SARS-CoV核蛋白和宿主细胞相互作用的初步研究

A Preliminary Study on the Interaction between SARS-CoV Nucleoprotein and the Host Cell

寻找与SARS-CoV核蛋白相互作用的宿主细胞蛋白,从而探索SARS-CoV的致病机理。可溶性表达SARS-CoV核蛋白,利用His标签和离子交换层析对表达的蛋白进行了纯化,获得较纯的可溶性核蛋白。再将SPR/BIA技术与MALDI-TOF MS技术结合起来,使用SPR生物传感芯片作为亲和吸附的表面,分别捕获2BS细胞和A549细胞裂解液中与SARS-CoV核蛋白相互作用的细胞蛋白,收集足够量的相互作用蛋白,再利用MALDI-TOF-MS分析获得蛋白的性质。结果鉴定出与SARS-CoV核蛋白相互作用的蛋白:26S蛋白酶调节亚单位S10B(蛋白酶体亚单位p42)(蛋白酶体26S亚单位ATPase 6)(P62333),属于泛素/蛋白酶体系统;目前国内外尚未见类似报道。此研究初步发现了一种与SARS-CoV核蛋白在细胞外相互作用的蛋白,但这种相互作用在SARS-CoV感染及SARS的发生发展中发挥的作用还有待于深入研究和探索。

To find out the proteins of host cell interacting with SARS-CoV nucleoprotein （NP） for probing some important clues of SARS pathogenesis, the soluble NP of SARS CoV with his tag was expressed in E. coli BL21,then purified by Ni^2＋ chelating chromatography and ion exchange chromatography. The surface plasmon resonance biomolecular interaction analysis （SPR/BIA） and matrix-assistant laser desorption /ionization time of flight mass spectrometry （MALDI-TOF-MS） were used to find out the proteins in host cells （2BS cell and A549 cell） binding to SARS CoV NP. The purified NP was coupled on the surface of the CM5 biosensor chip. When the host cell lysate was driven across the flowcell of the biosensor chip, NP could capture the proteins in lysate solution which interacted with it. The biosensor chip was eluted to collect the stripped proteins for MALDI-TOF MS. After searching database, one protein interacting with SARS-CoV NP was identified, which is 26S protease regulatory subunit S10B （Proteasome subunit p42） （Proteasome 26S subunit ATPase 6） （P62333）. Up to date, there is no such report yet. Though we have found p42 interacting with SARS-CoV NP in vitro the first time, there is still a long way to elucidate the mechanism of SARS-CoV infection, the interaction between virus and host cell needs to be confirmed by the other way.