ErbB3和ErbB4特异性配体筛选系统的建立及neuregulin-1突变体的筛选

doi:10.3724/SP.J.1005.2010.01247

遗传 ›› 2010, Vol. 32 ›› Issue (12): 1247-1255.doi: 10.3724/SP.J.1005.2010.01247

ErbB3和ErbB4特异性配体筛选系统的建立及neuregulin-1突变体的筛选

戚英^{1, 2}, 魏东芝¹, 刘喜富², 周明东²

1. 华东理工大学生物工程学院生物反应器工程国家重点实验室鲁华生物技术研究所, 上海 200237;
2. 上海泽生科技开发有限公司, 上海 201203

收稿日期:2010-03-29 修回日期:2010-06-12 出版日期:2010-12-20 发布日期:2010-12-20
通讯作者: 魏东芝；周明东 E-mail:dzhwei@ecust.edu.cn；mdzhou@zensun.com

Establishment of an ErbB3- and ErbB4-specific ligand screening system, and a screen of neuregulin-1 mutants

QI Ying^{1, 2}, WEI Dong-Zhi¹, LIU Xi-Fu², ZHOU Ming-Dong²

1. Newworld Institute of Biotechnology, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China;
2. Zensun (Shanghai) Science and Technology, Ltd., Shanghai 201203, China

Received:2010-03-29 Revised:2010-06-12 Online:2010-12-20 Published:2010-12-20
Contact: WEI Dong-Zhi;ZHOU Ming-Dong2 E-mail:dzhwei@ecust.edu.cn；mdzhou@zensun.com

摘要/Abstract

摘要： Neuregulin-1(NRG1, 纽兰格林)通过活化ErbB2/ErbB4二聚体具有治疗心衰的作用, 目前已完成临床二期。为避免作为心衰治疗药物时同时激活ErbB3并产生副作用, 因此用NRG1变异体的方法寻找能对ErbB4专一性激活的配体。文章构建了带有不同筛选标记的ErbB2、ErbB3、ErbB4细胞表达质粒, 将ErbB2/ErbB3、ErbB2/ErbB4质粒共转染至CHO细胞, 建立了ErbB2/ErbB3特异性表达和ErbB2/ErbB4特异性表达的细胞株。通过与新生大鼠原代心肌细胞比较, 证明ErbB2/ErbB4细胞株信号传导功能与心肌细胞相似, NRG1可以激活下游的AKT信号途径、PI3K信号途径, 并表现出良好的剂量效应。因此可以通过检测与心肌功能密切相关的下游信号AKT磷酸化水平快速筛选抗心衰药物, 并通过与ErbB2/ErbB3信号激活水平比较鉴定其对心肌细胞的特异性。文章还构建了31个不同的NRG1突变体并在大肠杆菌中成功的表达和纯化。将这些突变体用于刺激两个细胞株, 通过检测AKT磷酸化水平, 发现这些突变体对ErbB2/ErbB3与ErbB2/ErbB4受体的激活能力不同。进一步检测其中5个ErbB2/ErbB4激活特异性发生改变的突变体与两对受体的亲和力, 发现这些突变体和ErbB2/ErbB4与ErbB2/ErbB3受体亲和力的变化有一致性。最终筛选到了4个可以更特异性激活ErbB2/ErbB4受体的突变体作为更有效治疗心衰的候选药物

关键词: 纽兰格林(NRG1)突变体, 心衰, 表皮生长因子受体家族, 稳定细胞株, 磷酸化AKT

Abstract: Neuregulin-1 (NRG1), now in a phase II clinical trial, has beneficial effects on heart failure patients through the activation of ErbB2/ErbB4 receptor pair. To decrease the side effect of NRG1 on activating ErbB3, a mutation screen was carried out to get NRG1 mutants, which have more specific binding to ErbB2/ErbB4 receptor pair. Two CHO stable cell lines were constructed, which express ErbB2/ErbB3 or ErbB2/ErbB4 receptor pair, respectively. The ErbB2/ErbB4 cell line showed similar characteristics in ligand-binding activity and the activation of downstream signaling molecules, such as the AKT and PI3K to the primary neonatal rat ventricular myocytes (NRVM), which endogenously expresses ErbB2/ErbB4. Both cell lines have good dose-response. Thirty-one NRG1 mutants were successfully expressed in Escherichia coli and purified. Their ability to stimulate the downstream signaling was measured by detecting AKT phosphorylation. Some mu-tants showed more specific activation activity in ErbB2/ErbB4 cells. Further study on five of these mutants demonstrated that the change of the activation activity is associated with that of their binding activities to ErbB2/ErbB4 and ErbB2/ErbB3. Four of the candidates are more specific ligands for ErbB2/ErbB4 activation, and thus may serve as more potent drug can-didates for heart failure.

Key words: NRG1 mutant, heart failure, stable transfected cell line, ErbB family, p-AKT

戚英，魏东芝，刘喜富，周明东. ErbB3和ErbB4特异性配体筛选系统的建立及neuregulin-1突变体的筛选[J]. 遗传, 2010, 32(12): 1247-1255.

CU Yang, WEI Dong-Zhi, LIU Chi-Fu, ZHOU Meng-Dong. Establishment of an ErbB3- and ErbB4-specific ligand screening system, and a screen of neuregulin-1 mutants[J]. HEREDITAS, 2010, 32(12): 1247-1255.

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ErbB3和ErbB4特异性配体筛选系统的建立及neuregulin-1突变体的筛选

Establishment of an ErbB3- and ErbB4-specific ligand screening system, and a screen of neuregulin-1 mutants

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