长双歧杆菌抗毒素基因mazE细菌双杂交诱饵质粒的构建与鉴定

Construction and identification of a bacterial two -hybrid bait plasmid carrying mazE from Bifutobacterium longum

目的构建细菌双杂交系统中的诱饵载体pBT-mazE，为长双歧杆菌的压力调控机制研究奠定基础。方法PCR扩增抗毒素蛋白MazE的编码基因，插入到带有XcI基因的载体pBT构建诱饵质粒pBT-mazE。将pBT-mazE转化至大肠杆菌报告菌株XL-1 BlueMRF’Kan，检测诱饵蛋白maze-λcI的表达。将pBT-mazE和pTRG空载体共转化报告菌株XL-1 BlueMRF’Kan，通过3-氨基-1，2，4-三氮唑（3-amino-1，2，4-triazole，3-AT）选择性筛选平板检测诱饵质粒的自激活作用。结果酶切和测序结果显示MazE蛋白编码序列成功克隆入载体pBT，融合区读码框正确。诱饵质粒pBT-mazE能够表达诱饵蛋白maze-λcI。pBT-mazE和pTRG空载体共转化的报告菌株XL-1 BlueMRF’Kan在含有3-AT的选择性筛选平板上无菌落生成，而在no3-AT非选择性筛选平板上生长良好，表明诱饵质粒pBT-mazE在细菌双杂交系统中无自激活作用。结论所构建的诱饵载体pBT-mazE可以用于细菌双杂交的进一步实验。

Objective To construct a bait vector pBT - mazE in a bacterial two - hybrid system for research on the mechanism of stress regulation about Bifidobacterium longum. Methods The coding gene of MazE was amplified by PCR, which was then inserted into a vector pBT carrying XcI gene to establish the bait plasmid pBT - mazE. The ob- tained plasmid was transformed into a reporter strain of E. coli XL - 1 Blue MRF＇Kan and the level of the bait protein MazE -λcI was detected. Then, the resultant reporter strain co - transferred with both pBT - mazE and an empty vector pTRG was screened using the selective screening medium plates containing 3 - amino - 1,2,4 - triazole （3 - AT）. Re- suits According to enzymatic digestion and sequencing, the coding sequence of MazE was successfully cloned into the vector pBT with a right reading frame. The bait protein MazE -λcI can be expressed by the bait plasmid pBT - mazE. No reporter strain of XL - 1 Blue MRF＇Kan co - transferred with both pBT - mazE and pTRG vectors could grow on the selec- tive screening medium plates containing 3 - AT, while colonies were obtained on the selective screening medium plates without 3 - AT. It indicated that the bait plasmid pBT - mazE could not self - activate in a bacterial two - hybrid system. Conclusion The resultant bait vector pBT - maze can be used for further experiments in a bacterial two - hybrid sys- tem.