BDNF的酵母双杂交诱饵载体的构建和初步鉴定

Construction and transformation of yeast bait plasmids carrying BDNF cDNA fragment

目的：构建和转化脑源性神经营养因子(brain-derived neurotrophic factor, BDNF)的酵母诱饵重组质粒,为通过酵母双杂交研究BDNF的功能及作用机制奠定基础。方法：用PCR扩增BDNF基因cDNA中编码完整开放阅读框的基因片段；将该基因片段与plexA载体定向重组；用酶切和测序鉴定重组质粒；将核苷酸序列正确的重组质粒转化人EGY48(p8op-LacZ)酵母菌株。结果：成功构建pLexA-BDNF重组质粒。转化有重组质粒和pLexA空载体的两种EGY48(p8Op—LacZ)酵母都能在SD／Gal／Raf／-His／-Ura培养基中长成白色菌落(同时,转化pLexA-pos阳性对照质粒的酵母菌在相同条件下长成蓝色菌落),但都不能在SD／-His／-Leu／-Ura培养基中生长,在SD／-His／-Ura液中培养16h后,OD_(600)均值均为0.9±0.1。这表明重组质粒表达的融合蛋白没有激活LEU2和lacZ酵母报告基因表达的活性,电没有酵母毒性作用。结论：构建的诱饵重组质粒可以用于下一阶段的人胎脑cDNA文库筛选。

Objective： To construct and transform yeast bait plasmid carrying brain-derived neurotrophic factor （BDNF） cDNA fragment.Methods：Full fragment of ORF of BDNF cDNA was amplified using PCR and directly ligated to the pLexA vector.Insert-contained plasmid was confirmed by restriction endonuclease analysis and DNA sequencing and then transformed into EGY48 [p8op-LacZ] yeast strain. Results：recombinant pLexA-BDNF plasmid was successfully constructed.Two sorts of yeasts respectively transformed by recombinant plasmids and empty pLexA vector could grow white colonies on SD/Gal/Raf/-His/-Uar plates （while the yeasts transformed pLexA-pos,positive control plasmid were blue colonies） and none could survive on SD/His/-Leu/-Ura plates.After being cultured in SD/-His/-Ura liquid medium for 16h the OD600 of them were hoth 0.9±0.1 respectively.This indicated the fusion proteins expressed by recombinant plasmids did nut activate the expression of yeast reporter genes LEU2,and lacZ had no toxicity to yeast strain. Conclusion：The bait plasmids constructed can be used to study the function of BDNF in Yeast Two-Hybrid Screen,