Immune BlotAnalysis on Expression of the Mammalian Target of Rapamycin in Goat Fetal Fibroblasts with Recombinant Polyclonal Antibody

doi:10.1016/S1671-2927(00)8624

Journal of Integrative Agriculture ›› 2012, Vol. 12 ›› Issue (6): 1002-1008.DOI: 10.1016/S1671-2927(00)8624

Immune BlotAnalysis on Expression of the Mammalian Target of Rapamycin in Goat Fetal Fibroblasts with Recombinant Polyclonal Antibody

LIANGYan, WANGXiao-jing, YANGJiao-fu, HAOXi-yan, BAYin-ma, CHENXian-wei WANGZhi-gang

College of Life Science, Inner Mongolia University, Hohhot 010021, P.R.China

收稿日期:2011-02-11 出版日期:2012-06-01 发布日期:2012-07-20
通讯作者: WANG Zhi-gang, Mobile: 13190603899, E-mail: lswzg@imu.edu.cn
作者简介:LIANG Yan, Mobile: 15847152620, E-mail: xixils@163.com
基金资助:
This work supported by the National Natural Science Foundation of China (30860191), the Graduate Student Research Projects of Inner Mongolia University and Major Projects for New Varieties of Genetically Modified Organisms of China (2008ZX08008-002).

Immune BlotAnalysis on Expression of the Mammalian Target of Rapamycin in Goat Fetal Fibroblasts with Recombinant Polyclonal Antibody

LIANGYan, WANGXiao-jing, YANGJiao-fu, HAOXi-yan, BAYin-ma, CHENXian-wei WANGZhi-gang

College of Life Science, Inner Mongolia University, Hohhot 010021, P.R.China

Received:2011-02-11 Online:2012-06-01 Published:2012-07-20
Contact: WANG Zhi-gang, Mobile: 13190603899, E-mail: lswzg@imu.edu.cn
About author:LIANG Yan, Mobile: 15847152620, E-mail: xixils@163.com
Supported by:
This work supported by the National Natural Science Foundation of China (30860191), the Graduate Student Research Projects of Inner Mongolia University and Major Projects for New Varieties of Genetically Modified Organisms of China (2008ZX08008-002).

摘要/Abstract

摘要： To detect the mammalian target of rapamycin (mTOR) expressed in Cashmere goat fetal fibroblasts (GFb), mTOR gene was cloned from Inner Mongolia Cashmere goat (Capra hircus) and expressed in Escherichia coli followed by immunizing mice with the purified recombinant protein as an immunogen to produce the anti-goat mTOR recombinant polyclonal antibody. Antiserum was collected from the immunized mice after the fifth immunization and its titer was determined with enzyme-linked immunosorbent assay (ELISA). The results showed that the recombinant polyclonal antibody had a titer 1:200 000 and could react with the mTOR expressed in GFb cells with a specific and sensitive affinity. Western blot showed that mTOR expression and phospho-mTOR (Ser 2448) activity were inhibited when GFb cells were treated with CCI-779, an mTOR specific inhibitor.

关键词: prokaryotic expression, recombinant antibody, goat, mTOR, CCI-779

Abstract: To detect the mammalian target of rapamycin (mTOR) expressed in Cashmere goat fetal fibroblasts (GFb), mTOR gene was cloned from Inner Mongolia Cashmere goat (Capra hircus) and expressed in Escherichia coli followed by immunizing mice with the purified recombinant protein as an immunogen to produce the anti-goat mTOR recombinant polyclonal antibody. Antiserum was collected from the immunized mice after the fifth immunization and its titer was determined with enzyme-linked immunosorbent assay (ELISA). The results showed that the recombinant polyclonal antibody had a titer 1:200 000 and could react with the mTOR expressed in GFb cells with a specific and sensitive affinity. Western blot showed that mTOR expression and phospho-mTOR (Ser 2448) activity were inhibited when GFb cells were treated with CCI-779, an mTOR specific inhibitor.

Key words: prokaryotic expression, recombinant antibody, goat, mTOR, CCI-779

LIANGYan, WANGXiao-jing, YANGJiao-fu, HAOXi-yan, BAYin-ma, CHENXian-wei WANGZhi-gang . Immune BlotAnalysis on Expression of the Mammalian Target of Rapamycin in Goat Fetal Fibroblasts with Recombinant Polyclonal Antibody[J]. Journal of Integrative Agriculture, 2012, 12(6): 1002-1008.

LIANGYan , WANGXiao-jing , YANGJiao-fu , HAOXi-yan , BAYin-ma , CHENXian-wei WANGZhi-gang . Immune BlotAnalysis on Expression of the Mammalian Target of Rapamycin in Goat Fetal Fibroblasts with Recombinant Polyclonal Antibody[J]. Journal of Integrative Agriculture, 2012, 12(6): 1002-1008.

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Immune BlotAnalysis on Expression of the Mammalian Target of Rapamycin in Goat Fetal Fibroblasts with Recombinant Polyclonal Antibody

Immune BlotAnalysis on Expression of the Mammalian Target of Rapamycin in Goat Fetal Fibroblasts with Recombinant Polyclonal Antibody

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