人FL在大肠杆菌中的表达及表达产物的纯化和生物学活性测定  

Expression of human FL in E.coli and the purification as well as bioassay of its expression product

作　　者：张友鸿[1] 周希亚[1] 陈松森[1] 娄可佳 刘深基[1] 杨克恭[1] 何维[1] 

机构地区：[1]中国医学科学院中国协和医科大学基础医学研究所,北京100005

出　　处：《基础医学与临床》2002年第2期125-129,共5页Basic and Clinical Medicine

摘　　要：建立人FL(humanflt3ligand ,hFL)在大肠杆菌中的高效表达系统 ,分离纯化重组hFL为其功能和应用研究打下基础。根据大肠杆菌偏爱密码子人工合成hFL基因膜外区DNA片段 ,由PCR方法获得的hFL基因膜外区DNA片段 ,经测序证明序列正确。构建表达载体pET30a Trx hFL并转化大肠杆菌BL2 1(DE3) ,IPTG诱导表达 ,hFL融合蛋白的表达占菌体总蛋白的 6 0 %以上 ,并以包涵体形式存在。融合蛋白经离子交换层析、分子筛层析纯化 ,并用FXa切割 ,切割效率 >80 %。切割产物经亲和层析纯化。纯化产物进行Westernblot鉴定。纯化的rhFL(recombinanthFL)与GM CSF及TNFα联合作用 ,具有良好的刺激DC增殖活性 ,其增殖能力是GM CSF +TNFα的 2 5倍左右。本文以大肠杆菌为宿主 ,成功地表达了融合蛋白Trx hFL。经纯化的rhFL在体外与GM CSF及TNFα联合使用能有效地刺激人DC的增殖。Human FL(flt3 ligand) is expressed in E.coli for clinical application and functional study of hFL. An artificial DNA fragment encoding FL outmemberance region was synthesized by using favored genetic codons in E.coli. The sequence of hFL DNA fragment prepared by PCR is confirmed to be correct. Recombinant expressing vector pET30a-Trx-hFL was transformated into E.coli BL21(DE3). The yield of Trx-hFL is over 60% of total cellular protein and expressed as inclusion bodies. Trx-hFL was purified with anion-exchange and gel filtration. The fusion protein was cleavaged by FXa with cleavage efficiency over 80%. rhFL was purified by affinity chromatography. The purified protein was identified by Western blot. The biological activity of rhFL in combination with GM-CSF and TNFα was examined by stimulating proliferation of dendritic cells from cord blood. The purified rhFL has biological activity in stimulating dendritic cell proliferation in combination with GM-CSF and TNFα in vitro.

关 键 词：大肠杆菌 FL 融合表达 FXa裂解 纯化 

分 类 号：R341[医药卫生—基础医学]