应用酵母双杂交系统筛选与蛋白激酶Pkmyt1相互作用的候选分子

doi:10.16098/j.issn.0529-1356.2017.01.009

解剖学报 ›› 2017, Vol. 48 ›› Issue (1): 48-53.doi: 10.16098/j.issn.0529-1356.2017.01.009

应用酵母双杂交系统筛选与蛋白激酶Pkmyt1相互作用的候选分子

刘超¹孟智超² 栾治东¹任丽莉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; 2. 基础医学实验教学中心; 3. 神经生物学教研室; 4. 锦州医科大学教务处，辽宁锦州 121001

收稿日期:2016-07-14 修回日期:2016-09-29 出版日期:2017-02-06 发布日期:2017-02-06
通讯作者: 肖建英 E-mail:xiaojianying@lnmu.edu.cn
基金资助:
国家自然科学基金面上项目;辽宁省科学技术计划项目

Screening of candidate molecules interacting with protein kinase Pkmyt1 using yeast two hybrid technique

LIU Chao¹MENG Zhi-chao² LUAN Zhi-dong¹REN Li-li³ LIU Yi-meng¹XIAO Jian-ying^4*

1. Department of Developmental Biology; 2. Experimental Teaching Center of Basic Medicine; 3. Department of Neurobiology; 4. Teaching Affairs Department of Jinzhou Medical University, Liaoning Jinzhou 121001, China

Received:2016-07-14 Revised:2016-09-29 Online:2017-02-06 Published:2017-02-06
Contact: XIAO Jian-ying E-mail:xiaojianying@lnmu.edu.cn

摘要/Abstract

摘要：

目的蛋白激酶Pkmyt1负调控小鼠1-细胞期受精卵有丝分裂的进程，但具体调控机制还不清楚。因此，利用酵母双杂交系统从人卵巢cDNA文库中筛选与Pkmyt1相互作用的候选蛋白，为研究Pkmyt1调控小鼠受精卵早期发育提供新线索。方法以小鼠卵巢组织cDNA为模板，构建pGBKT7-Pkmyt1诱饵质粒，转化酵母感受态细胞后，分别接种于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，Western blotting法检测pGBKT7-Pkmyt1在酵母细胞中的表达。将人卵巢cDNA文库与含有pGBKT7-Pkmyt1的酵母感受态细胞融合，PCR筛选出阳性克隆，提取质粒，测序鉴定，再次检测其对酵母自激活能力，利用生物信息学分析筛选蛋白与胚胎发育的关系。结果酶切鉴定和Blast分析表明pGBKT7-Pkmyt1质粒构建成功。将该质粒转入Y2H golden中，在SDO板上克隆均匀生长，在SDO/X/A平皿上无克隆生长，Western blotting 检测Pkmyt1在酵母细胞中有表达。PCR验证融合后的阳性克隆质粒有插入片段。通过初步筛选得到182种能够与Pkmyt1相互作用的蛋白质，经回复性实验进一步验证有46个与Pkmyt1之间存在相互作用的蛋白。结论通过筛选发现46个蛋白可能通过与Pkmyt1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: Pkmyt1, 酵母双杂交, 蛋白-蛋白相互作用, 免疫印迹法, 小鼠

Abstract:

Objective Protein kinase Pkmyt1 negatively regulates the mitosis of mouse 1-cell stage fertilized eggs, but the specific mechanism is not clear. Therefore, the yeast two hybrid system was used to screen the candidate proteins interacting with Pkmyt1 from human ovary cDNA library in order to provide a new clue for the study of Pkmyt1 regulating the early development of mouse fertilized eggs. Methods Mice ovarian tissue cDNAs was used as a template to build pGBKT7-Pkmyt1 bait plasmid, and pGBKT7-Pkmyt1 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) medium to detect the toxicity and self-activation ability of yeast and detect its expression in yeast using Western blotting method. The pGBKT7-Pkmyt1 containing yeast cells was fused with human ovary cDNA library, followed by screening positive clones by PCR, plasmid extraction, DNA sequencing and detection the yeast self-activated ability again, the use of bioinformatics analysis of selected proteins and embryo development. Results The enzyme digestion analysis and Blast analysis showed that the pGBKT7-Pkmyt1 plasmid was successfully constructed. When the plasmid was transferred into golden Y2H, the SDO medium was evenly grown, and no clones were grown on the SDO/X/A plate, and the protein was detected by Western blotting to detect the expression of Pkmyt1 in the yeast cells. After PCR validation, the positive clones were inserted into fragments. 182 proteins which interact with Pkmyt1 were obtained by preliminary screening, and 46 proteins were further verified by the recovery experiment with Pkmyt1. Conclusion Totally 46 proteins were found through the selection of the interaction between Pkmyt1 and they regulated in mouse oocyte maturation and early development of fertilized eggs.

Key words: Pkmyt1, Two hybrid system, Protein-protein interaction, Western blotting, Mouse

刘超孟智超栾治东任丽莉刘乙蒙肖建英. 应用酵母双杂交系统筛选与蛋白激酶Pkmyt1相互作用的候选分子[J]. 解剖学报, 2017, 48(1): 48-53.

LIU Chao MENG Zhi-chao LUAN Zhi-dong REN Li-li LIU Yi-meng XIAO Jian-ying. Screening of candidate molecules interacting with protein kinase Pkmyt1 using yeast two hybrid technique[J]. Acta Anatomica Sinica, 2017, 48(1): 48-53.

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应用酵母双杂交系统筛选与蛋白激酶Pkmyt1相互作用的候选分子

Screening of candidate molecules interacting with protein kinase Pkmyt1 using yeast two hybrid technique

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