[发明专利]抗日本血吸虫单抗NP11-4嵌合Fab抗体及其制备方法、应用

摘要

本发明抗日本血吸虫单抗NP11-4嵌合Fab抗体及其制备方法、应用涉及基因工程技术及制备血吸虫病治疗药物领域。应用定位于日本血吸虫成虫表膜、尾蚴膜、童虫膜、虫卵卵壳及卵内毛蚴膜的单抗NP11-4，利用基因工程技术提取杂交瘤细胞株总RNA，RT-PCR方法扩增V_H、V_L基因，将鼠源V_H、V_L分别与人IgG₁的C_H1、C_L通过PCR扩增Fd及全长L，再利用重叠延伸PCR以Fd和L基因为模板扩增嵌合Fab基因，将嵌合Fab基因与载体pComb3XSS酶切、连接，克隆至大肠杆菌Top10F’，诱导目的蛋白可溶性表达。表达的抗日本血吸虫单克隆抗体NP11-4嵌合Fab抗体N端从第1个氨基酸到第215个氨基酸为嵌合Fab抗体的L链，从第216个氨基酸到第488个氨基酸为嵌合Fab抗体的Fd段，两者之间由二硫键相连，嵌合Fab抗体全长488个氨基酸。

主权项

1、一种抗日本血吸虫单抗NP11-4嵌合Fab抗体，即抗日本血吸虫单克隆抗体NP11-4嵌合Fab抗体N端从第1个氨基酸到第215个氨基酸为嵌合Fab抗体的全长L链，从第216个氨基酸到第488个氨基酸为嵌合Fab抗体的Fd段，两者之间由二硫键连接，全长488个氨基酸，其氨基酸序列为：Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Gln Val Ser Ile Ser Cys ArgSer Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln LysPro Gly Gln Ser Pro Lys Pro Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val ProAsp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val GluAla Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly Ser His Val Pro Tyr Thr Phe GlyGly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro ProSer 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 GlnGlu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu ThrLeu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln GlyLeu Ser Leu Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Lys Tyr Leu Leu Pro ThrAla Ala Ala Gly Leu Leu Leu Leu Ala Ala Gln Pro Ala Met Ala Leu Glu Val GlnLeu Gln Gln Ser Gly Pro Glu Leu Val Arg Pro Gly Ser Ser Val Lys Ile Ser Cys LysAla Ser Gly Tyr Thr Phe Thr Asn Phe Trp Leu Gly Trp Val Lys Gln Arg Pro Gly HisGly Leu Glu Trp Ile Gly Asp Ile Tyr Pro Gly Gly Val Tyr Ile Asn Tyr Asn Glu LysPhe Lys Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr Met Gln LeuSer Ser Leu Thr Ser Glu Asp Val Phe Val Tyr Phe Cys Ala Arg Val Gly Tyr Asp GlySer Ser Gln Ser Leu Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Ser ThrLys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Lys Thr Ser Gly Gly Thr AlaAla Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn SerGly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu TyrSer Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile CysAsn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser CysAsp Lys Thr Ser Gly Gln Ala Gly Gln His His His His His His Gly Ala Tyr Pro TyrAsp Val Pro Asp Tyr Ala Ser。