Olig_CDR1
CTGCGTCTCTCCTGTGCAGCCTCCGGAKWTANSNTTANCNMTNASDHTRSCNNTTGGGTCCGCCAGGCTCCAGGGAA;
Olig_CDR2
GGGTCTAGAGTGGGTATCARSCNNKRNSVVWCGTAGCGGTAGCACATACTACGCAGACTCCGTG:
Olig_CDR3a
GACACCGCGGTATATTATTGCGCGGRSWNVSTHTNNKNNKNNKNNKNNKNNKNNKNNKNNKNNKNNKNNKNNKNNKTGGGGTCAGGGAACCCTGGTCACCGTC:
Olig_CDR3b
CGAGGACACCGCGGTATATTATTGCGCGGRSWNVSTHTNNKNNKNNKNNKNNKNNKNNKNNKNNKTGGGGTCAGGGAACCCTGGTCACCGTCACC。
寡核苷酸链的磷酸化
将人工合成的寡核苷酸链加入下列100μl,50mM的Tris-HCl溶液中,在pH为7.5,含有5U T4多核苷酸激酶,10mM MgCl2,1mM ATP及5mM DTT的体系中:,在37℃条件下静置1小时后,磷酸化好的寡核苷酸链用PCR纯化试剂盒(购自天根)纯化。
磷酸化寡核苷酸与ssDNA退火结合
将磷酸化寡核苷酸和Uracilated ssDNA(磷酸化寡核苷酸:ssDNA=3:1,ssDNA由1.1.2.2中制备所得)溶于含10mM MgCl2的50mM Tris-HCl,pH为7.5的缓冲液中,在90℃条件下加热2分钟后,以1℃/min的降速降至25℃。
dsDNA的合成
将0.55mM ATP,0.8mM dNTPs,5mM DTT,15u T4 DNA合成酶及15u T7 DNA合成酶等材料加入退火结合的磷酸化寡核苷酸与ssDNA复合物中:在20℃下保温3小时后,用Qiaquick PCR纯化试剂盒(购自Qiagen)纯化。将纯化后的DNA待转化至电转感受态TG1中。
1.2 BM13辅助噬菌体以及噬菌体文库的增值
BM13辅助噬菌体的增值
从基本琼脂培养基平板上挑取一个大肠杆菌TG1(购于武汉淼灵)的新鲜单菌落,接种到20ml 2×TY培养基中,温和摇动,37℃条件下培养至OD600约为0.8备用。将1.1.1中制备的原始BM13辅助噬菌体用2×TY培养基制备一系列10倍稀释的BM13噬菌体。各稀释度分别取10μl与200μl TG1(OD600=0.8)菌液混匀,振荡器温和震荡3s,并与上层琼脂培养基混匀,倾倒在预先平衡室温的TY平板上。转动平板以确保菌体和上层琼脂分布均匀。待上层培养基凝固后,于37℃生化培养箱倒置培养过夜。
次日挑选分离良好的单个噬菌体接种于装有2~3ml含25μg/ml卡那霉素的2×TY培养基的15ml培养管中。在37℃,250rpm条件下震荡培养12~16h。将感染上清液转移至1.5ml无菌微量离心管,在微量离心机上,以最大转速,4℃离心2min。上清液转移至新管中4℃保存。
噬菌体文库的增值
将制备好的噬菌体文库接种至100ml含60μg/ml氨苄青霉素的2×TY培养基中,在37℃,250rpm条件下震荡培养至OD600为0.8时,加入BM13至浓度为2×107pfu/ml。在37℃,300rpm条件下培养1h,加入25μg/ml卡那霉素,在37℃条件下继续培养14~18h。将菌液离心,上清液用5%PEG沉淀,然后重悬于5%MPBS中备用。
1.3筛选HLA-A2/NLVPMVATV复合物的单域抗体
1.3.1生物淘选
链酶亲和素免疫磁珠(购自Invitrogen cat no.SKU#112-05D)取25μl,加入一定量的生物素化的HLA-A2/NLVPMVATV,室温结合5min。PBST及PBS洗2~3次。MPBS封闭2h,PBST及PBS洗2~3次。将噬菌体文库加入磁珠中,室温孵育2h。
移走未结合文库,磁珠用PBST及PBS各洗10次。向磁珠中加入0.01%胰酶溶液,室温孵育1H,得胰酶洗脱液。
将胰酶洗脱液加入到30ml TG1(OD600=0.5)中,按1.2中方法增值第一次洗脱文库。
如此进行第二轮、第三轮淘选。挑取三轮淘选后所得的单菌落至96孔板中。按1.2中文库增值方法制备出培养物上清液。
1.3.2 ELISA鉴定
取一定量上清液做ELISA鉴定。见图1A和图2A。
ELISA步骤如下:用包被缓冲液将已知抗原稀释至1~10μg/ml,每孔加0.1ml,4℃过夜;次日洗涤3次;加一定稀释的待检样品0.1ml于上述已包被之反应孔中,置37℃孵育1小时,洗涤;加入新鲜稀释的酶标第二抗(辣根过氧化物酶HRP标记的抗噬菌体的抗体,1∶5000)体0.1ml,37℃孵育60分钟,洗涤;最后一遍用DDW洗涤。于各反应孔中加入临时配制的TMB底物溶液0.1ml,在37℃条件下静置10~30分钟。用高级读板机在650nm波长下读板。
ELISA鉴定的结果如图1A和图2A所示,各孔光吸收值如图1B和图2B所示。其中,A:ELISA所有孔均包被抗原HLA-A2/NLVPMVATV;每孔中抗体各不相同。B:数据分析,纵坐标为各孔在650nm下的光吸收值,横坐标为96个孔,其中,1-8为A1、B1、C1、D1、E1、F1、G1、H1,9-16为A2、B2、C2、D2、E2、F2、G2、H2,依次类推,89-96为A12、B12、C12、D12、E12、F12、G12、H12。
选择图1和2中A650nm在0.8以上的克隆进行测序,得到多个不同的氨基酸序列,SEQ ID NO.7(M4-F4),SEQ ID NO.8(M4-G5)。
两种不同单域抗体对不同抗原的特异性和亲和力的ELISA鉴定,见图3A。加入TMB20min后用高级读板机在650nm波长下读板,数据整理如图3B。
其中,图3A中ELISA酶标板A、B两行包被抗原HLA-A2/ITDQVPFSV,C、D两行包被抗原HLA-A2/NLVPMVATV,E、F两行包被抗原HLA-A2/RMFPNAPYL,G、H两行包被抗原HLA-A2/SLLMWITQC;每个孔中所加入的单域抗体均以培养上清液的形式加入。其中,Line 1的八个孔中均孵育一抗为的M4-F4,line 2为HLA-A2/RMFPNAPYL的抗体,做阳性对照;line 3为某一个对四种抗原有交叉反应的抗体MA;line 4为M4-G5。每种抗体对一种抗原的亲和检测反应均有两个重复孔。图3B为数据分析,纵坐标为650nm下的光吸收值,图中显示A650均为两个重复孔的平均值,横坐标1,2,3,4为四种抗原HLA-A2/ITDQVPFSV,HLA-A2/NLVPMVATV,HLA-A2/RMFPNAPYL,HLA-A2/SLLMWITQC。a,b,c,d依次为M4-F4,M5对照1,MA对照2,M4-G5。
结果显示筛选得到阳性克隆对HLA-A2/NLVPMVATV表现特异性高亲和力,对其它三个抗原基本不结合。
实施例2、融合蛋白M4-G5-FC在pcDNA3.1中的表达
为形成双价抗体,单域抗体基因融合人源FC,并在抗体前面引入Kozak序列及信号肽后克隆到pcDNA3.1上,质粒图谱见图4。单域抗体与FC之间引入通过BamHI酶切位点,单域抗体前使用HindIII限制性内切酶,FC后使用XbaI限制性酶切。
将该重组质粒瞬时转染到293F中,培养4天后离心,收集上清液,并用Protein A纯化。
其中,M4-G5-FC融合蛋白纯化后跑SDS-PAG,见图5。其中,图5A为融合蛋白M4-G5-FC的还原电泳图,图5B为融合蛋白M4-G5-FC非还原电泳图。Marker的条带从小到大依次为14,25,30,40,50,70,100,120,160KD。Linel为还原态M4-G5-FC,大约43KD,Line2为非还原态M4-G5-FC,大约100KD。
实施例3、融合蛋白M4-G5-FC特异性识别HLA-A2/NLVPMVATV复合物
ELISA检测融合抗体M4-G5-FC对不同抗原的亲和力,数据整理后见图6。其中,纵坐标为650nm下的光吸收值,横坐标1,2,3,4为四种抗原HLA-A2/ITDQVPFSV,HLA-A2/NLVPMVATV,HLA-A2/RMFPNAPYL,HLA-A2/SLLMWITQC。样品为pcDNA3.1中纯化后的M4-G5-FC融合蛋白,MB为HLA-A2/RMFPNAPYL的一种抗体,做阳性对照。
结果显示融合蛋白M4-G5-FC对HLA-A2/NLVPMVATV表现较高亲和力,对其它三个抗原基本不结合。
以上实施例仅为本发明的示例性实施例,不用于限制本发明,本发明的保护范围由权利要求书限定。本领域技术人员可以在本发明的实质和保护范围内,对本发明做出各种修改或等同替换,这种修改或等同替换也应视为落在本发明的保护范围内。
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Claims (13)
1.一种识别HLA-A2/NLVPMVATV的单域抗体,其特征在于,所述单域抗体的氨基酸序列包含互补决定区CDR1为SEQ ID NO.1,CDR2为SEQ ID NO.2和CDR3为SEQ ID NO.3;
或,所述单域抗体的氨基酸序列包含互补决定区CDR1为SEQ ID NO.4,CDR2为SEQ IDNO.5和CDR3为SEQ ID NO.6。
2.权利要求1中所述的单域抗体,其特征在于,所述抗体为单区抗体或重链变区或轻链变区。
3.权利要求1中所述的单域抗体,其特征在于,所述抗体为scFv或Fc融合蛋白或完整抗体的一部分。
4.权利要求1中所述的单域抗体,其特征在于,所述单域抗体氨基酸序列包括SEQ IDNO.7或SEQ ID NO.8。
5.权利要求1中所述的单域抗体,其特征在于,包含权利要求1中所述互补决定区中的一个或者两个以上的氨基酸序列,且与所包含的氨基酸序列具有最低79%同源性。
6.权利要求1中所述的单域抗体,其特征在于,所述单域抗体的氨基酸序列包含权利要求4中所述框架中的一个或者两个以上的氨基酸序列,且至少与一个氨基酸序列最低具有90%同源性。
7.一种融合蛋白,其特征在于,使用权利要求1所列氨基酸序列与至少包含一个具治疗功能的多肽制备的融合蛋白。
8.权利要求7中所述的融合蛋白可以与各具识别功能多肽直接融合,也可以是通过接头肽连接的含1个或1个以上的识别功能多肽分子融合蛋白。
9.一种多特异或多功能分子,其特征在于,包含权利要求1所述的单域抗体。
10.一种核酸分子,其特征在于,编码权利要求1-6任一所述的单域抗体、或权利要求7-8任一所述的融合蛋白、或权利要求9所述的多特异或多功能分子的氨基酸序列。
11.一种载体,其特征在于,包含权利要求10所述核酸序列,由于遗传密码子具有兼并性,该核酸序列可以根据不同的应用目的不同。
12.权利要求11所述的一种核酸分子表达的蛋白质,其特征在于,其所述核酸序列或者至少部分序列可以通过合适的表达***进行表达,以得到相应的蛋白质或多肽;所述表达***包括细菌,酵母菌,丝状真菌,哺乳动物细胞,昆虫细胞,植物细胞,或无细胞表达***。
13.一种宿主细胞,其特征在于,包含可表达权利要求1-6任一所述的单域抗体、权利要求7或8所述的融合蛋白、权利要求9所述的多特异或多功能分子、权利要求10所述的核酸分子、权利要求11所述的载体或权利要求12所述的蛋白质或多肽的宿主细胞。
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