噬菌体抗体库的构建及人源抗内毒素类脂A抗体的筛选  

Study of anti-lipid A antibodies of bacterial endotoxin from phage antibody library

作　　者：韩晓芳[1] 王希良[2] 郭力军[1] 冯笑梅[1] 赵建平[1] 杜英[1] 高玉明[1] 

机构地区：[1]内蒙古自治区医院,呼和浩特010017 [2]重庆第三军医大学全军免疫研究所

出　　处：《中华传染病杂志》2001年第4期236-239,共4页Chinese Journal of Infectious Diseases

基　　金：内蒙古自然科学基金资助项目 ( 97130 3 3)

摘　　要：目的　寻找一种治疗内毒素血症及其并发症较为有效的途径。方法　从感染革兰阴性杆菌J7患者体内含有内毒素类脂A抗体的淋巴细胞中提取mRNA ,经反转录再从IgG重链Fd两端及轻链通用引物分别扩增Fab基因片段 ,依次插入到噬菌体载体 pCOMB3中 ,电穿孔转入大肠杆菌XL1 blue ,经辅助病毒VCSM13感染 ,重组噬菌体溶源裂解。结果　Fab抗体表达于噬菌体CPⅢ的N端 ,噬菌体抗体库的容量为 4 .8× 10 6 ,筛选出抗内毒素类脂A抗体 ,经LPS蛋白对噬菌体抗体库进行3轮淘选 ,使抗内毒素类脂A的特异性抗体得到 10 0倍的富集。结论　通过直接和竞争ELISA实验筛选出 3株结合活性好的特异性抗体 ,为下一步应用研究奠定了基础。Objective To study the treatment of sepsis caused by G - bacteria, anti-lipid A antibodies of bacterial endotoxin were screened from phage antibody library. Methods The mRNA was extracted from human B-lymphocytes against lipid A of bacterial endotoxin, reversely transcripted and amplified by polymerase chain reaction using general primers scanning Fd and light chain of IgG. The amplified fragments were inserted into pCOMB3 vector and electrotransfected competent E.coli XL 1-blue cells. Furthermore, the recombinant phage was lysed by coculture with helper VCSM13. Results Fab displayed on the surface as fusion protein with the N terminal of coat protein Ⅲ, and 4.8×10 6 clone library was established. Antibodies against lipid A of bacterial endotoxin were screened. Specific antibodies against lipid A of bacterial endotoxin were enriched by 100 times after three rounds of panning with lipid A.Conclusions Three clones exhibited specific binding to lipid A is identified by direct and competitive ELISA methods. The succcess of isolating anti-lipid A proves the usefulness of phage display system in human McAb preparation. The result shows that we have got the recombinant phage antibody.

关 键 词：噬菌体抗体库 内毒素 FAB抗体 脓毒血症 内毒素血症 

分 类 号：R392-33[医药卫生—免疫学]