For expressing the recombinant S protein in spodoptera fragiperda（Sf9） cells, the full-length cDNA of S protein was firstly amplified from plasmid pET-14b/S by means of PCR and subcloned into baculovirus transfer ve...For expressing the recombinant S protein in spodoptera fragiperda（Sf9） cells, the full-length cDNA of S protein was firstly amplified from plasmid pET-14b/S by means of PCR and subcloned into baculovirus transfer vector pFastBacTM 1, forming a recombinant plasmid pFastBacTM 1/S. It was then transposited with baculovirus shuttle vector （Bacmid） in the Max efficiency DH10BAC component cells by homologous recombination to construct the expression vector Bacmid/S. The Bacmid/S, after identified by PCR, was used to transfect Sf9 cells to express the target protein. The expressed recombinant S protein was analyzed by Western blot with human anti-SARS-CoV antiserum. The results showed that the recombinant S protein had been expressed correctly and efficiently in insect Sf9 cells and was purified by Ni-NTA affinity chromatography. （Western） blot showed that recombinant S protein could be recognized by human anti-SARS-CoV antiserum.展开更多
目的:研究SARS冠状病毒S蛋白诱导呼吸道上皮细胞合成释放IP-10(interferon-gamma induc-ible protein 10)的信号分子机制。方法:通过基因芯片检测SARS冠状病毒的S蛋白作用于人支气管上皮细胞16HBE后信号通路基因表达谱的变化;采纳RT-PCR...目的:研究SARS冠状病毒S蛋白诱导呼吸道上皮细胞合成释放IP-10(interferon-gamma induc-ible protein 10)的信号分子机制。方法:通过基因芯片检测SARS冠状病毒的S蛋白作用于人支气管上皮细胞16HBE后信号通路基因表达谱的变化;采纳RT-PCR、EMSA、Western blotting等方法进一步分析JAK-STAT通路中信号分子的磷酸化、IRF-1和IP-10基因表达的变化及其相应信号分子抑制剂对表达水平的影响。结果:S蛋白作用于人支气管上皮细胞16HBE诱导了JAK-STAT信号通路涉及的重要转录因子基因IRF-1的表达,该信号通路的转录因子STAT1在刺激后15 min发生磷酸化,2 h即可检出IP-10基因的表达,IP-10的表达可以完全被STAT1、JAK2抑制剂阻断。EMSA显示:支气管上皮细胞在S蛋白的作用下,其核蛋白能够特异性与ISRE和GASDNA基序相结合,而不能与NF-κB的DNA基序相结合。结论:SARS-CoV的S蛋白通过激活JAK-STAT信号转导通路诱导IP-10在宿主细胞的生成。提示病毒诱导的JAK-STAT信号通路激活在病毒感染相关的急性肺损伤发生中具有重要地位。展开更多
文摘For expressing the recombinant S protein in spodoptera fragiperda（Sf9） cells, the full-length cDNA of S protein was firstly amplified from plasmid pET-14b/S by means of PCR and subcloned into baculovirus transfer vector pFastBacTM 1, forming a recombinant plasmid pFastBacTM 1/S. It was then transposited with baculovirus shuttle vector （Bacmid） in the Max efficiency DH10BAC component cells by homologous recombination to construct the expression vector Bacmid/S. The Bacmid/S, after identified by PCR, was used to transfect Sf9 cells to express the target protein. The expressed recombinant S protein was analyzed by Western blot with human anti-SARS-CoV antiserum. The results showed that the recombinant S protein had been expressed correctly and efficiently in insect Sf9 cells and was purified by Ni-NTA affinity chromatography. （Western） blot showed that recombinant S protein could be recognized by human anti-SARS-CoV antiserum.
