启动子控制严密性对抗体库多样性影响的研究  

Influence of the tightness of promoter regulation on phage antibody library technology

作　　者：刘晓琳[1] 王刚[1] 化冰[1] 王琰[1] 

机构地区：[1]海军总医院中心实验室,北京100037

出　　处：《中华微生物学和免疫学杂志》2001年第6期623-626,共4页Chinese Journal of Microbiology and Immunology

基　　金：全军医药卫生重点课题资助项目 ( 0 1Z0 15 )

摘　　要：目的　比较阿拉伯糖 (arabinose,Ara)启动子和Lac启动子的控制严密性 ,探讨启动子控制严密性对抗体库多样性的影响 ,寻找适合构建抗体库的启动子。方法　 1 以Ara启动子和Lac启动子驱动抗HBs、TNF α及角蛋白的人Fab段 ,比较其本底表达。 2 比较 2种启动子表达载体对宿主菌生长的影响。 3 观察 2种启动子在抗体库扩增过程中对含有抗体基因的克隆重组率的影响。结果1 本底表达在Lac启动子载体远高于Ara启动子载体 ;2 经相应诱导物诱导后二者表达水平相似 ;3 相同条件下 ,含有Lac启动子的抗体表达载体的细菌与Ara启动子相比处于生长劣势。 4 在抗体库扩增过程中 ,含Lac启动子的抗体库中抗体基因克隆的重组率逐渐下降 ,而使用Ara启动子基本无变化。结论　启动子控制严密性差会导致抗体克隆重组率下降而影响抗体库的多样性 ,Ara启动子更适合于构建噬菌体抗体库。Objective To compare the tightness of Ara promoter and Lac promoter regulation, and to investigate the influence of the tightness on phage antibody library technology. Methods 1. Human anti HBsAg, TNF α and keratin Fabs were expressed with the control by Ara promoter or Lac promoter, and their background expression levels were compared. 2. The influences of the different expression vectors on the proliferation of host bacteria were examined. 3. The effects of the two promoters on phage antibody library during amplification were studied. Results 1. The background expression of Fabs with Lac promoter was obviously higher than that with Ara promoter. 2. Fab expression driven by promoter Lac and Ara was similar when induced with corresponding inducers. 3. Bacteria carrying Lac promoter based Fab expression vectors grew much slower than bacteria containing the Ara promoter counterpart. 4. During the amplification of phage antibody libraries Ab gene containing clones decreased rapidly with Lac promoter based libraries, while in Ara promoter based libraries Ab gene containing rate remained basically unchanged. Conclusion Promoter tight regulation is essential for maintaining antibody library diversity, and Ara promoter is superior than Lac promoter. [

关 键 词：抗体库 阿拉伯糖启动子 Lac启动子 

分 类 号：R392[医药卫生—免疫学]