Flt-1 Gene Cloning And Expression Of Extracellular III Preparation Of Its Monoclonal Antibody

The local invasion and metastasis of tumors are one of major causes of clinical tumor therapy failures, so the research on inhibiting tumor growth and metastasis has been one focus of intense researches about theoretical and clinical tumor therapies. In the past decades, many researches showed that tumor growth, invasion and metastasis are angiogenesis-dependent, this also promoted the studies on the realm of angiogensis. With the revealing of the mechanism of angiogensis, the notion of inhibiting tumor growth by suppressing tumor angiogensis has been verified by the increasing evidence from theoretical and clinical researches.Angiogenesis is a tightly regulated multiple-step process that plays a key role in the growth and metastasis of tumors, including the endothelial cell differentiation, proliferation, migration and remodeling. Vascular endothelial growth factor (VEGF) is one of the most potent and specific positive regulator of angiogenesis both in the solid tumors and hematological malignancies. VEGF receptor-1 (fms-like tyrosine kinase, Flt-1/VEGFR1) is one of the VEGFRs, which has high affinity with VEGF. VEGFR1 is expressed on many cells, not only on vascular endothelial cell, glomerular membrane cell, placenta tissues and monocyte-macrophages, but also highly expressed on many solid tumor cells and many hematopoietic tumor cells. Now it is believed that Flt-1 has dual functions during the angiogenesis, one is physiological, the other is pathological. In early embryogenesis, Flt-1^-/-mice die from vascular disorganization, but during adult angiogenesis Flt-1 can enhance vessel permeability, activate extravasation and stimulate the chemotaxis of monocyte-macrophages. Flt-1 also stimulates stem-cell recruitment by interacting with PIGF. So tumor angiogenesis can be potentially inhibited by blocking the interaction between VEGF and Flt-1.Belonging to the family of receptor tyrosine kinase, Flt-1 is a high-affinity receptor for VEGF. The receptor consists of seven extracellular immunoglobulin(lg)-like domains followed by a membrane-spanning domain and a conserved intracellular tyrosine kinase domain interrupted by a kinase insertion sequence. Among the seven extracellular Ig-like domains, I -III is sufficient to interact with VEGF. We aimed to express extracellular III domain of Flt-1, analyze its biological activity, produce the monoclonal antibody (McAb) against extracellular IIIdomains of Flt-1, determine its biological activity and prepare for the engineering antibody. In our test, total RNA was extracted from human umbilical vein endothelial cell (HUVEC), and RT-PCR was used to clone cDNA fragment of extracellular domain III of Flt-1, and the expression vector of Flt-1 was constructed by cloning Flt-1 EU DNA fragment into the expression vector pAYZ. Flt-1 DI was induced to express in E.coli 16c9. The fusion protein was expressed soluble in periplasmic space, which was purified by E-tag affinity chromatography. BCA assay showed that the yield of the soluble fusion protein is about 1.10mg/L Enzyme-Linked Immunosorbent Assay (ELISA) revealed that the Flt-1 domain III was able to bind VEGF₁₆₅ dose-dependently. Monolayer denudation assay and Transwell assay showed that the fusion protein could inhibit HUVECs migration induced by conditional medium with 50ng/ml VEGF₁₆₅ and 100ng/ml bFGF.After purification, the fusion protein was used to immunize Balb/c mice. hybridoma cell lines secreting monoclonal antibodies against Flt-1 IIIwere screened by cell fusion and subcloning approaches. ELISA and fluorescence-activated cell sorting (FACS) was used to select the positive clone and FACS and Western blot was used to identify the specific affinity of McAb. We have obtained two hybridomal cells, which can steadily secrete the McAb against Flt-1 III and have proved that the McAb has high affinity to K562/A02.