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

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）比较两种启动子表达载体对宿主菌生长的影响。（３）观察两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；证实（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步下降．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 under the control of 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 observed. (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 of 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 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.