SARS冠状病毒未知蛋白Orf 9b的克隆、表达和结构分析  

The Cloning,Expression and Structural Analysis of Putative Unknown Protein Orf 9b in SARS-CoV

作　　者：李文[1] 刘阳[2] 兰蕾[2] 刘铭楠[2] 

机构地区：[1]首都医科大学基础医学院细胞生物学系,北京100069 [2]中国科学院生物物理研究所脑与认知研究中心,北京100101

出　　处：《病毒学报》2009年第4期274-278,共5页Chinese Journal of Virology

摘　　要：从SARS病毒基因组中克隆Orf 9b基因并构建pET32-Orf 9b原核表达质粒,在原核细胞中表达目的重组蛋白并将其纯化,经肠激酶酶切后,得到分子量为11 kD的Orf 9b蛋白。ELISA检测表明重组Orf 9b蛋白可以与SARS康复病人血清反应而不与健康人血清反应。用圆二色光谱和红外光谱初步分析了重组Orf 9b蛋白的二级结构组成。圆二色光谱显示重组Orf 9b蛋白中α-螺旋占12.5%,β-折叠占40%,无规则卷曲占47.5%,红外光谱显示重组Orf 9b蛋白中α-螺旋占13.7%,β-折叠占47.5%,无规则卷曲占37.9%,两种检测方法对重组Orf 9b蛋白结构分析的结果基本一致,提示Orf 9b蛋白富含β折叠,而α-螺旋含量较少。获得该重组蛋白及对其结构的初步分析为进一步研究Orf 9b蛋白的功能奠定基础。Off 9b was amplified by PCR from SARS-CoV genome and cloned into the Nco I and Barn HI sites of the pET32c expression vector, and then recombinant plasmid pET32c-Orf 9b was constructed. The recombinant fusion protein Orf 9b was expressed by IPTG induction and purifed. After being cleaved by rEK, Orf 9b protein with MW 11 kD was separated and collected. It was demonstrated by ELISA that the purified Orf 9b protein could react with sera of SARS rehabilitaion patients but not with sera from healthy donors. CD and Infrared spectroscopy were used to predict the secondary structure of the purified Orf 9b protein . The distribution percentages for the the secondary structures of a-helix, β-sheet, and random coil in the Orf 9b protein estimated by CD were 12.5%, 40%, and 47.5%, respectively, while the same parameters estimated by Infrared spectroscopy were 13.7%, 47.5%, and 37.9%, respectively. The results obtained by the two methods were substantially identical and showed that the structure of the Orf 9b protein consisted mostly of β-sheet, and comprised only few a-helix. The acquisition of purified protein and the structural information presented herein may provide foundation for further functional study.

关 键 词：SARS-COV ORF 9b 基因克隆 蛋白表达 二级结构 

分 类 号：R373.1[医药卫生—病原生物学]