【摘要】 构建和转化神经营养素3(neurotrophin 3,NT3)的酵母诱饵重组质粒,为通过酵母双杂交研究NT3的功能及作用机制奠定基础。用PCR扩增NT3基因cDNA中编码完整开放读框的基因片段;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入EGY48[p8op LacZ]酵母菌株。结果成功构建pLexA NT3重组质粒。转化有重组质粒和pLexA空载体的二种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,OD600均值均为0.8±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,也没有酵母毒性作用。因此,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。更多还原

【Abstract】 To construct and transform yeast bait plasmid carrying neurotrophin-3( NT3) cDNA fragment.The full fragment of ORF of NT3 cDNA was amplified using PCR and it was directly ligated to the pLexA vector. Insert-contained plasmid was confirmed by restriction endonuclease analysis and DNA sequencing. Then the vector was transformed into EGY48 yeast strain. recombinant pLexA-NT3 plasmid successfully constrcted. Two sorts of yeasts respectively transformed recombinant plasmids and empty pLexA vector could grow white colonies on SD/Gal/Raf/-His/-Ura plates(while the yeasts transformed pLexA-pos,positive control plasmid grew blue colonies) and none could survive on SD/-His/-Leu/-Ura plates. After being cultured in SD/- His/-Ura liquid medium for 16 hours, the OD600 of them were both 0.8±0.1 respectively. This indicated that the fusion proteins expressed by recombinant plasmids did not activate the expression of yeast reporter genes LEU2 and lacZ.In addition,it had no toxicity to yeast strain. The bait plasmids constructed can be used to study the function of NT3 in Yeast Two-Hybrid Screen.更多还原