【摘要】 应用PCR技术从含有丙型肝炎病毒(HCV)全长开放阅读框的质粒pBRTM/HCV 1~3 011中获得NS5A全长基因片段,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及测序鉴定证实,NS5A基因已正确插入到pcDNA3.1(-)中。再利用脂质体介导转染Huh7细胞,30h后收获细胞,经Western blot验证,证实HCV的NS5A基因在Huh7细胞中已经获得表达。在培养条件完全一致的条件下,表达NS5A基因的Huh7细胞与pcDNA3.1(-)转染的细胞在转染30h后被收集起来,乙醇固定,PI染色后利用流式细胞仪检测细胞周期变化。G0/G1期由60.6%下降到49.7%,S期由23.9%上升到32.7%,而转染pcDNA3.1(-)细胞的细胞周期与正常的Huh7细胞则差别不大。从而证明HCV NS5A蛋白对Huh7细胞周期具有调节作用。更多还原

【Abstract】 Full-length NS5A gene of hepatitis C virus was amplified by PCR, using 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 recombinant vector was identified by restriction enzyme digestion, polymerase chain reaction and direct sequencing. Then both the recombinant vector pcDNA3.1（-）-NS5A and the control vector pcDNA3.1（-） were used to transfect Huh7 cell with Lipofectin 2000. After 30h, the cells were collected and HCV NS5A gene expression was proved by Western blot analysis. Under the same culture conditions, both of the Huh7 cell transfected with pcDNA3.1（-）-NS5A and pcDNA3.1（-） were collected after 30h postransfection, fixed by ethanol and stained by propidine iodide. Then the cell cycle was detected by flow cytometry. We found the G₀/G₁ phase was decreased from 60.6% to 49.7%, the S phase increased from 23.9% to 32.7%, while there was no significant difference found between the control cells and the cells transfected with pcDNA3.1（-）. These results indicated that NS5A could affect the cell cycle of Huh7 cell.更多还原