目的:在酵母细胞中构建LRP16诱饵质粒,以期从人cDNA文库中钓取可与LRP16相互作用的蛋白。方法:pLPC-LRP16质粒经酶切、琼脂糖凝胶电泳、回收,获得LRP16的基因片段,将其连接至酵母双杂交系统诱饵载体pGBKT7中,经酶切验证其正确插入方向后,将重组诱饵质粒转化酵母菌AH109和Y187,并检测其在酵母中有无自激活作用和毒性。随后提取转化后的酵母总蛋白,经Westernblot检测诱饵质粒在酵母中的表达情况,以鉴定其作为诱饵蛋白的可行性。结果:重组诱饵质粒经酶切验证,片段大小和插入方向均正确,将其转化人酵母菌后无自激活作用和毒性,Westernblot证实在酵母中重组诱饵质粒可正确表达人LRP16蛋白。结论:构建的重组质粒pGBKT7-LRP16能够在酵母细胞中正确表达,可作为酵母双杂交系统的诱饵质粒。
Objective: To construct a bait plasmid of LRP16 gene in yeast cells for probing the proteins that could interact with LRP16 by screening in human cDNA library. Methods: The LRP16 fragment that could encode full-length protein sequence was recovered by restriction digestion from pLPC-LRP16 plasmid, and subsequent electrophoresis and reclamation. The fragment was inserted into bait vector pGBKT7 of yeast two-hybrid system at the EcoR I sites. Following confirming the correct insertion di- rection by the appropriate restriction enzymes, the recombined construct was transformed into yeast cells AH109 and Y187, re- spectively. The toxicity and the self-activating transcriptional activation of human LRP16 protein in these two yeast cells were firstly detected. The ectopic expression of human LRP16 protein in the total yeast protein extracts was also measured by western blot analysis. Results: The insertion direction and size of human LRP16 in the recombined plasmid were both correct, the toxicity and self-activating transcriptional activation ot: human LRP16 protein were not observed in yeast cells. Importantly, the human LRP16 protein could be effectively expressed in transformed yeast. Conclusions: The construct of pGBKTT-LRP16 can correctly encode human LRP16 protein in yeast cells, and can be used as a bait in yeast two-hybrid system.