Chen Ying-yu, Shuang Bao, Tan Ya-xia, Meng Min-jie, Han Pu, Mo Xiao-ning, Song Quan-sheng, Qiu Xiao-yan, Luo Xin, Gan Qi-ni, Zhang Xin, Zheng Ying, Liu Shun-ai, Wang Xiao-ning, Zhong Nan-shan, Ma Da-long
Center for Human Disease Genomics, Peking University, Beijing 100083, China.
Chin Med J (Engl). 2005 Feb 20;118(4):267-74.
The genome of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) includes sequences encoding the putative protein X4 (ORF8, ORF7a), consisting of 122 amino acids. The deduced sequence contains a probable cleaved signal peptide sequence and a C-terminal transmembrane helix, indicating that protein X4 is likely to be a type I membrane protein. This study was conducted to demonstrate whether the protein X4 was expressed and its essential function in the process of SARS-CoV infection.
The prokaryotic and eukaryotic protein X4-expressing plasmids were constructed. Recombinant soluble protein X4 was purified from E. coli using ion exchange chromatography, and the preparation was injected into chicken for rising specific polyclonal antibodies. The expression of protein X4 in SARS-CoV-infected Vero E6 cells and lung tissues from patients with SARS was performed using immunofluorescence assay and immunohistochemistry technique. The preliminary function of protein X4 was evaluated by treatment with and over-expression of protein X4 in cell lines. Western blot was employed to evaluate the expression of protein X4 in SARS-CoV particles.
We expressed and purified soluble recombinant protein X4 from E.coli, and generated specific antibodies against protein X4. Western blot proved that the protein X4 was not assembled in the SARS-CoV particles. Indirect immunofluorescence assays revealed that the expression of protein X4 was detected at 8 hours after infection in SARS-CoV-infected Vero E6 cells. It was also detected in the lung tissues from patients with SARS. Treatment with and overexpression of protein X4 inhibited the growth of Balb/c 3T3 cells as determined by cell counting and MTT assays.
The results provide the evidence of protein X4 expression following SARS-CoV infection, and may facilitate further investigation of the immunopathological mechanism of SARS.
严重急性呼吸综合征相关冠状病毒（SARS-CoV）的基因组包含编码假定蛋白X4（开放阅读框8，开放阅读框7a）的序列，该蛋白由122个氨基酸组成。推导的序列包含一个可能的切割信号肽序列和一个C端跨膜螺旋，表明蛋白X4可能是一种I型膜蛋白。本研究旨在证明蛋白X4是否表达及其在SARS-CoV感染过程中的重要功能。
构建原核和真核表达蛋白X4的质粒。使用离子交换色谱从大肠杆菌中纯化重组可溶性蛋白X4，并将该制剂注射到鸡体内以产生特异性多克隆抗体。使用免疫荧光测定法和免疫组织化学技术检测蛋白X4在SARS-CoV感染的Vero E6细胞和SARS患者肺组织中的表达。通过在细胞系中用蛋白X4处理和过表达来评估蛋白X4的初步功能。采用蛋白质印迹法评估蛋白X4在SARS-CoV颗粒中的表达。
我们从大肠杆菌中表达并纯化了可溶性重组蛋白X4，并产生了针对蛋白X4的特异性抗体。蛋白质印迹证明蛋白X4未组装在SARS-CoV颗粒中。间接免疫荧光测定显示，在SARS-CoV感染的Vero E6细胞感染后8小时检测到蛋白X4的表达。在SARS患者的肺组织中也检测到了该蛋白。通过细胞计数和MTT测定法确定，用蛋白X4处理和过表达可抑制Balb/c 3T3细胞的生长。
这些结果提供了SARS-CoV感染后蛋白X4表达的证据，并可能有助于进一步研究SARS的免疫病理机制。
