人神经菌毛素1 b结构域单克隆抗体的制备及其抑制血管生成作用

Generation of monoclonal antibodies against human neuropilin 1 b domain and its inhibitory function on angiogenesis

目的:制备抗人神经菌毛素1(human neuropilin 1,Hu NRP1)b结构域单克隆抗体(monoclonal antibodies,m Ab),为靶向抑制肿瘤血管生成提供生物材料。方法:运用杂交瘤技术,将Hu NRP 1 b结构域重组蛋白(TF-Hu NRP1b)免疫BALB/c鼠,共免疫3次,取免疫鼠的脾脏细胞与sp2/0细胞进行融合,采用间接免疫荧光法(immunofluorescent assay,IFA)筛选稳定分泌抗Hu NRP1bm A,以Western blot、IFA、流式细胞术分析m Ab的特异性。以MTT法及小室迁移实验分析m Ab抑制血管生成的能力。结果:获得1株稳定分泌抗Hu NRP1bm Ab的细胞株4F11。Western blot结果表明,本研究所获得的m Ab 4F11只与重组Hu NRP1b发生反应,与对照蛋白不发生反应;IFA及流式细胞分析结果表明,所获得的m Ab能够识别肿瘤细胞MDA-MB-231、Hep G2以及人脐静脉内皮细胞表达的天然Hu NRP1蛋白;MTT法及小室迁移实验表明,所获得的m Ab 4F11具有抑制人脐静脉内皮细胞增殖、迁移的能力。结论:成功制备出抗Hu NRP1b的m Ab,该m Ab的获得将为进一步研究Hu NRP1的功能及靶向抑制肿瘤血管生成提供生物材料。

Objective：To develop monoclonal antibodies（m Ab）against human neuropilin 1（Hu NRP1）and provide useful materials to inhibit tumor angiogenesis by targeting Hu NRP1. Methods：Using lymphocyte hybridoma technique,8-week-old BALB/c mice were immunized with recombinant protein,b domain of human neuropilin 1（TF-Hu NRP1 b）derived from E. Coli,then splenocytes of the immunized mice were prepared and collected,with the addition of sp2/0 myeloma cells,all the mixed cells were fused. Positive hybridoma cells were screened by indirect immunofluorescent assay（IFA）established with native Hu NRP1 expressed by breast tumor cell line MDA-MB-231. The specificity of m Ab was identified by IFA,Western blot and flow cytometry（FCM）. The inhibitory ability of m Abs against tumor angiogenesis was identified by methyl thiazolyl tetrazolium（MTT）and chamber migration test. Results：One hybridoma cell line producing m Ab against TF-Hu NRP1 bwere obtained and named as 4 F11. Western blot analysis showed that m Abs could recognize only recombinant Hu NRP1 bnot the other proteins produced in BL21（DE3）（p Cold-Hu NRP1 b）and BL21（DE3）（p Cold）. The results of IFA and FCM showed that m Ab could recognize native Hu NRP1 expressed on human umbilical vein endothelial cells（HUVEC）,tumor cell line MDA-MB-231 and HepG2. The results of MTT assay and chamber migration test further showed that m Ab 4 F11 had strong inhibitory effect on migration and proliferation of HUVEC. Conclusion：m Ab against Hu NRP1 bwas successfully obtained which will be useful in the functional study of Hu NRP1 and provide good materials for the inhibition of tumor angiogenesis.