以报告基因荧光素酶研究HPRE功能元件与IFN-α应答的关系  

Study on relationship between HBV post-transcriptional regulatory element and response to IFN-α by using a reporter gene luciferase

作　　者：姚文娟[1] 刁振宇[2] 邓小昭[2] 孔晶[3] 周宗安[2] 高健[2] 张云[2] 

机构地区：[1]南京师范大学生命科学学院,江苏南京210097 [2]南京军区军事医学研究所,江苏南京210002 [3]中国药科大学,江苏南京210009

出　　处：《细胞与分子免疫学杂志》2006年第5期575-578,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：江苏省自然科学基金资助项目(BK2004011;BK2005004)

摘　　要：目的:以荧光素酶(luciferase,LUC)基因为报告基因,探讨HBV转录后调节序列(HPRE)的功能元件(α、β1和β2)对IFNα作用的影响。方法:用PCR法从载体pGEMluc中扩增LUC基因,并克隆入真核表达载体pcDNA3.0中。以含乙肝病毒(HBV)基因组的质粒A01为模板,扩增HPRE(完整的HPRE)、HPREαβ1及HPREβ1β2片段,并分别插入LUC基因的下游。利用脂质体介导的转染法,将重组质粒分别导入人肝癌细胞株HepG2中,用LUC检测系统检测IFNα作用前后LUC表达活性的变化。结果:经测序证实,重组质粒pcDNA3.0luc、pcDNA3.0lucHPRE、pcDNA3.0lucHPREαβ1及pcDNA3.0lucHPREβ1β2构建成功。检测结果显示,IFNα作用前,HPRE、HPREαβ1和HPREβ1β2均能提高LUC的活性,IFNα作用后,HPRE和HPREβ1β2能明显降低LUC的活性,而HPREαβ1对其表达则没有显著影响。结论:HPRE的功能元件β2与IFNα作用的关系最为密切,而功能元件α和β1在IFNα应答中作用甚小,提示IFNα诱导产生的HPRE抑制性结合蛋白很可能是与β2结合的,为进一步研究IFNα在治疗HBV感染和慢性肝炎中的作用机制,以及HPRE抑制性结合蛋白的作用提供了一定的实验依据。AIM： To identify the function of HBV post-transcriptional regulatory element（HPRE） in response to IFN-α by using the reporter gene luciferase （LUC）. METHODS： The gene encoding LUC was obtained by PCR from the vector pGEM-luc and then cloned into the eukaryotic expression vector pcDNA3.0. The HPRE, HPREαβ1, and HPREβ1β2 fragments were amplified by PCR from HBV genome and then cloned into the recombinant vector pcDNA3.0-luc. These constructed plasmids were transfected into the human hepatoma cell line HepG2. The expression of LUC before and after the addition of I FN-α was detected by LUC assay system. RESULTS： The DNA sequencing analysis showed that the recombinant plasmids pcDNA3.0-luc, pcDNA3.0-luc-HPRE, pcDNA3.0-luc-HP- REαβ1 and pcDNA3.0-luc-HPREβ1β2 were successfully constructed. The results of LUC detection assay showed that HPRE, HPREαβ1, and HPREβ1β2 could enhance the expression of LUC before the addition of IFN-α. When IFN-α was added, only the fragment of HPRE, HPREβ1β2 could significantly reduce the expression of LUC, but the expression of LUC was not influenced by HPREαβ1. CONCLUSION： The functional element β2 of HPRE plays a more important part in response to IFN-α than the element α and β1, which may be valuable for further research on mechanisms of IFN-α therapy in HBV infection and function of HPRE binding protein.

关 键 词：HPRE IFN-Α 荧光素酶 转染 

分 类 号：R392.11[医药卫生—免疫学]