Analysis of proteins that interact with nucleocapsid protein of SARS-CoV using 15-mer phage-displayed library  

作　　者：LIU ZhengXue WANG ZhanHui LIU YingLe DONG Wei QI YiPeng 

机构地区：[1]State Key Laboratory of Virology, College of Life Science, Wuhan University, Wuhan 430072, China [2]Biology Department, Chongqing Three Gorges University, Chongqing 404000, China

出　　处：《Chinese Science Bulletin》2007年第15期2072-2080,共9页

基　　金：the Science and Technology Project of Chongqing Municipal Educa- tion Commission, China (Grant No. KJ071109);the Key Project of the Ministry of Science and Technology of China (Grant No. 2003CB514120)

摘　　要：Analysis of proteins that interact with N protein of SARS-CoV using 15-mer phage-displayed library will help to explore the virus pathogenesis and to develop new drugs and vaccines against SARS. In this study,we cloned,expressed and purified N protein of SARS-CoV. This 46-kD N protein was verified by SDS-PAGE and Western-blot. Then,the peptides binding-specific to N protein were identified using 15-mer phage-displayed library. Surprisingly,all of the 89 clones from monoclonal ELISA were positive (S/N>2.1) and the result was further confirmed experimentally once again. Six N protein-binding pep-tides,designated separately as SNA1,SNA2,SNA4,SNA5,SNA9 and SNG11,were selected for se-quencing. Sequence analysis suggested that SNA5 shared approximatively 100% sequence identity to SNA4,SNA2,SNA9 and SNA1. In addition,the binding specificity of the 15-mer peptides with the SARS-CoV N protein was further demonstrated by blocking ELISA using the synthetical 15-mer peptide according to the deduced amino acid sequence of SNA5. Also,the deduced amino sequence of SNA5 was compared with proteins in translated database using the tblastx program,and the results showed that the proteins with the highest homology were Ubiquinol-cytochrome c reductase iron-sulfur sub-units (UCRI or UQCR),otherwise known as the Rieske iron-sulfur proteins (RISP). Notablely,in the 2Fe-2S redox centre of UCRI,there were 6 residues GGW(Y)F(Y)CP compatible to the residues (po-sition 2→7,GGWFCP7) of the NH2-terminal of the 15-mer peptide,which indicated higher binding specificity between the N protein of SARS-CoV and the redox centre of UCRI to some extent. Here,the possible molecular mechanisms of SARS-CoV N protein in the pathogenesis of SARS are discussed.Analysis of proteins that interact with N protein of SARS-CoV using 15-mer phage-displayed library will help to explore the virus pathogenesis and to develop new drugs and vaccines against SARS. In this study, we cloned, expressed and purified N protein of SARS-CoV. This 46-kD N protein was verified by SDS-PAGE and Western-blot. Then, the peptides binding-specific to N protein were identified using 15-mer phage-displayed library. Surprisingly, all of the 89 clones from monoclonal ELISA were positive （S/N〉2.1） and the result was further confirmed experimentally once again. Six N protein-binding peptides, designated separately as SNA1, SNA2, SNA4, SNA5, SNA9 and SNG11, were selected for sequencing. Sequence analysis suggested that SNA5 shared approximatively 100% sequence identity to SNA4, SNA2, SNA9 and SNA1. In addition, the binding specificity of the 15-mer peptides with the SARS-CoV N protein was further demonstrated by blocking ELISA using the synthetical 15-mer peptide according to the deduced amino acid sequence of SNA5. Also, the deduced amino sequence of SNA5 was compared with proteins in translated database using the tblastx program, and the results showed that the proteins with the highest homology were Ubiquinol-cytochrome c reductase iron-sulfur subunits （UCRI or UQCR）, otherwise known as the Rieske iron-sulfur proteins （RISP）. Notablely, in the [2Fe-2S] redox centre of UCRI, there were 6 residues [GGW（Y）F（Y）CP] compatible to the residues （position 2→7, GGWFCP7） of the NH2-terminal of the 15-mer peptide, which indicated higher binding specificity between the N protein of SARS-CoV and the redox centre of UCRI to some extent. Here, the possible molecular mechanisms of SARS-CoV N protein in the pathogenesis of SARS are discussed.

关 键 词：非典型肺炎 核蛋白壳 抗菌素 细胞凋亡 发病机制 

分 类 号：R563.1[医药卫生—呼吸系统]