人白细胞介素24蛋白酵母双杂交诱饵载体的构建及其自激活检测

Construction of Human Interleukin-24 Yeast Two-hybrid Vector and Characterization of its Self-activation in Yeast Cells

构建人白细胞介素24蛋白酵母双杂交诱饵载体,并检测其自激活作用。采用PCR技术特异性扩增人白细胞介素24基因IL-24,将其克隆入酵母双杂交诱饵质粒pGBKT7中,通过PCR扩增、限制性酶切鉴定及序列测定,获得质粒pGBKT7-IL-24。采用PEG/L iAc法将其转入酵母AH109中,经表型筛选检测其自激活作用,同时利用对照质粒组筛选组氨酸(H is)本底表达抑制剂3-AT的合适工作浓度。结果获得了无自激活作用的诱饵载体pGBKT7-IL-24。确定了组氨酸(H is)本底表达抑制剂3-AT的最佳工作浓度。结论:诱饵质粒pGBKT7-IL-24可以用于酵母双杂交实验,为进一步运用酵母双杂交技术在人类组织cDNA文库中筛选与之相互作用的蛋白奠定了基础。

To construct a bait vector of IL-24 of Homo sapiens to evaluate its self-activation activity.Full-length of IL-24 gene was amplified by polymerase chain reaction（PCR） and confirmed by sequencing.A fragment of the gene was then subcloned into MCS of pGBKT7 vector to construct the bait vector according to the reading frame.The reconstructed bait vector pGBKT7-IL-24 was transformed into the yeast strain AH109 by PEG/LiAc method and its self-activation was tested by the phenotype assay.We selected the appropriate working concentration of 3-AT（3-amino-1,2,4-triazole） which could inhibit the background expression of his amino acid.Results：The reconstructed bait vector of pGBKT7-IL-24 would not self-activate the reporter gene.An appropriate working concentration ofhistidine（His） background expression inhibitor 3-AT was identified.Conclusion：Bait plasmid pGBKT7-IL-24 can be used in yeast two-hybrid system,which layed the foundation of using yeast two-hybrid to screen protein interaction.