抗人整合素β3亚基单克隆抗体的制备及其抗肿瘤活性的研究  

Preparation of anti-human integrin β3 monoclonal antibody and study on its antitumor activity

作　　者：马淑花[1] 甘愉[2] 张毅[2] 朱明洁[2] 胡晶莹[2] 杨瑶琴[2] 赵新泰[2] 

机构地区：[1]复旦大学医学院 [2]上海市肿瘤研究所细胞与分子免疫学研究室,上海200032

出　　处：《细胞与分子免疫学杂志》2007年第5期453-456,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(30271221);上海市科委资助项目(04ZD19108)

摘　　要：目的制备抗人整合素β3亚基的单克隆抗体(mAb),研究其对肿瘤治疗的作用。方法用RT-PCR方法扩增人β3胞膜外基因,将其克隆至原核表达载体pQE30中,获得含β3胞膜外基因的高效表达载体pQE30-β3,将其转化大肠杆菌M15,通过诱导表达及纯化,获得β3蛋白。将此蛋白免疫BALB/c小鼠,应用B淋巴细胞杂交瘤技术制备抗β3亚基mAb,Western blot鉴定mAb的特异性。通过小鼠体内抑制黑色素瘤生长实验筛选出具有抑制肿瘤生长作用的mAb。进一步用MTT法及流式细胞术观察该mAb体外抑制人脐静脉内皮细胞(HUVEC)增殖及诱导其凋亡的作用。结果扩增获得了β3胞膜外基因,构建了β3蛋白的原核表达载体,表达纯化了β3蛋白,成功制备了8株mAb,Western blot证明了其特异性。经过体内抑瘤实验筛选出一株具有显著抑制肿瘤生长的mAb4F12。该mAb可抑制HUVEC细胞增殖并诱导其凋亡。结论成功表达了β3蛋白,并制备了有活性的mAb,抗体具有抑制肿瘤生长的作用,这可能是通过抑制血管内皮细胞的增殖和诱发凋亡实现的。AIM： To prepare the monoclonal antibody （mAb） against human integrin β3, and explore its role in tumor therapy. METHODS： Total RNA was isolated from human lymphocytes, and the DNA fragment encoding extracellular domain of human integrin β3 was amplified by RT-PCR. The integrin β3 gene was cloned into the prokaryotic expression vector pQE30, and the expression plasmid pQE30-β3 was transformed into E. coil M15. The expression of β3 protein was induced by IPTG, and the expressed β3 protein was purified by Ni-affinity agarose. BALB/c mice were immunized with the purified β3 protein, and mAb was prepared by hybridoma technique. The specificity of the mAbs was identified by Western blot. The mAbs were screened by their inhibitory effect on the tumor growth in vivo. The inhibition of the mAb to HUVEC cell growth was detected by MTT assay. The role of the mAb in inducing HUVEC apoptosis was analyzed by flow cytometry （FCM）. RESULTS： The extra-cellular gene fragment of human integrin β3 was amplified by RT-PCR, and the expression plasmid pQE30-β3 was constructed. Human integrin β3 protein was expressed and purified, and used for immunization. Eight clones of mAb against human integrin β3 were successfully prepared. The mAb 4F12 was found to inhibit tumor growth in vivo and reduce blood vessels in tumor. Furthermore, the mAb 4F12 could inhibit HUVEC growth and induce apoptosis in vitro. CONCLUSION： The human integrin β3 protein was obtained and the mAbs against it have been prepared successfully. The mAb 4F12 can inhibit tumor growth in vivo and reduce blood vessels in tumor. One of the mechanisms of the antitumor effect is inhibition of growth and induction of apoptosis of endothelial cells of blood vessels in tumor.

关 键 词：整合素Β3 单克隆抗体 肿瘤治疗 

分 类 号：R392.11[医药卫生—免疫学]