基于酵母双杂交技术的PRR11相互作用蛋白筛选与初步分析

Screening and preliminary analysis of PRR11 interacting proteins based on the yeast two-hybrid technique

摘要:

目的：采用酵母双杂交技术，筛选PRR11的相互作用蛋白，为深入研究其分子行为机制奠定基础。方法：构建用于酵母双杂交筛选的PRR11诱饵质粒并检测其自激活作用，根据结果进一步构建突变体获得具有较低自激活作用的突变体。对人胎脑cDNA文库进行筛选，获得初始阳性克隆。通过LacZ报告基因检测、回转验证、阳性克隆测序及BLAST序列比对分析，排除假阳性克隆和重复克隆，确定PRR11的候选相互作用蛋白。结果：PRR11具有自激活作用，构建了一个较低自激活作用的PRR11突变体。通过酵母双杂交筛选，获得102个初始阳性克隆转化子。经LacZ报告基因检测，将阳性克隆锁定至39个。通过测序和序列比对分析，证实这39个克隆属于编码15种不同蛋白的基因。最终有RNF41等4个蛋白通过回转验证。结论：RNF41、SCG5、BCYRN1和PRR13是PRR11的潜在相互作用蛋白。

Abstract:

Objective：To screen PRR11 interacting proteins by the yeast two-hybrid technique，and to provide a basis for further stud－ies of its molecular mechanism of action. Methods：PRR11 bait plasmids were made for the yeast two-hybrid system and tested for auto-activation. Mutants were constructed to obtain lower auto-activation. The human fetal brain cDNA library was screened to obtain initial positive clones. The lacZ reporter gene assay，retransformation，positive clone sequencing，and BLAST sequence alignment analysis were used to rule out false positive and repeated clones and identify candidate PRR11 interacting proteins. Results：PRR11 showed auto-activation and a PRR11 mutant with lower auto-activation was constructed. The yeast two-hybrid screening resulted in 102 initial positive clones. The lacZ reporter gene assay further validated 39 positive clones. According to gene sequencing and se－quence alignment analysis，the 39 clones were from genes encoding 15 different proteins. Finally，four candidate proteins including RNF41 were verified by retransformation. Conclusion：RNF41，SCG5，BCYRN1，and PRR13 are potential PRR11 interacting proteins.