神经突起因子酵母双杂交诱饵质粒构建和转化

Construction and transformation of yeast bait plasmids carrying synapse growth factor Neuritin cDNA fragment

目的构建和转化神经突起因子(Neuritin)的酵母诱饵重组质粒,为通过酵母双杂交研究Neuritin的功能及作用机制奠定基础。方法用PCR扩增neuritin全长cDNA中编码开放读框的基因片段;将该基因片段与pLex-A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-neuritin重组质粒。转化有重组质粒和pLexA空载体的2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16 h后,A600均值均为0.9。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。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 the synapse growth factor neuritin eDNA fragment. Methods Fragment of ORF of full neuritin eDNA was amplified using PCR and directly ligated to the pLexA vector. Insert-contained plasmid was confirmed by restriction analysis and DNA sequencing and then transformed into EGY48 [p8op-LacZ]yeast strain. Results Recombinant pLexA-neuritin plasmid was successfully constrcted. Two sort 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 were blue colonies） and none could survive on SD/ His/-Leu/-Ura plates. After being cultured in SD/-His/-Ura liquid medium for 16 h, the A600 of them were both 0.9 respectively. This indicated that the fusion proteins expressed by recombinant plasmids did not activate the expression of yeast reporter genes LEU2 and lacZ, and had no toxicity to yeast strain. Conclusion The bait plasmids constructed can be used to study the function of neuritin in Yeast Two-Hybrid Screen.