摘要
缺乏合适的ＨＣＶ感染细胞及小动物模型，是抗ＨＣＶ药物研究和开发的主要障碍之一。建立一种ＨＣＶ５＇ＮＣＲ调控荧光素酶基因的转基因细胞模型，可为以ＨＣＶ５＇ＮＣＲ及Ｃ基因５＇端为靶的反义寡核苷酸及特异性核酶等抗ＨＣＶ药物的评价及筛选创造条件。将ＨＣＶ５＇ＮＣＲ－Ｃ基因与荧光素酶基因的融合基因片段插入ｐＣＩ－ｎｅｏ表达载体，通过ＰＣＲ扩增、酶切反应、质粒大小检测及荧光素酶瞬间表达活性鉴定等试验，获得ＨＣＶ５＇ＮＣＲ调控荧光素酶的稳定表达质粒ｐＨＣＶ－ｎｅｏ４。将ｐＨＣＶ－ｎｅｏ４转染ＨｅｐＧ２细胞，经Ｇ４１８筛选，从１５０个克隆中获得一个荧光素酶活性表达为１４４３ＭＶ的细胞株（ＨｅｐＧ２．９７０６）。该株细胞内ＨＣＶ片段的ＤＮＡ及ｍＲＮＡ检测均呈阳性，接种培养板后，细胞荧光素酶活性表达持续１４４小时，无明显降低。该株细胞已传６０代。
Control of HCV infection by antiviral agents is at present limited because of lacking small animal and cultured cell model system. Our aim is to establish a cultured HCV stable expressing transgenic cell line to facilitate the evaluation and screening of antisense oligodeoxynucleotide drugs targeted at HCV 5′NCR and C regions. Expression plasmid (pHCV-neo4) of luciferase controlled by HCV 5′NCR was confirmed by PCR, endonuclease digestion as well as luciferase transient expression. HepG2 cells was transfected with pHCV-neo4 using lipofectin reagent and selected with G418 (700μg/ml). A highly stable expressing cell line was obtained from 150 clones. The luciferase activity expressed by the cell line can reach 1443MV. Transgenic HCV DNA and mRNA were detected by PCR and RT-PCR in HepG2.9706 cells. The luciferase expession of the cell line can persist 144 hours after plating into multi-well culture plate. The cell line has a stable luciferase expressing activity for 6 months.
出处
《病毒学报》
CAS
CSCD
北大核心
1998年第4期296-301,共6页
Chinese Journal of Virology
基金
国家"863"基金
国家自然科学基金
军队"九五"重点课题基金
