酵母双杂交技术的影响因素及其实验策略

Factors effecting on Yeast Two-Hybrid experiments and their responding Strategies

目的 为了研究蛋白质分子间的相互作用 ,以大T抗原和P5 3为阳性对照 ,建立酵母双杂交实验系统。方法 通过比较共转化、顺序转化和交配实验三种方法的优劣 ,分析了影响酵母转化效率的因素 ,与此同时还分析了酵母体内 β 半乳糖苷酶的活性。 结果 ①顺序转化比共转化转化效率高 ;②质粒纯度是影响酵母转化效率的一个主要因素 ,与经典碱裂解法提取质粒相比 ,试剂盒提取质粒的转化效率明显高 ;③PEG浓度对转化效率也有一定影响 ;④β 半乳糖苷酶定量分析能够区分蛋白质分子间的强相互作用与弱相互作用 ,实验结果更可靠。结论 在此基础上 ,我们提出了一种共转化与交配实验有机结合的有效的酵母双杂交新实验策略 ,并将之成功运用于筛选NGAL(neutrophilgelatinaseassociatedlipocalin ,NGAL)相互作用蛋白实验。

Objective To study the proteins interaction, interaction between the SV40 large T antigen and the p53 protein as a positive control was established for the yeast two hybrid system. Methods Important factors influencing the yeast two hybrid experiments were probed into from the aspects of the transformation efficiencies of three methods containing the yeast simultaneous cotransformation, yeast sequence transformation and yeast mating, and the β Gal assay in yeast. Results It was found in the experiment that the transformation efficiency of the sequence transformation was better than that of the simultaneous cotransformation, and the purity of the plasmid and the concentration of the PEG were the important factors influencing the transformation efficiency. The plasmid extracted by QIAprep Spin Miniprep Kit was more fitted for the transformation than that extracted by Alkaline lysis methods. The β Gal quantitative assay was better than the β Gal qualitative assay in distinguishing between the strong proteins interaction and the weak proteins interaction and was more credible. Conclusion Based on these results, we reported a screening protocol based on the genetic method followed by a cotransformation verification, which has been used in screening a cDNA library to find some proteins interacted with the neutrophil gelatinase associated lipocalin(NGAL).