噬菌体展示重组人淋巴毒素突变体库及受体亲和筛选  

Phage Display of Recombinant Human Lymphotoxin Mutation Libraries and Receptor Affinity Screening

作　　者：沈毅珺[1,2] 潘卫[1] 许燕 王征 杨彤 谭靖伟 吴劲松 吴芳 曹峰 刘彦君 

机构地区：[1]第二军医大学基础医学部微生物学教研室 [2]上海复旦张江生物医药股份有限公司,上海201203 [3]上海复旦张江生物医药股份有限公司

出　　处：《生物化学与生物物理进展》2005年第1期75-80,共6页Progress In Biochemistry and Biophysics

基　　金：国家高技术"863"研究发展计划资助项目(2001AA215051;2002AA2Z3309).~~

摘　　要：淋巴毒素(lymphotoxin,LT) 通过TNFR1受体传递凋亡信号,从而发挥抗肿瘤活性. 对LT的受体结合区域进行多点随机突变,利用噬菌体展示重组人淋巴毒素(rhLT) R46,S106,L130三点随机突变组合文库和S106～F110区域随机突变体库. 噬菌体库与固相化TNFR1受体进行亲和筛选,富集了能与TNFR1受体结合的rhLT突变体. 随机挑选20个单克隆噬菌体进行ELISA受体结合鉴定,其中80%的克隆与TNFR1受体特异性结合,有4个克隆与TNFR1受体的结合能力高于野生型序列的rhLT. 将这4个结合力高的突变体克隆于pET32a(+)载体,经过大肠杆菌表达和纯化操作后,检测这些突变体蛋白与TNFR1受体的结合活性和对L929细胞的杀伤活性,发现3个克隆的受体结合性质与其展示于噬菌体上时基本吻合,其中C199克隆与TNFR1的结合活性比野生型序列的rhLT提高了近30%,且其对L929细胞的杀伤活性提高了近90%. 应用噬菌体展示技术对淋巴毒素进行体外进化研究的尝试,为下一步的蛋白质结构和功能研究提供了思路,可成为大分子药物开发的有效工具.Lymphotoxin(LT) delivers the signal of apoptosis by TNFR1, sequentially develops anti-tumor activity. Several receptor binding sites of LT were mutated randomly, then the R46, S106, L130 combined site-directed random mutation library and S106-F110 region random mutation library were displayed on a filamentous phage surface. By using TNFR1 as solid phase molecule, the phage libraries were biopanned and positive mutant clones were enriched. 20 monoclonal phages were picked randomly to detect receptor binding by ELISA assay, 80% of them can bound TNFR1 specifically, and TNFR1 binding ability of 4 clones was higher than rhLT with wild type sequence. 4 mutants with high binding ability were subcloned to pET32a(+) vector, after expression in E.coli and purification process, the binding ability and the cytotoxicity in L929 mouse fibroblast cells of these mutants were examined. 3 mutants were having similar binding ability compared with monoclonal phage, the binding ability to TNFR1 and the cytotoxicity to L929 of C 199 clone have been enhanced near 30% and 90% respectively. The effort to proceed molecular evolution study of lymphotoxin in vitro by phage display not only can be very useful for the following research involved in the relation between structure and function, but also provides potent tool for macromolecule drug development.

关 键 词：淋巴毒素 随机突变 噬菌体展示 亲和筛选 

分 类 号：Q78[生物学—分子生物学]