论文部分内容阅读
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。
Objective: To develop human monoclonal antibody against human VEGF165 (mAb) and provide the basis for the study of the biological activity of VEGF165. Methods: Human VEGF165 gene was cloned from human umbilical vein endothelial cells by RT-PCR and cloned into prokaryotic expression vector pGEX-6P1 to obtain recombinant expression vector pGEX-6P1-VEGF165. The recombinant plasmid was transformed into E. coli BL21 and induced by IPTG to obtain recombinant human VEGF165 protein , BALB / c mice were immunized after the recombinant protein was purified, and the high titer of human VEGF165 mAb was prepared by the hybridoma technique. The obtained human VEGF165-specific mAb was further identified by chicken embryo angiogenesis inhibition assay, HUVEC migration inhibition assay and HUVEC angiogenesis inhibition assay. Results: Human VEGF165 gene was successfully cloned from human umbilical vein endothelial cells and expressed in E. coli expression system. The purified recombinant protein was used as immunogen to immunize mice, and 5 human VEGF165-specific mAbs Hybridoma cell lines were named 5A6,3F5,6H3,7D10,7A10, 5A6,3F5,6H3,7D10 secreted mAb subclass IgG2a, 7A10 secreted mAb subclass IgG2b, the antibody light chain are κ chain . All 5 mAbs could inhibit the formation of chicken embryo vasculature, inhibit HUVEC migration and angiogenesis. Conclusion: The obtained mAb has the advantages of high potency and strong activity, and provides an important basis for further study on the biological role of VEGF165 in anti-tumor vascular research.