SARS—CoVSars7a和EGFP融合蛋白真核表达载体构建及其表达

根据SARS-CoV sars7a基因设计并化学合成部分重叠引物，经二轮PCR获得sars7a基因片段，以此片段为模板并利用一对带有Kozak序列及删除终止密码的引物进行PCR，获得产物与pEGFP-N1载体连接，使sars7a基因位于．EGFP的基因上游，得到含编码Sars7a-EGFP融合蛋白基因的哺乳动物细胞表达载体。采用细胞核转染技术将重组表达载体转染K562细胞，以流式细胞仪和共聚焦显微镜分析，可检测到EGFP的绿色荧光，表明Sars7a—EGFP得到表达，该蛋白分布于整个细胞，提示Sars7a并非膜蛋白，更可能是胞浆蛋白。此外，该蛋白的表达对K562细胞凋亡无明显影响。

Construction of an Eukaryotic Expression Vector for SARS-CoV Sars7a and EGFP Fusion Protein Expression

Partial overlapping primers were designed based on the severe acute respiratory syndrome coronavirus (SARS-CoV) sars7a gene and were chemically synthesized. The sars7a gene fragment was obtained by two-round of PCR and this fragment was used as template for a further round of PCR by using a pair of primers to introduce Kozak sequence and to delete the stop codon. The mammalian cell expression vector for Sars7a and enhanced green fluorescent protein (EGFP) fusion protein was generated by inserting the PCR product into pEGFP-N1 vector, with sars7a gene upstream to EGFP gene. K562 cells were transfected by the expression vector and the green fluorescence of EGFP could be detected with flow cytometry and confocal microscopy, indicating the expression of Sars7a-EGFP fusion protein. This fusion protein was distributed in the whole cells, which suggested that Sars7a was probable a cytosolic protein rather than a membrane protein. Besides, the expression of Sars7a had no significant effect on the apoptotic cell death of K562 cells.