CN102952191B - 全人源抗cd33单链抗体zjl101及其应用 - Google Patents
全人源抗cd33单链抗体zjl101及其应用 Download PDFInfo
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- CN102952191B CN102952191B CN201210343744.4A CN201210343744A CN102952191B CN 102952191 B CN102952191 B CN 102952191B CN 201210343744 A CN201210343744 A CN 201210343744A CN 102952191 B CN102952191 B CN 102952191B
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Abstract
本发明提供一种全人源抗CD33单链抗体ZJL101。通过噬菌体展示技术，以人CD33-ECD蛋白做为靶抗原从高容量的全人源白血病噬菌体抗体库中经多轮筛选，并在大肠杆菌中诱导表达出单链抗体ZJL101，并经过Ni柱亲和纯化而获得。本发明获得的全人源抗CD33单链抗体ZJL101能够特异抑制CD33阳性的白血病细胞株HL-60和Kasumi-1的生长，该单链抗体本身及其可变区序列经过基因工程改造成其它的抗体形式后，可作为治疗药物或药物运载体用于CD33阳性的白血病的靶向治疗。该单链抗体具有结构简单、免疫源性低、特异性强、相对分子量小、穿透性强等优点，是一种理想的靶向药物和药物运载体。
Description
技术领域
本发明属基因工程，涉及全人源抗CD33单链抗体的筛选、鉴定和原核表达，及其在CD33阳性白血病靶向药物制备中的应用。
背景技术
噬菌体展示抗体技术可以制备全人源的抗体，是将蛋白分子或肽段的基因克隆到丝状噬菌体的基因组DNA中，与噬菌体的外壳蛋白形成融合蛋白，从而使异源分子展示于噬菌体表面。主要特点是将特定的分子的基因型和表达型同时出现在一个噬菌体颗粒内，通过DNA测序可以获得特定展示蛋白的序列信息。
噬菌体抗体库技术模拟了人类B细胞的分化成熟过程，首先通过分子生物学手段将人类抗体的重链和轻链可变区基因连接起来形成单链抗体(Single-chain variable fragment, scFv)，插入噬菌体载体上，使单链抗体scFv展示在噬菌体表面，再用特定的抗原筛选出特异性的噬菌体抗体，并用大肠杆菌表达可以表达大量单链抗体。
噬菌体可以表达抗体结合片段(Fragment antigen binding, Fab)、单链抗体(scFv)、双链抗体(Diabody)和双特异性抗体(Bispecific scFv, BsAb)等。其中单链抗体(scFv)是目前报道最多的一种小分子抗体，是由抗体识别抗原的可变区Fv段组成。Fv由重链可变区VH和轻链可变区VL通过连接肽（linker）连接构成scFv。所加的linker很重要，必须不影响Fv的构象，目前最常用的是由四个甘氨酸和一个丝氨酸组成的重复3次的序列(GGGGS)3。将scFv基因克隆在丝状噬菌体载体pIII基因先导序列和pIII基因之间，以融合蛋白的形式被导入细菌膜间隙，并组装成scFv，在加入辅助噬菌体M13K07后，scFv以融合蛋白的形式表达在噬菌体表面。pCANTAB-5E载体的特点是，在scFv基因后面含有一段编码Tag尾肽(E-Tag)的序列，在E-Tag后面有一个琥珀(Amber)终止密码，位于scFv基因与pIII基因之间，在抑制性细菌TG1中，这种琥珀密码子只有20%有效，所以在蛋白翻译过程中可以被通读而形成scFv-pIII融合蛋白；而在非抑制性菌株中，如HB2151，此终止子被识别，同时scFv基因在pIII基因前终止，形成独立的抗体蛋白，滞留于细胞膜间隙，并在长时间培养后释放于培养液中，形成可溶性表达。另外，用分子克隆的方法，也可以将scFv基因转移进入pET表达体系，并在大肠杆菌中实现高表达而获得单链抗体。
单链抗体具有结构简单、相对分子量小、穿透性强、免疫源性低等优点，是一种理想的载体。构建全人源抗体库目的是寻找一种能与特定白血病相关抗原（如CD33等）特异性结合的单链抗体scFv，这类特异识别肿瘤细胞的单链抗体本身或者经过基因工程改造成其它的抗体形式后，可以作为抗肿瘤药物进行开发；其次可以作为药物载体，如偶联药物、毒素或放射性同位素，进行白血病的靶向治疗。
发明内容
本发明的目的是提供一种全人源抗CD33单链抗体ZJL101，具有结构简单、相对分子量小、穿透性强、免疫源性低等优点，是一种理想的药物运输载体。SEQ ID NO 1是该单链抗体的DNA序列：
atggcccagg tccagcttgt gcagtctggg actgtagtga agaagcctgg gtcctcggtg
aaggtctcct gcaagacttc tggaggcacc ctccccaacc atgccatcaa ctgggtgcga
caggcctctg gacaagggct tgagtggatg ggatggatga gccctaacac tggtgacaca
ggctatgcac ggaagttcca gggcagagtc accatgacca gggacacctc cataagcaca
gcctacatgg aactgagcag cctgagatct gacgacacgg ccatatatta ctgtgcgacg
ggcgaccccc gggttggtca ttcgtggggc cagggcaccc tggtcactgt ctcctcaggt
ggtggtggtt ctggcggcgg cggctccggt ggtggtggat ctgacatcca gatgacccag
tctccaccct ccctgtctgc atctgtagga gacagagtca ccatcacttg ccgggcaagt
caaaccattg gcagtcattt aaattggtat cagcagaaac cagggaaagc ccctaaactc
ctgatctatg gtgcatccag tttgcaaagt ggggtccccg caaggttcac tggcagtgga
tttgggacag atttcactct caccatcagc agtctacaag ttgaagactt ggcaacttac
tcctgtcaac agacttacaa taccccccgg acgttcggcc aagggaccaa ggtggaaatc
aaa
总长度为723 bp；
SEQ ID NO 2是该单链抗体的氨基酸序列：
Met Ala Gln Val Gln Leu Val Gln Ser Gly Thr Val Val Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Leu Pro Asn His Ala Ile Asn Trp Val Arg Gln Ala Ser Gly Gln Gly Leu Glu Trp Met Gly Trp Met Ser Pro Asn Thr Gly Asp Thr Gly Tyr Ala Arg Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Thr Gly Asp Pro Arg Val Gly His Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Pro Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Gly Ser His Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Thr Gly Ala Ser Ser Leu Gln Ser Gly Val Pro Ala Arg Phe Thr Gly Ser Gly Phe Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Val Glu Asp Leu Ala Thr Tyr Ser Cys Gln Gln Thr Tyr Asn Thr Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
其分子量为25212，含有完整的抗体重链可变区VH和轻链可变区VL ，中间连接肽为（Gly Gly Gly Gly Ser）3；
所述单链抗体的重链可变区（VH）具有SEQ ID NO 3的的氨基酸序列：
Met Ala Gln Val Gln Leu Val Gln Ser Gly Thr Val Val Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Leu Pro Asn His Ala Ile Asn Trp Val Arg Gln Ala Ser Gly Gln Gly Leu Glu Trp Met Gly Trp Met Ser Pro Asn Thr Gly Asp Thr Gly Tyr Ala Arg Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Thr Gly Asp Pro Arg Val Gly His Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
含119个氨基酸，其分子量为12764；
所述单链抗体的轻链可变区（VL）具有SEQ ID NO 4的的氨基酸序列：
Asp Ile Gln Met Thr Gln Ser Pro Pro Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Gly Ser His Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Thr Gly Ala Ser Ser Leu Gln Ser Gly Val Pro Ala Arg Phe Thr Gly Ser Gly Phe Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Val Glu Asp Leu Ala Thr Tyr Ser Cys Gln Gln Thr Tyr Asn Thr Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
含107个氨基酸，其分子量为11519。
本发明的另一个目的是提供全人源抗CD33单链抗体ZJL101在制备白血病靶向药物中的应用。本发明的单链抗体ZJL101可以对CD33阳性的白血病细胞，如髓系白血病HL-60和Kasumi-1细胞，产生生长抑制作用。
本发明的优点是：（1）提供的全人源单链抗体ZJL101结构简单，是抗体重链可变区VH和轻链可变区VL由连接肽(GGGGS)3连接而成，MW为25.212 kD，且含有完整抗原结合部位；（2）本发明的单链抗体ZJL101可以对CD33阳性的白血病细胞，如髓系白血病HL-60和Kasumi-1细胞，产生生长抑制作用；（3）本发明的单链抗体ZJL101分子量小、穿透性强、免疫源性低，可作为药物或药物载体运输药物、同位素或毒素分子，可以治疗CD33阳性的白血病。
附图说明
图1是ELISA检测噬菌体单链抗体的结合活性。
图2 A是噬菌体单链抗体ZJL101结构VH 片段序列分析图。
图2 B是噬菌体单链抗体ZJL101结构VL片段序列分析图。
图3是阳性噬菌体（展示单链抗体ZJL101）对白血病细胞株的特异抑制作用。
图4是用SDS-PAGE电泳分析单链抗体ZJL101在大肠杆菌HB2151中的表达。
图5是大肠杆菌HB2151中表达的单链抗体ZJL101的Western Blot分析图。
图6是用SDS-PAGE电泳分析单链抗体ZJL101在大肠杆菌BL21中的表达和纯化过程。
图7是单链抗体ZJL101对白血病细胞的特异抑制作用测定。
具体实施方式
本发明结合以下实施例及附图作进一步的说明。
实施例1： 噬菌体抗体库的筛选
实验方法：使用基因重组表达的CD33胞外区（CD33-ECD）抗原筛选全人源白血病抗体库。用Na2CO3/NaHCO3将抗原稀释后，加入96孔ELISA板，4℃包被过夜。次日，封闭液封闭1 h，加入噬菌体抗体库，37℃孵育2 h。TBS洗涤（第一轮5遍，第二轮10遍，第三、四轮10遍）。用甘氨酸-盐酸（pH 2.2）洗脱并收集噬菌体，Tris-HCl中和至pH 7.0，侵染对数期E. coli TG1，37℃静置20 min，取10 mL测滴度。其余转至20 mL 2×YT-A-G，37℃振摇培养至OD600nm达到0.5，然后加入辅助噬菌体, 37℃振摇1 h，离心，沉淀重悬于200 mL 2×YT-AK，30℃振摇过夜，次日，收集噬菌体。
实验结果：噬菌体抗体库经四轮筛选后，得到105倍的富集，见表1。
结果说明：第四轮筛选的效率（2.0×10-6）是第一轮筛选的效率（1.9×10-8）的105倍，表明与靶抗原CD33-ECD特异结合的噬菌体已经得到有效富集。
实施例2： 抗体库多样性分析
实验方法：随机挑选筛选到的200个单克隆送南京金斯瑞生物科技有限公司，进行插入区域的DNA序列测序。将能正确编码氨基酸序列的单克隆DNA序列翻译成氨基酸，经ClustaIX软件进行序列比对分析(http://www.ncbi.nlm.nih.gov/igblast/)。
实验结果：序列分析结果表明，有8株噬菌体单克隆的插入区域的DNA可以正确编码氨基酸，氨基酸序列比对结果见表2，其中3号和4号两株噬菌体插入区域的DNA序列完全相同, 其序列为SEQ ID NO 1。
结果说明：筛选出的8株单克隆噬菌体中，除3号和4号两株噬菌体插入区域的DNA序列完全相同（序列为SEQ ID NO 1），其余均有良好的多样性。
实施例3： ELISA鉴定单链抗体的抗原结合活性
实验方法：挑选筛选四轮后获得的噬菌体单克隆，送测序。将含测序能通读序列的单克隆，制备单克隆重组噬菌体。用PBS稀释抗原（CD33-ECD），加入到96孔培养板中，4℃包被过夜。取出后，用封闭液37℃封闭1h，将待测的单克隆重组噬菌体加入96孔板中，37℃温育2 h。以PBS作为阴性对照，加入HRP/anti-M13 抗体，37℃温育1 h，加底物TMB-H2O2显色，酶标仪测定OD450nm 值。
实验结果：用单克隆噬菌体做ELSIA验证，结果参见图1，其中9和10，分别为噬菌体M13阴性对照和PBS对照组，OD450nm值<0.2； 3号和4号噬菌体的序列完全相，OD450nm值约为1.6；1号、2号和5号噬菌体的OD450nm值在0.8-1.2之间；7号噬菌体的OD450nm为0.5，6号和8号OD450nm值<0.2。
结果说明：3号和4号噬菌体抗原结合呈强阳性，1号、2号和5号噬菌体为阳性，7号噬菌体为弱阳性，6号和8号噬菌体为阴性。
实施例4：阳性噬菌体单链抗体DNA序列测定和结构分析
实验方法：3号和4号噬菌体插入区域（单链抗体）进行DNA序列分析；根据DNA序列分析结果，取编码区序列，输入VBASE2数据库（http://www.vbase2.org/），分析获得单链抗体结构分析图。
实验结果：3号和4号噬菌体插入区域（单链抗体）的DNA序列完全相同，其序列为SEQ ID NO 1，分析结果参见图2A和图2B，其中图2A为VH 片段序列（包括linker）、图2B为VL片段序列图，图中可见重链和轻链分别具有FR1、FR2、FR3、FR4、CDR1、CDR2和CDR3结构域。
结果说明：该阳性株单链抗体具有完整的抗原结合区，为正确的单链抗体结构，命名为ZJL101。
实施例5： 噬菌体展示的单链抗体ZJL101对白血病细胞的特异抑制作用测定
实验方法：选用CD33阳性的髓系白血病细胞株HL-60和Kasumi-1以及CD33阴性的白血病细胞Jurkat株，在37℃和5%CO2的条件下培养至对数期；将细胞培养悬液，离心，弃上清，收集细胞；用细胞培养液悬浮细胞，并以5.0×103-1.0×104 cells/孔的密度接种于96孔细胞培养板，在37℃和5% CO2的条件下培养24 h；将上述ELISA鉴定阳性的3号噬菌体扩增，得到噬菌体浓度为7.5x 1011 cfu/mL；设置四个浓度梯度，分别加入噬菌体至各细胞孔，使噬菌体终浓度分别为0, 7.5x107 cfu/mL，7.5x108 cfu/mL，7.5x109 cfu/mL，7.5x1010 cfu/mL；然后，将细胞孵育72 h，每孔设4个复孔，以PBS稀释的M13噬菌体做阴性对照；加cck-8溶液，37℃孵育1 h；在470 nm波长下测定吸光度值，并计算细胞在不同浓度的噬菌体（展示有单链抗体ZJL101序列）作用下的生长抑制率。
实验结果：参见图3，该噬菌体展示的单链抗体ZJL101，在各剂量组，均可对CD33 阳性的Kasumi-1和HL-60细胞产生抑制作用，其中对Kasumi-1细胞的抑制作用最高可达50%，而对HL-60细胞可达30%。用Jurkat细胞做阴性对照，仅有轻微抑制作用，而且不呈剂量依赖关系，与上述两种髓系白血病细胞有显著性差异（P< 0.05）。
结果说明：该噬菌体展示的单链抗体ZJL101可对CD33阳性的HL-60和Kasumi-1细胞产生明显生长抑制作用，而对CD33阴性的Jurkat细胞无明显抑制作用。
实施例6：单链抗体ZJL101在大肠杆菌HB2151中的可溶性表达
实验方法：取阳性克隆的重组噬菌体侵染对数生长期的大肠杆菌HB215l，接种于SOBAG-嘧啶酸板上，30℃孵育过夜；挑取一个克隆，于2×YT-AG中，30℃培养过夜；次日将过夜菌液1：10加入50 mL 2×YT-AG中，30℃培养l h，离心，弃去上清，50 mL 2×YT-Amp-IPTG重悬沉淀，30℃，培养6~7 h后，离心，沉淀用2.5 mL含20%蔗糖的25 mM Tris-HCl (pH 7.3)重悬，充分悬浮，冰浴30 min；重新离心收集菌体，加入5 mL双蒸水重悬，冰浴30 min，离心后，取上清即周质蛋白进行常规的12% SDS-PAGE电泳分析。
实验结果：经IPTG诱导，SDS-PAGE电泳检测参见图4：M，为分子量标准；1，为未诱导全细胞；2，为诱导组周质蛋白；3，为诱导组全细胞，4，为培养物上清。2号和3号均可见分子量27 kD左右目的条带（带His标签），1号和4号中无该条带。
结果说明：经IPTG诱导，单链抗体ZJL101可以在细菌周质蛋白和全细胞中表达，而在培养物上清中未见表达。
实施例7： Western blot鉴定单链抗体ZJL101
实验方法：上述表达的蛋白样品，进行SDS-PAGE电泳后，取下电泳后的丙烯酰胺凝胶，转膜，100V恒压，转膜90 min；取下PVDF膜，封闭液(TBST-5%脱脂奶粉)封闭PVDF膜，缓慢摇1 h；稀释一抗Rabbit Anti-6×His tag antibody ，取出PVDF膜于封闭液中，缓慢摇1 h，用TBST再洗涤5 min × 5次；封闭液稀释二抗Goat anti-rabbit IgG-FIFC conjugate，缓慢摇1h，用TBST洗涤5 min × 5次；化学发光ECL显色，拍照记录结果。
实验结果：Western blot检测结果参见图5，1号为诱导组细胞周质蛋白，2号为培养物上清，3号为诱导组全细胞。 可见诱导组的细胞周质蛋白、全细胞液和培养物上清中均有27 kD的条带（带His标签）。
结果说明：经IPTG诱导，单链抗体ZJL101在细胞周质蛋白和全细胞液中有所表达，在培养物上清中也有少量表达。
实施例8：单链抗体ZJL101在大肠杆菌BL21中的表达和纯化
实验方法：取ELISA检测阳性最高的3号噬菌体进行PCR扩增目的DNA片段，用XhoⅠ和NdeⅠ双酶切后克隆进入表达载体pET30a(+) ，在大肠杆菌BL21中进行表达。加入IPTG至终浓度为0.5 mM，在30℃条件下诱导表达5 h,超声破碎后，经过12000 rpm/10 min离心，取上清过Ni柱进行纯化，用含100 mM咪唑的平衡缓冲液洗脱目的蛋白，用12% SDS-PAGE电泳分析表达和纯化产物。
实验结果：电泳分析结果参见图6，M，为分子量标准；1，是诱导前对照；2，是诱导后全菌；3号是超声后上清，4、5号是样品上柱后未结合的杂蛋白，6号是纯化后的目的蛋白，呈单一条带，MW 27 kD（带His标签），与预期的一致。
结果说明：单链抗体ZJL101可以在大肠杆菌中进行表达，可以用Ni柱进行纯化，可以制备高纯度的单链抗体ZJL101。
实施例9： 单链抗体ZJL101对白血病细胞的特异抑制作用测定
实验方法：选用CD33阳性的髓系白血病细胞株HL-60和Kasumi-1以及CD33阴性的白血病细胞Jurkat，在37℃和5%CO2的条件下培养至对数期；将细胞培养悬液离心，弃上清，收集细胞；用细胞培养液重悬细胞，并以5.0×103-1.0×104 cells/孔的密度接种于96孔细胞培养板，在37℃和5% CO2的条件下培养24 h；加入不同浓度的单链抗体ZJL101，孵育72 h，每孔设3个复孔，以只加PBS的细胞做阴性对照；加cck-8溶液，37℃孵育1 h；在470 nm波长下测定吸光度值，并计算细胞在不同浓度的单链抗体ZJL101作用下的生长抑制率。
实验结果：参见图7，该单链抗体ZJL101可对HL-60和Kasumi-1细胞产生抑制作用，其中高浓度单链抗体（0.25 mg/mL）对Kasumi-1细胞的抑制作用达到50.15%，对HL-60细胞达到30.52%；中浓度单链抗体（0.12 mg/mL）对Kasumi-1细胞的抑制作用达到33.99%，对HL-60细胞达到20.35%；低浓度单链抗体（0.06 mg/mL）对Kasumi-1细胞的抑制作用达到23.39%，对HL-60细胞达到16.55%；在高、中浓度组均没有观测到对Jurkat细胞（阴性对照）的抑制作用，低剂量组观察到有轻微抑制作用，属于正常实验误差。
结果说明：该单克隆抗体ZJL101可对CD33阳性的HL-60和Kasumi-1细胞产生明显的生长抑制作用，而对CD33阴性的Jurkat细胞无抑制作用；表明其抑制作用具有特异性。
综上所述，我们从全人源抗体库中筛选得到了一种能特异与CD33分子结合的噬菌体单链抗体ZJL101，经DNA序列测定、ELISA分析和电泳鉴定证明其结构正确，并可以经大肠杆菌表达而制备，在培养的CD33阳性的白血病细胞中，可以引起特异的抑制作用，该单链抗体单独或者经过基因工程等方法改造成其它的抗体形式后，可以作为抗肿瘤药物或药物载体，如偶联药物、毒素或放射性同位素，进行CD33阳性白血病的靶向治疗。
无需进一步详细阐述，相信采用前面所公开的内容，本领域技术人员可最大限度地应用本发明。因此，前面的优选具体实施方案应理解为仅是举例说明，而非以任何方式限制本发明的范围。
<110>浙江大学
<120> 全人源抗CD33单链抗体ZJL101及其应用
<160> 4
<210> 1
<211> 723
<212> DNA
<213> 人工序列
<220>
<223> 含连接肽的抗CD33单链抗体ZJL101的DNA序列
<400>1
atggcccagg tccagcttgt gcagtctggg actgtagtga agaagcctgg gtcctcggtg 60
aaggtctcct gcaagacttc tggaggcacc ctccccaacc atgccatcaa ctgggtgcga 120
caggcctctg gacaagggct tgagtggatg ggatggatga gccctaacac tggtgacaca 180
ggctatgcac ggaagttcca gggcagagtc accatgacca gggacacctc cataagcaca 240
gcctacatgg aactgagcag cctgagatct gacgacacgg ccatatatta ctgtgcgacg 300
ggcgaccccc gggttggtca ttcgtggggc cagggcaccc tggtcactgt ctcctcaggt 360
ggtggtggtt ctggcggcgg cggctccggt ggtggtggat ctgacatcca gatgacccag 420
tctccaccct ccctgtctgc atctgtagga gacagagtca ccatcacttg ccgggcaagt 480
caaaccattg gcagtcattt aaattggtat cagcagaaac cagggaaagc ccctaaactc 540
ctgatctatg gtgcatccag tttgcaaagt ggggtccccg caaggttcac tggcagtgga 600
tttgggacag atttcactct caccatcagc agtctacaag ttgaagactt ggcaacttac 660
tcctgtcaac agacttacaa taccccccgg acgttcggcc aagggaccaa ggtggaaatc 720
aaa 723
<210> 2
<211> 241
<212> PRT
<213> 人工序列
<223> 含连接肽的抗CD33单链抗体ZJL101的多肽链序列
<400>2
Met Ala Gln Val Gln Leu Val Gln Ser Gly Thr Val Val Lys Lys
1 5 10 15
Pro Gly Ser Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr
20 25 30
Leu Pro Asn His Ala Ile Asn Trp Val Arg Gln Ala Ser Gly Gln
35 40 45
Gly Leu Glu Trp Met Gly Trp Met Ser Pro Asn Thr Gly Asp Thr
50 55 60
Gly Tyr Ala Arg Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp
65 70 75
Thr Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser
80 85 90
Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Thr Gly Asp Pro Arg Val
95 100 105
Gly His Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly
110 115 120
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp
125 130 135
Ile Gln Met Thr Gln Ser Pro Pro Ser Leu Ser Ala Ser Val Gly
140 145 150
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Gly Ser
155 160 165
His Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
170 175 180
Leu Ile Thr Gly Ala Ser Ser Leu Gln Ser Gly Val Pro Ala Arg
185 190 195
Phe Thr Gly Ser Gly Phe Gly Thr Asp Phe Thr Leu Thr Ile Ser
200 205 210
Ser Leu Gln Val Glu Asp Leu Ala Thr Tyr Ser Cys Gln Gln Thr
215 220 225
Tyr Asn Thr Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
230 235 240
Lys
241
<210> 3
<211> 119
<212> PRT
<213> 人工序列
<223> 抗CD33单链抗体ZJL101的重链的多肽链序列
<400>3
Met Ala Gln Val Gln Leu Val Gln Ser Gly Thr Val Val Lys Lys
1 5 10 15
Pro Gly Ser Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr
20 25 30
Leu Pro Asn His Ala Ile Asn Trp Val Arg Gln Ala Ser Gly Gln
35 40 45
Gly Leu Glu Trp Met Gly Trp Met Ser Pro Asn Thr Gly Asp Thr
50 55 60
Gly Tyr Ala Arg Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp
65 70 75
Thr Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser
80 85 90
Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Thr Gly Asp Pro Arg Val
95 100 105
Gly His Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
110 115 119
<210> 4
<211> 107
<212> PRT
<213> 人工序列
<223> 抗CD33单链抗体ZJL101的轻链的多肽链序列
<400>4
Asp Ile Gln Met Thr Gln Ser Pro Pro Ser Leu Ser Ala Ser Val
1 5 10 15
Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Gly
20 25 30
Ser His Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
35 40 45
Leu Leu Ile Thr Gly Ala Ser Ser Leu Gln Ser Gly Val Pro Ala
50 55 60
Arg Phe Thr Gly Ser Gly Phe Gly Thr Asp Phe Thr Leu Thr Ile
65 70 75
Ser Ser Leu Gln Val Glu Asp Leu Ala Thr Tyr Ser Cys Gln Gln
80 85 90
Thr Tyr Asn Thr Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu
95 100 105
Ile Lys
107
Claims (2)
1.一种全人源抗CD33单链抗体ZJL101，SEQ ID NO 1是该单链抗体的DNA序列，SEQ ID NO 2是该单链抗体的氨基酸序列，该单链抗体的分子量为25212，含有完整的抗体重链可变区VH和轻链可变区VL ，中间连接肽为（Gly Gly Gly Gly Ser）3，其重链可变区VH的氨基酸序列为SEQ ID NO 3，含119个氨基酸，其分子量为12764，轻链可变区VL的氨基酸序列为SEQ ID NO 4，含107个氨基酸，其分子量为11519。
2.根据权利要求1所述的一种全人源抗CD33单链抗体ZJL101在制备白血病靶向药物中的应用，其特征在于，在制备抑制CD33阳性的HL-60和Kasumi-1白血病细胞的靶向治疗药物中的应用。
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