Stable Expression of NS5A Gene of HCV in Hela Cells and Inhibition to the Growth of Hela Cells

YANG Xiao-jun; YE Lin-bai*; GAO Jin-rong; LIU Jing; YANG Fan; YE Li; SHE Ying-long; LIAO Qing-jiao; WU Zheng-hui; ZHENG Yi

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August 2004

YANG Xiao-jun, YE Lin-bai*, GAO Jin-rong, LIU Jing, YANG Fan, YE Li, SHE Ying-long, LIAO Qing-jiao, WU Zheng-hui and ZHENG Yi. Stable Expression of NS5A Gene of HCV in Hela Cells and Inhibition to the Growth of Hela Cells[J]. Virologica Sinica, 2004, 19(4): 340-344.

Citation: YANG Xiao-jun, YE Lin-bai*, GAO Jin-rong, LIU Jing, YANG Fan, YE Li, SHE Ying-long, LIAO Qing-jiao, WU Zheng-hui, ZHENG Yi. Stable Expression of NS5A Gene of HCV in Hela Cells and Inhibition to the Growth of Hela Cells .VIROLOGICA SINICA, 2004, 19(4) : 340-344.

HCV 5A基因在Hela细胞中的稳定表达及对细胞生长的抑制现象

1.
武汉大学生命科学学院病毒研究所，湖北武汉，430072

通讯作者： 叶力, ;

摘要

应用PCR技术从含有HCV (Hepatitis C virus)全长开放阅读框的质粒pBRTM/HCV 1-3011中获得NS5A全长基因片断，利用基因重组技术将其克隆至真核表达载体pcDNA3.1(-)中。通过酶切、PCR及测序鉴定NS5A基因已正确插入到pcDNA3.1(-)中，再利用脂质体介导转染Hela细胞，48h后传代并利用pcDNA3.1(-)质粒上的neo抗性基因加入G-418进行筛选。大约两周后，获得稳定表达的细胞株。经RT-PCR及western blot验证，证实HCV的NS5A基因在Hela细胞中已经获得了表达。在培养条件完全一致的条件下，表达NS5A基因的Hela细胞与pcDNA3.1(-)转染的细胞相比，生长速度明显变慢，其倍增时间约为35-36h，比对照组细胞增加了约50%，而转染pcDNA3.1(-)的细胞的倍增时间与正常Hela细胞则无明显差别，都为23-24h。从而证明HCV的NS5A蛋白具有抑制Hela细胞生长的作用。
- HCV NS5A
- , Hela细胞
- , 基因表达
- , 抑制细胞生长

Stable Expression of NS5A Gene of HCV in Hela Cells and Inhibition to the Growth of Hela Cells

1.
Institute of Virology, Wuhan University, Wuhan 430072, China

Corresponding author: YE Li,

Abstract

Full-length NS5A gene of Hepatitis C virus（HCV） was amplified by PCR, using the plasmid pBRTM/HCV 1-3011 containing HCV full-length open reading frame（ORF）as template, and cloned into the eukaryotic expressing plasmid pcDNA3.1（-）by DNA recombination technique. The recombin- ant vector was identified by digestion with restriction enzymes and polymerase chain reaction and by directly sequencing. Then both the recombinant vector pcDNA3.1（-）-NS5A and the control vector pcDNA3.1（-）were transfected Hela cells using LipoVecTM. The cells expressing NS5A stablely were selected by G-418 and further proved by RT-PCR and Western blot analysis. We found the growth of Hela cells expressing NS5A was slower than the cells transfected by pcDNA3.1（-）in the same culture condition, and the population doubling time of Hela cells expressing NS5A gene is increased about 50%(about 35-35 hours). There was no significant difference between the control cells and the cells transfected with pcDNA3.1(-) (about 23-24 hours). The results indicate that NS5A can inhibit the proliferation of Hela cells.
- HCV NS5A
- , Hela cell
- , Gene expression
- , Cell growth inhibition

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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

Stable Expression of NS5A Gene of HCV in Hela Cells and Inhibition to the Growth of Hela Cells

Corresponding author: YE Li,

1. Institute of Virology, Wuhan University, Wuhan 430072, China

Abstract: Full-length NS5A gene of Hepatitis C virus（HCV） was amplified by PCR, using the plasmid pBRTM/HCV 1-3011 containing HCV full-length open reading frame（ORF）as template, and cloned into the eukaryotic expressing plasmid pcDNA3.1（-）by DNA recombination technique. The recombin- ant vector was identified by digestion with restriction enzymes and polymerase chain reaction and by directly sequencing. Then both the recombinant vector pcDNA3.1（-）-NS5A and the control vector pcDNA3.1（-）were transfected Hela cells using LipoVecTM. The cells expressing NS5A stablely were selected by G-418 and further proved by RT-PCR and Western blot analysis. We found the growth of Hela cells expressing NS5A was slower than the cells transfected by pcDNA3.1（-）in the same culture condition, and the population doubling time of Hela cells expressing NS5A gene is increased about 50%(about 35-35 hours). There was no significant difference between the control cells and the cells transfected with pcDNA3.1(-) (about 23-24 hours). The results indicate that NS5A can inhibit the proliferation of Hela cells.