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

doi:10.13481/j.1671-587x.20160505

吉林大学学报(医学版) ›› 2016, Vol. 42 ›› Issue (05): 866-871.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

刘超¹, 任丽莉², 栾治东¹, 孟智超³, 刘乙蒙¹, 肖建英⁴

1. 锦州医科大学基础医学院发育生物学教研室, 辽宁锦州 121001;
2. 锦州医科大学基础医学院神经生物学教研室, 辽宁锦州 121001;
3. 锦州医科大学基础医学实验教学中心, 辽宁锦州 121001;
4. 锦州医科大学教务处, 辽宁锦州 121001

收稿日期:2016-02-22 出版日期:2016-09-28 发布日期:2016-09-29
通讯作者: 肖建英,教授,硕士研究生导师(Tel:0416-4673371,E-mail:xiaojianying@lnmu.edu.cn) E-mail:xiaojianying@lnmu.edu.cn
作者简介:刘超(1978-),女,辽宁省铁岭市人,副教授,理学博士,主要从事生殖生理学方面的研究。
基金资助:
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（2015020697）

Screening of candidate molecules interacting with protein kinase Wee1B from human ovary cDNA library and its regulation effect

LIU Chao¹, REN Lili², LUAN Zhidong¹, MENG Zhichao³, LIU Yimeng¹, XIAO Jianying⁴

1. Department of Developmental Biology, School of Basci Medical Sciences, Jinzhou Medical University, Jinzhou 121001, China;
2. Department of Neurobiology, School of Basic Medical Sciences, Jinzhou Medical University, Jinzhou 121001, China;
3. Experimental Teaching Center of Basic Medical Sciences, Jinzhou Medical University, Jinzhou 121001, China;
4. Deparment of Teaching Affairs, Jinzhou Medical University, Jinzhou 121001, China

Received:2016-02-22 Online:2016-09-28 Published:2016-09-29

摘要/Abstract

摘要：

目的：利用酵母双杂交系统筛选与蛋白激酶Wee1B相互作用的新候选分子，并用生物信息学方法分析其与Wee1B相互作用是否能调控小鼠受精卵早期发育。方法：以pcDNA3.1/V5 His TOPO Wee1B WT质粒为模板构建pGBKT7 Wee1B诱饵质粒；制备酵母感受态细胞，将诱饵质粒pGBKT7 Wee1B转化酵母感受态细胞后分别接种SD/Trp（SDO）、SD/Trp/X α Gal（SDO/X）和SD/Trp/X α Gal/AbA plates（SDO/X/A）培养板检测其对酵母的毒性和自身激活能力，利用Western blotting法检测pGBKT7 Wee1B在酵母中的表达情况；将含有pGBKT7 Wee1B的酵母感受态细胞与人卵巢cDNA文库相融合，利用酵母双杂交系统筛选出阳性克隆，提取阳性克隆的酵母质粒转化大肠杆菌后进行测序鉴定，最终再经酵母重新转化验证。在GenBank中对其进行BLAST分析，根据基因注释推断其在小鼠受精卵发育过程中可能发挥的作用。结果：双酶切鉴定和测序分析，pGBKT7 Wee1B诱饵质粒构建成功。将该质粒转入酵母感受态细胞后在SDO/X/A平皿上无克隆。将pGBKT7 Wee1B和pGBKT7空载体转入酵母感受态细胞中，菌液接种于SDO平皿，2个SDO平皿上克隆都均匀生长。从工作板SDO和阳性对照板上挑取阳性克隆扩大培养，裂解细胞提取蛋白，Western blotting法检测示pGBKT7 Wee1B对应位置出现条带。含诱饵质粒的酵母菌与人卵巢cDNA文库融合，PCR验证融合后的阳性克隆质粒有插入片段。将质粒测序并经BLAST分析筛选出9个与Wee1B蛋白激酶存在相互作用的蛋白，生物信息学分析可能与小鼠卵母细胞发育和受精卵发育密切相关的蛋白。结论：利用酵母双杂交系统从人卵巢cDNA文库中筛选出9个与Wee1B蛋白激酶相互作用的蛋白，这些筛选蛋白可能通过与Wee1B相互作用调控小鼠卵母细胞成熟和受精卵早期发育。

关键词: 蛋白激酶, Wee1B, 蛋白相互作用, 酵母双杂交, 小鼠受精卵

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.

Key words: protein kinase, Wee1B, protein-protein interaction, yeast two hybrid, mouse fertilized eggs

中图分类号:

Q132

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(05): 866-871.

LIU Chao, REN Lili, LUAN Zhidong, MENG Zhichao, LIU Yimeng, XIAO Jianying. Screening of candidate molecules interacting with protein kinase Wee1B from human ovary cDNA library and its regulation effect[J]. Journal of Jilin University Medicine Edition, 2016, 42(05): 866-871.

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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

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