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

作　　者：李斌元[1] 何淑雅[1] 王桂良[1] 马云[1] 孙春莉[1] 肖卫纯[1] 李洁[1] 闵凌峰[1] 彭丹妮[1] Nanbert Zhong 

机构地区：[1]南华大学生化与分子生物学研究室,湖南衡阳421001 [2]纽约州立基础研究所人类遗传研究室,美国纽约ny10314

出　　处：《南华大学学报（医学版）》2005年第3期299-302,共4页Journal of Nanhua University(Medical Edition)

基　　金：国家自然科学基金(30370795);湖南省卫生厅基金(Y02-072).

摘　　要：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.

关 键 词：脆性X综合征 FXR2基因 酵母双杂交 诱饵质粒 EGY48(p8op—lacZ) 

分 类 号：Q78[生物学—分子生物学]