抗VEGFR-2抗体嵌合Fab抗体片段及其制备方法、应用

摘要

本发明涉及的是抗VEGFR-2抗体嵌合Fab抗体片段及其制备方法、应用，利用基因重组技术，对抗VEGFR-2杂交瘤细胞株8H1进行改造，将鼠源性抗体的可变区与人IgG1的CH1、Cκ重组，以VH-linker-VL形式将其与原核表达载体连接，制备抗VEGFR-2抗体嵌合Fab抗体片段。该抗VEGFR-2抗体杂交瘤细胞株8H1已于2009年4月9日保藏于普通微生物菌种保藏管理中心，保藏编号为CGMCC No.3013。所述的抗VEGFR-2抗体嵌合Fab抗体片段在制备抗VEGFR-2的诊断性试剂、治疗性药物中的应用。

主权项

1、抗VEGFR-2抗体嵌合Fab片段，即鼠源性抗VEGFR-2抗体的重链和轻链可变区与人IgG1的重链部分恒定区(CH1)及轻链恒定区(Cκ)进行重组形成的融合蛋白；鼠源性抗VEGFR-2抗体的轻链可变区与人IgG1的轻链恒定区Cκ重组的嵌合L链及鼠源性抗VEGFR-2抗体的重链可变区VH与人IgG1的CH1重组的嵌合Fd段，两者中间通过前导序列pelB连接，嵌合Fd段、L链分泌到细菌周质腔时前导序列pelB被剪切，两者通过链间二硫键连接构成嵌合Fab抗体片段；嵌合L链的氨基酸序列为：Glu Leu Asp lle Val Met Thr Gln SerAla Phe Ser Asn Pro Val Thr Leu Gly Thr SerAlaSer Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr TrpTyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala SerGly Val Pro Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile SerArgVal Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala GlnAsn Leu Glu Leu Pro Leu Thr PheGly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr ValA la Ala Pro Ser Val Phe Ile Phe ProPro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe TyrPro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln GluSer Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu SerLys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser LeuPro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys ter Phe ter嵌合Fd段的氨基酸序列为：Val Lys Leu Val Glu Ser Gly Pro Gly Leu Val Gln Pro Ser、Gln Ser Leu Ser Ile Thr CysThr Val Ser Gly Phe Ser Leu Thr Ser His Gly ValHis Trp Val Arg Gln Ser Pro Gly LysGly Leu Glu Trp Leu Gly Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe IleSer Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Phe Lys Met Asn Ser LeuGln Ala Asn Asp Thr Ala Ile Tyr Tyr Cys Ala Arg Asn Arg Arg Gly Tyr Tyr Ala Met AspTyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val PhePro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val LysAsp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val HisThr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val ProSer Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser AsnThr LysVal Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr Ser Gly Gln Ala Gly Gln His HisHis His His His Gly Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser ter。