转录后调控元件部分或完全缺失对乙型肝炎病毒前基因组RNA核外输出的影响  

Influence of partial or complete deletion of post-transcriptional regulatory element on the extranuclear export of hepatitis B virus pregenomic RNA

作　　者：黄迎丽 顾文慧 朱慧慧 李亚茹 马园园 陈文文 孙锁锋 李修岭 HUANG Ying-li;GU Wen-hui;ZHU Hui-hui;LI Ya-ru;MA Yuan-yuan;CHEN Wen-wen;SUN Suo-feng;LI Xiu-ling(Department of Gastroenterology,Zhengzhou University People's Hospital,Henan Provincial People's Hospital,Zhengzhou,Henan 450003,China;Department of Gastroenterology,Henan University People's Hospital,Henan Provincial People's Hospital,Zhengzhou,Henan 450003,China;Grade 2020,Xinxiang Medical University,Xinxiang,Henan 453003,Chin)

机构地区：[1]郑州大学人民医院河南省人民医院消化内科,河南郑州450003 [2]河南大学人民医院河南省人民医院消化内科,河南郑州450003 [3]新乡医学院,河南新乡453003

出　　处：《中华实用诊断与治疗杂志》2022年第10期1017-1021,共5页Journal of Chinese Practical Diagnosis and Therapy

基　　金：河南省医学科技攻关计划省部共建项目(SBGJ202002006)。

摘　　要：目的探讨转录后调控元件(post-transcriptional regulatory element,PRE)部分或完全缺失对乙型肝炎病毒(hepatitis B virus,HBV)前基因组RNA(pregenomic RNA,pgRNA)核外输出的影响。方法以含HBV全基因组质粒为模板,采用PCR法构建PRE完全缺失质粒(HBVΔPRE)、PREα缺失质粒(HBVΔPREα)、PREβ缺失质粒(HBVΔPREβ)、SLα缺失质粒(HBVΔSLα)、SLβ缺失质粒(HBVΔSLβ)。对数生长期人肝癌HepG2细胞分为WT组(转染HBV全基因组质粒)、del-PREα组(转染HBVΔPREα)、del-PREβ组(转染HBVΔPREβ)、del-PRE组(转染HBVΔPRE),转染48h后采用反转录实时荧光定量PCR法检测4组细胞、细胞质、细胞核HBV pgRNA相对表达量,计算细胞质与细胞核HBV pgRNA比值(细胞质/细胞核HBV pgRNA);采用Western blot法检测WT组与del-PRE组细胞乙型肝炎表面抗原(hepatitis B surface antigen,HBsAg)、乙型肝炎核心抗原(hepatitis B core antigen,HBcAg)蛋白相对表达量。对数生长期HepG2细胞分为WT组(转染HBV全基因组质粒)、del-SLα组(转染HBVΔSLα)、del-SLβ组(转染HBVΔSLβ),转染48h后采用反转录实时荧光定量PCR法检测3组细胞、细胞质、细胞核HBV pgRNA相对表达量,计算细胞质/细胞核HBV pgRNA。结果WT组细胞、细胞质HBV pgRNA相对表达量(1.03±0.07、1.01±0.21)及细胞质/细胞核HBV pgRNA(1.01±0.01)均高于del-PREα组(0.74±0.04、0.57±0.01、0.41±0.00)、del-PREβ组(0.58±0.02、0.24±0.01、0.30±0.01)、del-PRE组(0.41±0.01、0.21±0.00、0.30±0.00)(P<0.05),del-PREα组均高于del-PREβ组、del-PRE组(P<0.05);del-PREβ组细胞、细胞质HBV pgRNA相对表达量均高于del-PRE组(P<0.05),细胞质/细胞核HBV pgRNA与del-PRE组比较差异无统计学意义(P>0.05)。del-PREα组细胞核HBV pgRNA相对表达量(1.38±0.01)均高于WT组(1.00±0.02)、del-PREβ组(0.79±0.01)、del-PRE组(0.71±0.01)(P<0.05),WT组均高于del-PREβ组、del-PRE组(P<0.05),del-PREβ组高于del-PRE组(P<0.05)。del-PRE组HBsAg蛋白相对表达量(0.66±0.00Objective To investigate the influence of partial or complete deletion of post-transcriptional regulatory element(PRE)on the extranuclear export of hepatitis B virus(HBV)pregenomic RNA(pgRNA).Methods Using the plasmid containing the whole genome of hepatitis B virus(HBV)as the template,PCR technique was used to construct PRE complete deletion plasmid(HBVΔPRE),PREαdeletion plasmid(HBVΔPREα)and PREβdeletion plasmid(HBVΔPREβ),SLαdeletion plasmid(HBVΔSLα),and SLβdeletion plasmid(HBVΔSLβ).HepG2cells in logarithmic growth phase were divided into WT group(transfected with HBV whole genome plasmid),del-PREαgroup(transfected with HBVΔPREα),del-PREβgroup(transfected with HBVΔPREβ),and del-PRE group(transfected with HBVΔPRE).After 48-h transfection,reverse transcription real-time fluorescence quantitative PCR was used to detect the relative expressions of HBV pgRNA in the cell,nucleus and cytoplasm in four groups,and the ratio of HBV pgRNA in the cytoplasm to nucleus was calculated.Western blot was used to detect the relative expressions of hepatitis B surface antigen(HBsAg)protein and hepatitis B core antigen(HBcAg)protein in WT group and del-PRE group.HepG2cells in logarithmic growth phase were divided into WT group(transfected with HBV whole genome plasmid),del-SLαgroup(transfected with HBVΔSLα),and del-SLβgroup(transfected with HBVΔSLβ).The relative expressions of HBV pgRNA in the cell,nucleus and cytoplasm were detected by reverse transcription real-time fluorescence quantitative PCR,and the ratio of HBV pgRNA in the cytoplasm to nucleus was calculated.Results The relative expressions of HBV pgRNA in the cell and cytoplasm and ratio of HBV pgRNA in the cytoplasm to nucleus were higher in WT group(1.03±0.07,1.01±0.21,1.01±0.01)than those in del-PREαgroup(0.74±0.04,0.57±0.01,0.41±0.00),del-βgroup(0.58±0.02,0.24±0.01,0.30±0.01)and del-PRE group(0.41±0.01,0.21±0.00,0.30±0.00)(P<0.05),and higher in del-PREαgroup than those in del-PREβgroup and del-PRE group(P<0.05).The relative expr

关 键 词：乙型肝炎病毒 转录后调控元件 茎环结构 核外输出 HEPG2细胞 

分 类 号：R373.21[医药卫生—病原生物学]