吉林大学学报（医学版）2016，Vol.42Issue(5)：866-871,6.DOI:10.13481/j.1671-587x.20160505
人卵巢 cDNA 文库中与蛋白激酶 Wee1B 相互作用的候选分子的筛选及其调控作用
Screening of candidate molecules interacting with protein kinase Wee1B from human ovary cDNA library and its regulation effect
摘要
Abstract
Objective:To screen the new candidate molecules interacting with protein kinase Wee1B by yeast two hybrid system, and to analyze their interaction with Wee1B in the early stage of mouse fertilized eggs by bioinformatics.Methods:The plasmid pcDNA3.1/V5-His-TOPO-Wee1B wild type encoding mouse Wee1B gene was used as template to construct bait plasmid pGBKT7 Wee1B and the bait plasmid pGBKT7-Wee1B was transformed into yeast competent cells at SD/Trp (SDO),SD/Trp/X-α-Gal (SDO/X)and SD/Trp/X α Gal/AbA plates (SDO/X/A)plates to detect the toxicity and self-activation ability of yeast and its expression in yeast using Western blotting method.The yeast cells containing pGBKT7-Wee1B were fused with human ovary cDNA library, the yeast plasmid transformation of Escherichia coli positive clones were sequenced after identified by yeast transformation.BLAST analysis was carried out in GenBank,and its effect on the development of mouse fertilized eggs was deduced according to the gene annotation.Results:The double enzyme digestion analysis and sequencing analysis results showed that the pGBKT7-Wee1B bait plasmid was successfully constructed.The plasmid was transformed into the yeast,and there were no clones in the SDO/X/A plates.The pGBKT7-Wee1B and pGBKT7 empty vectors were transformed into the yeast,the bacteria were inoculated on the SDO plates,and the clones were uniformly grown on the two SDO plates.The positive clones were picked and expanded in culture,the protein was extracted and Western blotting showed that pGBKT7 Wee1B was expressed in the yeast.The bait plasmids were fused with human ovary cDNA library and the positive clones inserted into the fragment were identified by PCR. Nine proteins which interacted with Wee1B protein kinase were screened out by sequencing and blast analysis,and the proteins which could be closely related to the development of mouse oocytes and the development of fertilized eggs were analyzed by bioinformatics analysis.Conclusion:Using the yeast two hybrid system from human ovary cDNA library,nine interacting proteins with Wee1B protein kinase are screened and these screened proteins may regulate mouse oocyte maturation and early embryo development through interacting with Wee1B.关键词
蛋白激酶/Wee1B/蛋白相互作用/酵母双杂交/小鼠受精卵Key words
protein kinase/Wee1B/protein-protein interaction/yeast two hybrid/mouse fertilized eggs分类
生物科学引用本文复制引用
刘超,任丽莉,栾治东,孟智超,刘乙蒙,肖建英..人卵巢 cDNA 文库中与蛋白激酶 Wee1B 相互作用的候选分子的筛选及其调控作用[J].吉林大学学报（医学版）,2016,42(5):866-871,6.基金项目
国家自然科学基金资助课题（81270698，31371173，81401199）；辽宁省科技厅科学技术计划项目资助课题 （）
