雷公藤内酯醇靶蛋白的筛选及其结合的初步验证

采用噬菌体展示肽库技术筛选雷公藤内酯醇的靶蛋白,得到了一个肽段,并通过酶联免疫吸附（ELISA）和免疫共沉淀验证了该片段对雷公藤内酯醇的结合特异性。用Basic Local Alignment Search Tool（BLAST）进行序列对比后,找到了77个匹配序列,其中最为匹配的序列是人类类固醇生成因子-1（hSF-1）,因此hSF-1可能是雷公藤内酯醇的一个潜在受体。在大肠杆菌中表达纯化了hSF-1的配体结合域（LBD）,荧光光谱实验表明雷公藤内酯醇对hSF-1-LBD有荧光淬灭作用、等温滴定量热（ITC）实验表明雷公藤内酯醇与hSF-1-LBD发生焓驱动的特异性结合,表面等离子体共振（S...

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Bibliographic Details
Published in分析化学 Vol. 40; no. 3; pp. 365 - 370
Main Author 杨旭光 徐晓煜 李家璜 姚其正 朱彤阳 华子春 郑伟娟
Format Journal Article
LanguageChinese
Published 南京大学医药生物技术国家重点实验室,生命科学学院,南京210093%中国药科大学药学院,南京,210009 2012
Subjects
噬菌体随机肽库
等温滴定量热
类固醇生成因子-1
荧光光谱
雷公藤内酯醇
噬菌体随机肽库
雷公藤内酯醇
荧光光谱
类固醇生成因子-1
等温滴定量热
Online AccessGet full text
ISSN0253-3820
DOI10.3724/SP.J.1096.2012.10481

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Summary:采用噬菌体展示肽库技术筛选雷公藤内酯醇的靶蛋白,得到了一个肽段,并通过酶联免疫吸附（ELISA）和免疫共沉淀验证了该片段对雷公藤内酯醇的结合特异性。用Basic Local Alignment Search Tool（BLAST）进行序列对比后,找到了77个匹配序列,其中最为匹配的序列是人类类固醇生成因子-1（hSF-1）,因此hSF-1可能是雷公藤内酯醇的一个潜在受体。在大肠杆菌中表达纯化了hSF-1的配体结合域（LBD）,荧光光谱实验表明雷公藤内酯醇对hSF-1-LBD有荧光淬灭作用、等温滴定量热（ITC）实验表明雷公藤内酯醇与hSF-1-LBD发生焓驱动的特异性结合,表面等离子体共振（SPR）实验表明雷公藤内酯醇可以与hSF-1-LBD剂量依赖性结合,这些都证实了hSF-1与雷公藤内酯醇存在特异性相互作用。
Bibliography:YANG Xu-Guang,XU Xiao-Yu,LI Jia-Huang,YAO Qi-Zheng, ZHU Tong-Yang,HUA Zi-Chun,ZHENG Wei-Juan 1（State Key Laboratory of Pharmaceutical Biotechnology, School of Life Science,Nanjing University,Nanjing 210093,China） 2（School of Pharmacy,China Phamaceutical University,Nanjing 210009,China）
Triptolide; Human steroidogenic factor-1; Phage display; Fluorescence spectrum; Isothermal titration calorimetry; Surface plasmon resonance
22-1125/O6
Triptolide is an extract from the Chinese herb Tripterygium wilfordii Hook f.We screened out a peptide from a C7 phage display library and presumed it was a potential peptide ligand for triptolide.The specificity of the selected clone for triptolide was confirmed by ELISA and immunoprecipitation.After DNA sequencing,a BLAST search was carried out and 77 matching sequences was retrieved.The most promising candidate is human steroidogenic factor-1（hSF-1）,a member of the nuclear receptor family that controls the synthesis of steroid hormones by regulating steroidogenic enzyme genes.We
ISSN:0253-3820
DOI:10.3724/SP.J.1096.2012.10481