FXR2酵母双杂交饵载体的构建及酵母菌转化

Construction and Transformation of Yeast Two-hybrid System “Bait” of FXR2

目的构建FXR2酵母双杂交系统的“饵”质粒,并转化酵母菌,为筛选FXR2P互作蛋白作基础。方法提取pMD18-T-FXR2,酶切后将FXR2基因连接到MATCHMAKERLexATwo-HybridSystem中的plexA质粒上,得到“诱饵”(Bait)-plexA-FXR2,导入大肠杆菌扩增,筛选阳性克隆并提取重组Bait质粒,再转入酵母菌EGY48(p8op-lacZ)。结果通过遗传学方法筛选得到构建成功的plexA-FXR2;通过营养缺陷筛选,证明Bait重组质粒转入酵母菌中。结论成功构建Bait质粒并使酵母菌转化。

Objective Fragile X syndrome is a form of hereditary mental retardation, usually results from lack of expression of the FMR1 gene (fragile X mental retardation 1).There are two autosomal homologues of FMR1, called FXR1 and FXR2 (fragile X-related gene 1 and 2) .In this paper we aim to construct bait of yeast two-hybrid system,and then transform it into yeast in order to screen the interactive protein with FXR2P. Methods Distill pMD18-T vector.,then slice off FXR2 from pMD18-T -FXR2 and clone it into plexA of MATCHMAKER LexA Two-Hybrid System. After getting (Bait)- plexA- FXR2,amplify it through transforming into E.coli.screen for positive clones and distill recombined Bait plasmid.at the last transform into yeast. Results FXR2 was confirmed had been constructed into expressing vector plex a successfully by genetics means;It is proved that Bait has been transformed into yeast successifully through nutrition absence substrate. Conclusions Bait has been constructed and transformed into yeast.