Kordi Shirafkan, Rahmati-Yamchi Mohammad, Asghari Vostakolaei Mehdi, Etemadie Ali, Barzegari Abolfazl, Abdolalizadeh Jalal
Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran AND Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.
Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.
Iran J Allergy Asthma Immunol. 2019 Jun 8;18(3):289-299. doi: 10.18502/ijaai.v18i3.1122.
Vascular endothelial growth factor receptor 2 (VEGFR-2) is known as one of the important antigens playing a vital role in angiogenesis. In this study, phage display technology (PDT) was used to produce a single-chain variable fragment (scFv) antibody against a region of the domain 3 in VEGFR-2 called kinase insert domain receptor 3 (KDR3). After designing the KDR3 peptide and biopanning, a colony was chosen for scFv antibody expression. Following expression and purification; western blotting, dot blotting and immunofluorescence (IF) were used to evaluate the antibody function. Surface plasmon resonance (SPR) was also employed to measure affinity of produced antibody. Once a colony was selected and transferred to the expression host, the scFv antibody was expressed in the expected range of 28 kDa. Using a designed chromatography column, antibody purification was found to be about 95%. In this study, a novel scFv with the capability of binding to KDR3 was isolated and purified and its intracellular function was investigated and verified.
血管内皮生长因子受体2（VEGFR-2）是已知在血管生成中起关键作用的重要抗原之一。在本研究中，噬菌体展示技术（PDT）被用于制备针对VEGFR-2中称为激酶插入结构域受体3（KDR3）的第3结构域区域的单链可变片段（scFv）抗体。在设计KDR3肽并进行生物淘选后，选择一个菌落用于scFv抗体表达。在表达和纯化后，使用蛋白质印迹、斑点印迹和免疫荧光（IF）来评估抗体功能。表面等离子体共振（SPR）也用于测量所产生抗体的亲和力。一旦选择一个菌落并转移到表达宿主中，scFv抗体就在预期的28 kDa范围内表达。使用设计的色谱柱，发现抗体纯化率约为95%。在本研究中，分离并纯化了一种具有结合KDR3能力的新型scFv，并对其细胞内功能进行了研究和验证。
