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

HE Xian-hui; XU Li-hui; LIU Yi; CAI Xiao-chang; ZENG Yao-ying

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June 2004

HE Xian-hui, XU Li-hui, LIU Yi, CAI Xiao-chang and ZENG Yao-ying. Construction of an Eukaryotic Expression Vector for SARS-CoV Sars7a and EGFP Fusion Protein Expression[J]. Virologica Sinica, 2004, 19(3): 209-213.

Citation: HE Xian-hui, XU Li-hui, LIU Yi, CAI Xiao-chang, ZENG Yao-ying. Construction of an Eukaryotic Expression Vector for SARS-CoV Sars7a and EGFP Fusion Protein Expression .VIROLOGICA SINICA, 2004, 19(3) : 209-213.

SARS-CoV Sars7a和EGFP融合蛋白真核表达载体构建及其表达*

何贤辉 ¹ ,
徐丽慧 ^1,2 ,
蔡小嫦 ^1,4 ,
曾耀英 ³ ,

1.
1. 暨南大学组织移植与免疫教育部重点实验室，广东广州 510632
2.
暨南大学生物工程研究所，广东广州 510632
3.
暨南大学第一附属医院皮肤科，广东广州 510632
4.
郑州大学第一附属医院皮肤科，河南郑州 450052

摘要

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。
- 严重急性呼吸综合征
- , 冠状病毒
- , Sars7a
- , 绿色荧光蛋白
- , 融合蛋白
- , 表达

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

HE Xian-hui ¹ ,
XU Li-hui ^1,2 ,
LIU Yi ^1,4 ,
CAI Xiao-chang ³ ,
ZENG Yao-ying ^1*,,

1.
1. Key Laboratory of Tissue Transplantation and Immunology, Ministry of Education, Jinan University, Guangzhou 510632
2.
Institute of Bioengineering, Jinan University, Guangzhou 510632
3.
Department of Dermatology, the First Affiliated Hospital, Jinan University，Guangzhou 510632, China
4.
Department of Dermatology, the First Affiliated Hospital, Zhengzhou University, Zhengzhou 450052, China

Corresponding author: ZENG Yao-ying,

Abstract

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.
- Severe acute respiratory syndrome
- , Coronavirus
- , Sars7a
- , Green fluorescent protein
- , Fusion protein
- , Expression

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

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

Corresponding author: ZENG Yao-ying,

1. 1. Key Laboratory of Tissue Transplantation and Immunology, Ministry of Education, Jinan University, Guangzhou 510632
2. Institute of Bioengineering, Jinan University, Guangzhou 510632
3. Department of Dermatology, the First Affiliated Hospital, Jinan University，Guangzhou 510632, China
4. Department of Dermatology, the First Affiliated Hospital, Zhengzhou University, Zhengzhou 450052, China

Abstract: 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.