Objective: To identify and verify the interacting protein of α-11 giardin, so as provide the experimental evidence for studies on the α-11 giardin function.
Methods: The yeast two-hybrid cDNA library of the Giardia lambia C2 strain and the bait plasmid of α-11 giardin were constructed. All proteins interacting with α-11 giardin were screened using the yeast two-hybrid system. α-11 giardin and all screened potential interacting protein genes were constructed into pBiFc-Vc-155 and pBiFc-Vn-173 plasmids, and co-transfected into the breast cancer cell line MDA-MB-231. The interactions between α-11 giardin and interacting proteins were verified using bimolecular fluorescence complementation (BiFC).
Results: The yeast two-hybrid G. lambia cDNA library which was quantified at 2.715 × 107 colony-forming units (CFU) and the bait plasmid containing α-11 giardin gene without an autoactivation activity were constructed. Following two-round positive screening with the yeast two-hybrid system, two potential proteins interacting with α-11 giardin were screened, including eukaryotic translation initiation factor 5A (EIF5A), calmodulin-dependent protein kinase (CAMKL) and nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH), hypothetical protein 1 (GL50803_95880), hypothetical protein 2 (GL50803_87261) and a protein from Giardia canis virus. The α-11 giardin and EIF5A genes were transfected into the pBiFc-Vc-155 and pBiFc-Vn-173 plasmids using BiFC, and the recombinant plasmids pBiFc-Vc-155-α-11 and pBiFc-Vn-173-EIF5A were co-tranfected into MDA-MB-231 cells, which displayed green fluorescence under a microscope, indicating the interaction between α-11 giardin and EIF5A protein in cells.
Conclusions: The yeast two-hybrid cDNA library of the G. lambia C2 strain has been successfully constructed, and six potential protein interacting with α-11 giardin have been identified, including EIF5A that interacts with α-11 giardin in cells.
［摘要］ 目的 鉴定并验证α-11贾第素的相互作用蛋白, 为其功能研究提供实验依据。方法 构建C2株蓝氏贾第鞭毛 虫酵母双杂交cDNA文库和α-11贾第素诱饵质粒, 通过酵母双杂交筛选出可能与α-11贾第素相互作用的蛋白。将α-11 贾第素和筛选出的可能互作蛋白基因分别构建入pBiFc-Vc-155、pBiFc-Vn-173质粒, 共转染乳腺癌细胞系MDA-MB-231, 通过双分子荧光互补 (bimolecular fluorescent complementation, BiFC) 实验验证两种蛋白的相互作用。结果 构建了库容 为2.715 × 107 细菌菌落总数 (CFU) 的蓝氏贾第鞭毛虫酵母双杂交cDNA文库和含有α-11 贾第素基因的无自激活活性的 诱饵质粒。通过酵母双杂交两轮阳性筛选, 共筛选出6个可能与α-11贾第素相互作用的蛋白, 分别为真核翻译起始因子 5A (eukaryotic translation initiation factor 5A, EIF5A) 、钙调蛋白激酶 (calmodulin-dependent protein kinase, CAMKL) 、烟酰胺 腺嘌呤二核苷酸磷酸特异性谷氨酸脱氢酶 (NADP-specific glutamate dehydrogenase, NADP-GDH) 、假设蛋白1 (GL50803_95880) 、假设蛋白2 (GL50803_87261) 和一段来自犬贾第虫病毒的蛋白。成功将α-11 贾第素基因与EIF5A 基因分别转入 BiFc系统质粒pBiFc-Vc-155与pBiFc-Vn-173, 将矫正突变的pBiFc-Vc-155-α-11与pBiFc-Vn-173-EIF5A重组质粒共转染 MDA-MB-231细胞后, 镜下观察可见绿色荧光, 证实α-11贾第素与EIF5A蛋白在细胞内存在相互作用。结论 成功构建 了C2株蓝氏贾第鞭毛虫酵母双杂交文库, 并鉴定出6个α-11贾第素可能的相互作用蛋白, 其中EIF5A与α-11贾第素在 细胞内存在相互作用。.
Keywords:
Bimolecular fluorescence complementation;