人抗内毒素类脂A抗体轻、重链可变区基因的克隆  

Cloning of human VH and VL genes anti-lipid A antibody of bacterial endotoxin

作　　者：雷曼苹[1] 府伟灵[1] 陈鸣[1] 华川[1] 李晶[1] 

机构地区：[1]第三军医大学西南医院 [2]成都军区昆明总医院 [3]解放军第252医院

出　　处：《中华医院感染学杂志》1998年第1期17-18,共2页Chinese Journal of Nosocomiology

基　　金：国家自然科学基金

摘　　要：目的为了寻找一种治疗内毒素血症及其并发症较为有效的途径，本研究进行了人抗内毒素类脂Ａ轻、重链可变区基因的克隆。方法从含有内毒素抗体的淋巴细胞中提取ｍＲＮＡ，反转录为ｃＤＮＡ，再使用设计合成的人抗体轻、重链引物。结果应用ＰＣＲ技术，扩增获得人抗内毒素抗体轻、重链可变区基因，琼脂糖凝胶电泳，得到两条约３４０ｂｐ和３２５ｂｐ的强荧光带。抗体的ＶＨ和ＶＬ基因经ＭｉｃｒｏＳｐｉｎＴＭ柱纯化后，将ＶＨ和ＶＬ基因用一个编码氨基酸序列为（Ｇｌｙ４Ｓｅｒ）３的基因连接子连接，最后将此物定向克隆进载体ｐＣＡＮＴＡＢ５中。结论抗体ＤＮＡ在噬菌体中的重组获得了成功，为下一步的筛选、表达打下了基础。Objective To study treatment of sepsis caused by G- bacteria, we describe the cloning of single chain antibody of human against lipid A of bacterial endotoxin. Methods RNA was isolated fron B-lymphocytes secreting anti-lipid A antibody and the first strand DNA was synthesized by reverse transcriptase from total RNA of B-lymphocytes. Using the primers of human light and heavy chain of antibody, we amplifed the VH and VL genes with PCR in two separate reactions. The purified heavy and light chain DNA were purified by microspinTM column. The purified heavy and light chain DNA products were joined with a specially constructed linker DNA to produce a single chain FV gene(ScFV). Then the assembled antibody ScFV DNA fragment was cloned into the phagemid vector. Results The vector, containing the phagemid DNA, analyzed 5μl of this products on a 1.5% agarose gel, and observed a predominant band of 750bp. Conclusion We have got the recombinant phage antibody.

关 键 词：抗体 细菌 基因重组 克隆 内毒素血症 外科感染 

分 类 号：R63[医药卫生—外科学] R392.11[医药卫生—临床医学]