拟南芥转录因子TDF1的原核表达及多克隆抗体制备

Prokaryotic expression of the arabidopsis transcription factor TDF1 and preparation of its polyclonal antibodies

在拟南芥花药发育过程中,MYB家族转录因子TDF1在调控绒毡层发育及后期功能上起到关键的作用.利用PCR方法扩增TDF1基因并克隆到原核表达载体pET-32a.将表达载体TDF1-pET32a转入大肠杆菌BL21(DE3),用IPTG成功诱导表达了分子量约为56KD的TDF1融合蛋白.该融合蛋白主要以包涵体形式存在,分离出包涵体后进行可溶性处理作为抗原免疫家兔.制备出的多克隆抗血清经ELISA测定效价为1∶2560,Western blot检测表明该抗血清与TDF1融合蛋白识别良好.TDF1抗体的制备有助于进一步从生化水平上研究TDF1在花药发育中的功能.

In Arabidopsis,TDF1 encodes a MYB transcription factor which plays a vital role in tapetal development and function during anther development. In this study, the TDF1 gene was obtained by PCR method and cloned into the expression vector pET- 32a. After the prokaryotic expression vector ＇TDF1-pET32a＇ introduced into E. coli BL21 （DE3）, the TDF1 recombinant protein of 56.2 KD was expressed by induction with IPTG. The fusion protein majorly existed in the inclusion body. After extracted from the inclusion body, the TDF1 protein was used as the antigen to immune rabbits. ELISA assay showed that the titer of the prepared polyelonal antibody was 1 ： 2560. The immunological specificity of the polyclonal antibody to TDF1 was confirmed by Western blot analysis. The prepared antibody in this work should facilitate the further functional investigation of TDF1 in biochemical levels in Arabidopsis anther development.