CAJ | 학술논문

目的：分析在不同宿主细胞的翻译体系下，缺失不同片段的HCV 5'UTR翻译启动活性的差异。方法以脂质体介导基因转染技术，将截短型HCV 5'UTR调控Fluc的真核表达质粒与Rluc真核表达质粒pRL-TK共转染至不同的细胞中，转染后36 h：①提取细胞RNA，半定量RT-PCR检测目的质粒的转录水平；②用双荧光素酶报告基因检测系统检测Fluc基因相对表达活性，分析HCV 5'UTR缺失不同结构域后在不同翻译体系中翻译启动活性的差异。结果⑴缺失5'端44个碱基的HCV 5'UTR的翻译启动活性分别与缺失前相比：在HeLa细胞和C6细胞中无明显影响，在L-02细胞中活性下降为46%，而在293T细胞则为缺失前的146%；⑵缺失5'端118个碱基后，HCV 5'UTR的活性分别与缺失前相比：在HeLa细胞中，缺失后活性仅为缺失前的49%，而在L-02细胞、C6细胞和293T细胞中，活性分别为缺失前的140%、160%和235%。在本研究使用的四种细胞中，pCNl的翻译启动活性的差异无统计学意义，pCNl-d2在293T细胞中活性最高，在L-02细胞中活性最低。pCNl-d3在293T、C6和L-2细胞中活性相近，但在HeLa细胞中的活性明显比其他细胞低。结论 HCV 5'UTR的DomainⅠ和DomainⅡ对其翻译启动活性的影响与宿主细胞种类相关，具细胞特异性。
목적：분석재불동숙주세포적번역체계하，결실불동편단적HCV 5'UTR번역계동활성적차이。방법이지질체개도기인전염기술，장절단형HCV 5'UTR조공Fluc적진핵표체질립여Rluc진핵표체질립pRL-TK공전염지불동적세포중，전염후36 h：①제취세포RNA，반정량RT-PCR검측목적질립적전록수평；②용쌍형광소매보고기인검측계통검측Fluc기인상대표체활성，분석HCV 5'UTR결실불동결구역후재불동번역체계중번역계동활성적차이。결과⑴결실5'단44개감기적HCV 5'UTR적번역계동활성분별여결실전상비：재HeLa세포화C6세포중무명현영향，재L-02세포중활성하강위46%，이재293T세포칙위결실전적146%；⑵결실5'단118개감기후，HCV 5'UTR적활성분별여결실전상비：재HeLa세포중，결실후활성부위결실전적49%，이재L-02세포、C6세포화293T세포중，활성분별위결실전적140%、160%화235%。재본연구사용적사충세포중，pCNl적번역계동활성적차이무통계학의의，pCNl-d2재293T세포중활성최고，재L-02세포중활성최저。pCNl-d3재293T、C6화L-2세포중활성상근，단재HeLa세포중적활성명현비기타세포저。결론 HCV 5'UTR적DomainⅠ화DomainⅡ대기번역계동활성적영향여숙주세포충류상관，구세포특이성。
Objective To investigate the roles of Domain I and Domain II of hepatitis C virus (HCV) 5' untranslated region (UTR) in the translation initiation activity of HCV 5'UTR in different host cell lines. Methods The eukaryotic expression plasmid pCMVNCRLuc (pCN1), in which full-length HCV 5'UTR regulates firefly luciferase expression, was modified by deleting Domain I and the downstream single-stranded sequence (43 bp in total) from the UTR (pCNl-d2), Domain I with the downstream single-stranded sequence and Domain II (118 bp in total) from the UTR (pCNl-d3), or the total UTR (pCNl-d5). The modified plasmids were transfected via liposome into different cell lines with pRL-TK plasmid co-transfected as the normalization control. At 36 h after the transfection, the total cellular RNA was harvested for semi-quantitative RT-PCR, and the relative expression activities of luciferase were assayed with a dual luciferase reporter gene assay system. The translation initiation activities of the truncated HCV 5'UTRs in different translation systems were analyzed. Results Deletion of Domain I and the downstream single-stranded sequence caused no significant changes of the translational activity of HCV 5'UTR in Hela or C6 cells, but decreased the translational activity by 46%in L-02 cells and increased the translational activity by 46%in 293T cells. Deletion of both Domain I and Domain II resulted in decreased translational activity of HCV 5'UTR by 51%in HeLa cells, but increased the translational activity by 40%in L-02 cells, 60%in C6 cells and 135%in 293T cells. Conclusions Domain I and Domain II of HCV 5'UTR perform cell type-specific roles in HCV IRES-driven translation initiation.