体外重组表达血管生长因子VEGF165单克隆抗体的制备及鉴定

Monoclonal antibody preparation and identification of vascular endothelial growth factor 165 expressed in vitro

目的:研制人VEGF165单克隆抗体(mAb),为研究VEGF165的生物学活性提供基础。方法:应用RT-PCR从脐静脉内皮细胞中克隆人VEGF165基因,克隆入原核表达载体pGEX-6P1中获得重组表达载体pGEX-6P1-VEGF165,转化大肠杆菌BL21,经IPTG诱导表达获得重组人VEGF165蛋白,将重组蛋白纯化后免疫BALB/c小鼠,通过杂交瘤技术,制备人VEGF165高效价的mAb。通过鸡胚血管形成抑制实验、HUVEC迁移抑制实验以及HUVEC血管形成抑制实验,对获得的人VEGF165特异性mAb进行进一步鉴定。结果:成功地从脐静脉血管内皮细胞中克隆出人VEGF165基因,并在大肠杆菌表达系统中获得高效表达,以纯化重组蛋白作为免疫原免疫小鼠,筛选获得5株分泌人VEGF165特异性mAb的杂交瘤细胞株,分别命名为5A6、3F5、6H3、7D10、7A10,其中5A6、3F5、6H3、7D10分泌的mAb亚类为IgG2a,7A10分泌的mAb亚类为IgG2b,抗体轻链均为κ链。5株mAb均能抑制鸡胚血管形成、抑制HUVEC迁移和及血管形成。结论:所获得的mAb具有效价高,活性强的优点,为抗肿瘤血管研究中进一步研究VEGF165的生物学作用提供了重要的基础。

AIM： To prepare a monoclonal antibody against human vascular endothelial growth factor （VEGF165）, for further study the VEGF165 in the tumorigenesis, tumor cell migration and the tumor cells escape from the immune response. METHODS： VEGF165 gene was cloned from the human umbilical vein endothelial cells （HUVEC） by RT-PCR, and then cloned into the pGEX-6P1, constructed the prokaryotic expression of pGEX-6P1-VEGF165. The fusion -protein of VEGF165 was expressed in E.coli （BL21） induced by the 1.0 mmol/L IPTG at 37℃ after 4 h. The fusion-protein was purified by the MicroSpin GST purification kit for immunized the BALB/c mouse. The monoclonal antibodies （mAbs） against the VEGF165 were prepared by hybridoma technique, and ELISA and Western blot identified their immunoglobulin subclass and specificity. And we used the inhibition the embryo angiogenesis assay, inhibition the HUVEC migration assay and inhibition the HUVEC tubule information assay to study the bioactivity of the mAbs of VEGF165. RESULTS： The sequence of the VEGF165 is agreed to the GenBank, and we obtained five species VEGF165 mAbs, and the titer of the antibody is high, and we named, they are 5A6, 3F5, 6H3, 7D10 and 7A10. Our study showed that the 5A6, 3F5, 6H3, 7D10 were classified to IgG2a, 7A10 was classified to IgG2b, and the light chain is κ. Meanwhile the purified mAbs inhibited formation of chicken embryo blood vessels, and inhibited tubule formation of the HUVEC and inhibited migration of the HUVEC. CONCLUSION： mAbs against human VEGF165 have the effective bioactivity, which would play a significant role for further study the mechanism of VEGF165.