摘要
噬菌体展示技术筛选缔组织生长因子(CTGF,connective tissue growth factors)蛋白表位。用含有30 mmol/L葡萄糖的DMEM培养液干预人肾小球系膜细胞2 d后,提取CTGF天然蛋白,应用Western blot-ting方法鉴定CTGF单克隆抗体的特异性;以CTGF单克隆抗体为筛选工具,对噬菌体随机12肽库进行筛选,逐步增加选择压力,经过3轮筛选后随机挑取4个克隆测序,用竞争抑制和间接ELISA方法检测噬菌体阳性克隆的特异性。Western blotting分析显示,CTGF单克隆抗体能与CTGF天然蛋白及重组蛋白特异性结合。噬菌体文库经过3轮筛选后得到的阳性克隆,测序结果表明4个克隆的序列高度一致,推导获得小肽序列,命名为ZD521;竞争抑制和间接ELISA分析与预期结果一致。本研究用噬菌体展示技术成功获得与CTGF单抗高度结合的小肽,为CTGF功能研究以及进一步以该肽为疫苗预防和治疗纤维化疾病打下基础。
To screen antigenic epitope of CTGF, total CTGF protein was extracted from human renal mesangial cells which were cultured in Dulbecco Modified Eagle Medium（DMEM） containing 30 mmol/L glucose for 2 days. The specificity of the monoclonal antibody against CTGF was identified by the method of Western blotting using recombinant and native CTGF proteins. A 12-peptide library was screened with monoclonal antibody against CTGF by the phage display. 4 of positive clones were sequenced, which were isolated after 3 circles screening by increasing choice pressure. The specificity of positive clones was determined by the methods of indirected ELISA and competitive inhibition ELISA. The monoclonal antibody against CTGF can specially react with recombinant and native CTGF proteins. The sequence analysis of the isolated 4 clones showed that the epitopes shared a highly consensus sequence （named ZD521）. The results of ELISAs were in accordance with those expected. A peptide specially combined with anti-CTGF antibody was obtained successfully, and it is valuable for the studies of CTGF function and vaccine design in future.
出处
《药物生物技术》
CAS
CSCD
2006年第4期255-259,共5页
Pharmaceutical Biotechnology
