HCV E2 Prodaryotic system Gene expression Purification

ZHANG Dong—wei; LIANG Bu—feng; LI Wei-dong; QI Zi—bai; LING Shi—gan

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February 2003

ZHANG Dong—wei, LIANG Bu—feng, LI Wei-dong, QI Zi—bai and LING Shi—gan. HCV E2 Prodaryotic system Gene expression Purification[J]. Virologica Sinica, 2003, 18(1): 1-4.

Citation: ZHANG Dong—wei, LIANG Bu—feng, LI Wei-dong, QI Zi—bai, LING Shi—gan. HCV E2 Prodaryotic system Gene expression Purification .VIROLOGICA SINICA, 2003, 18(1) : 1-4.

HCV E2区基因在原核系统中的表达

张东伟梁布锋李东祁自柏凌世淦 ,

1.
1．中国科学院武汉病毒研究所，湖北武汉430071
2.
中国药品生物制品检定所，北京100050
3.
军事医学科学院，北京100850

摘要

用提取的重组表达载体pET-E2转化BL21(DE3)感受态细胞,经IPTG绣导,再进行SDS-PAGE,可得到有一条约34 kDa的表达带,与理论推测的蛋白分子量一致,通过Western-blot鉴定,证明此带即为目的蛋白带。该产物有一个六聚组氨酸尾,主要以包涵体形式存在;计算机扫描分析考马斯亮兰染色后的蛋白胶显示:目的蛋白占整个菌体蛋白的36%以上,经Ni-柱纯化的E2蛋白纯度可达95%以上;以纯化的E2蛋白为抗原,用ELISA方法检测了20份抗HCV阴阳性血清,结果表明15份抗HCV阳性血清中检出5份E2抗体阳性血清,而5份抗HCV阴性血清中没有检测到E2抗体。
- HCV
- , E2
- , 原核系统
- , 基因表达
- , 纯化

HCV E2 Prodaryotic system Gene expression Purification

1.
1．Wuhan Institute of Virology，Academia Sinica，Wuhan 430071，．China
2.
Department ofHepatitis，National Institute for the Control ofPharmaceutical and Biology Products，Beijing 100050，China
3.
Military Medical Academia Sinica， Beijing 100850，China

Corresponding author: LIANG Bu—feng,

Abstract

Rceomdinant plasmid pET-E2 was transformed into BL21 (DE3) competent cell, then induced the amplified culture with IPTG. There was a 34 kDa band in SDS-PAGE gel. The molecular weight of the expressed protein was comsistent to that of deduced E2 protein. Tested by western-blot, It showed that the product was HCV E2 protein. It contained a six-Histidine tag and aggregated into the inclusion body. Computer scan analysis showed the protein interested took the percentage more than 36 of the total bacterial proteins and the rate of purification was higher than 95% after purified by Ni-column. The activity of pruified E2 protein was tested by ELISA. The result showed that the 5/15 HCVpositive sera had anti-E2 antibody, and the other 5 negative HCV sera hadn’t anti-E2 antibody
- HCV E2 Prodaryotic system Gene expression Purification

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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

HCV E2 Prodaryotic system Gene expression Purification

Corresponding author: LIANG Bu—feng,

1. 1．Wuhan Institute of Virology，Academia Sinica，Wuhan 430071，．China
2. Department ofHepatitis，National Institute for the Control ofPharmaceutical and Biology Products，Beijing 100050，China
3. Military Medical Academia Sinica， Beijing 100850，China

Abstract: Rceomdinant plasmid pET-E2 was transformed into BL21 (DE3) competent cell, then induced the amplified culture with IPTG. There was a 34 kDa band in SDS-PAGE gel. The molecular weight of the expressed protein was comsistent to that of deduced E2 protein. Tested by western-blot, It showed that the product was HCV E2 protein. It contained a six-Histidine tag and aggregated into the inclusion body. Computer scan analysis showed the protein interested took the percentage more than 36 of the total bacterial proteins and the rate of purification was higher than 95% after purified by Ni-column. The activity of pruified E2 protein was tested by ELISA. The result showed that the 5/15 HCVpositive sera had anti-E2 antibody, and the other 5 negative HCV sera hadn’t anti-E2 antibody