免疫共沉淀筛选牛卵泡CART相互作用蛋白的研究

doi:10.11843/j.issn.0366-6964.2015.12.007

畜牧兽医学报 ›› 2015, Vol. 46 ›› Issue (12): 2169-2175.doi: 10.11843/j.issn.0366-6964.2015.12.007

免疫共沉淀筛选牛卵泡CART相互作用蛋白的研究

李鹏飞¹，孟金柱²，刘岩²，黄洋²，陈建伟²，姜晓龙²，曹霞²，姚晓磊²，赵妙妙²，吕丽华^2*

(1.山西农业大学生命科学学院，太谷 030801；2.山西农业大学动物科技学院，太谷 030801)

收稿日期:2015-03-26 出版日期:2015-12-23 发布日期:2015-12-23
通讯作者: 吕丽华，E-mail：lihualvsxau@126.com
作者简介:李鹏飞（1978-），男，山西偏关人，博士，副教授，主要从事动物生殖生理方面的研究，E-mail：adamlpf@126.com
基金资助:
国家自然科学基金（31172211）；农业部948项目（2010-Z43）；山西省横向协作与委托项目（2010HX54）；山西省回国留学人员科研资助项目（2014-重点5）；山西省科技攻关项目（20130311027-2）；山西省人事厅人才引进项目；山西农业大学引进人才博士科研启动费(2014ZZ04)；科研管理费资助重大项目和标志性成果培育项目（71060003）

Screening Proteins Interacting with Cocaine-and Amphetamine-Regulated Transcript in Bovine Follicles by Co-immunoprecipitation

LI Peng-fei¹，MENG Jin-zhu²，LIU Yan²，HUANG Yang²，CHEN Jian-wei²，JIANG Xiao-long²，CAO Xia²，YAO Xiao-lei²，ZHAO Miao-miao²，LÜ Li-hua^2*

（1.College of Life Science，Shanxi Agricultural University，Taigu 030801，China；2.College of Animal Science and Veterinary Medicine，Shanxi Agricultural University，Taigu 030801，China）

Received:2015-03-26 Online:2015-12-23 Published:2015-12-23

摘要/Abstract

摘要：

旨在研究牛卵泡发育过程中可卡因-苯丙胺调节转录肽（Cocaine-and amphetamine-regulated transcript，CART）相互作用蛋白（Protein-protein interaction，PPI）。选择4头10月龄同期发情的健康母牛，B超检测卵泡达8~12 mm，屠宰并采集、分离卵泡颗粒细胞（Granulosa cells，GCs）后，进行总蛋白提取，兔抗CART一抗沉淀CART及其相互作用蛋白，经Protein G-Agarose磁珠吸附分离，多次洗涤去除盐分和杂蛋白，洗脱蛋白复合体并酶解，LC-ESI-Q-TOF质谱分析(Mass spectrometry，MS)及数据库比对。结果显示：共鉴定蛋白质组分111个；经Cytoscape V3.1.0分析，有81个蛋白符合功能模块，并构建蛋白相互作用网络图；同时对相互作用蛋白进行功能富集性分析，共分为3大类34组：其中分子功能占11.73%，生物学过程占46.93%，细胞组分占41.34%。α2巨球蛋白（A2M）和波形蛋白（VIM）与CART相互作用的相关系数最大； CART与NALP1、 SERPINH1和PDIA6具有负调控应答的功能，提示可能参与牛卵泡发育过程的调控。

Abstract:

The study is focused on screening the proteins interacting with cocaine-and amphetamine-regulated transcript(CART) during follicle development in bovine.Four 10-month-old healthy cows，treated for estrus synchronization，were monitored by B-ultrasonic machine to find the ovarian follicles with the diameter of 8-12 mm，which were collected followed by cows being slaughtered.The granulosa cells(GCs) were isolated from those ovarian follicles and then total protein was extracted.CART and the binding protein were deposited by primary antibodies(Rabbit anti CART) using Protein G-Agarose magnetic beads，followed by salt and miscellaneous protein being removed by washing repeatedly.Protein complexes was analyzed by LC-ESI-Q-TOF mass spectrometry(MS) and alignment followed by being eluted and digested by trypsin.Results showed that 111 proteins were identified，in which 81 proteins were functional in conformity with the interaction network modules by Cytoscape V3.1.0 analysis.For the function enrichment analysis，those 81 proteins were classified into 34 groups in 3 categories：molecular function(11.73%)，biological processes(46.93%)，cell component(41.34%).The correlation coefficient of the interaction between A2M，VIM and CART was maximum.It was also found CART was negatively regulated by NALP1，SERPINH1 and PDIA6，which suggested that they might participate in regulating follicle development in bovine.

中图分类号:

S823.2

李鹏飞，孟金柱，刘岩，黄洋，陈建伟，姜晓龙，曹霞，姚晓磊，赵妙妙，吕丽华. 免疫共沉淀筛选牛卵泡CART相互作用蛋白的研究[J]. 畜牧兽医学报, 2015, 46(12): 2169-2175.

LI Peng-fei，MENG Jin-zhu，LIU Yan，HUANG Yang，CHEN Jian-wei，JIANG Xiao-long，CAO Xia，YAO Xiao-lei，ZHAO Miao-miao，Lü Li-hua. Screening Proteins Interacting with Cocaine-and Amphetamine-Regulated Transcript in Bovine Follicles by Co-immunoprecipitation[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(12): 2169-2175.

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免疫共沉淀筛选牛卵泡CART相互作用蛋白的研究

Screening Proteins Interacting with Cocaine-and Amphetamine-Regulated Transcript in Bovine Follicles by Co-immunoprecipitation

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