中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2008年
4期
314-319
,共6页
王林%黄清玲%郭丹华%陈婉南%林建银%林旭
王林%黃清玲%郭丹華%陳婉南%林建銀%林旭
왕림%황청령%곽단화%진완남%림건은%림욱
乙型肝炎病毒%RNA剪接%α-干扰素%DNA聚合酶
乙型肝炎病毒%RNA剪接%α-榦擾素%DNA聚閤酶
을형간염병독%RNA전접%α-간우소%DNA취합매
Hepatitis B virus%RNA splicing%Interferon-α%DNA polymerase
目的 研究乙型肝炎病毒(HBV)458 nt～1308nt剪接特异性蛋白TSR'r'(源于HBVDNA聚合酶读码框,T代表TP区,S为Spacer区,R'为截短的RT区,r'为截短的RNaseH区)抗α-干扰素(IFN-α)作用并分析其功能区域.方法 PCR扩增获得HBV 458 nt～1308 nt剪接变异体剪接特异性基因TSR'r'及其缺失突变体,并克隆于pcDNA3.1/HisC.重组载体以FuGENE6转染Huh7肝细胞,通过融合表达的多肽表位抗体,Western blot检测目的蛋白表达.TSR'r'及其缺失突变体重组载体分别与IFN-α反应报告载体p6-16CAT共转染Huh7肝细胞,转染后48h给予终浓度为100 IU/ml的IFN-α2a刺激,作用24 h后裂解细胞,检测胞内氯霉素乙酰基转移酶(CAT)含量.所有实验数据采用单因素方差分析法进行统计分析.结果 构建TSR'r'及其缺失突变体重组真核表达载体,Westernblot显示各基因片段在Huh7细胞中均表达相应蛋白.p6-16CAT共转染结果显示,随着TSR'r'重组表达载体转染量的递增,Huh7胞内CAT值逐渐降低.此外,转染TP+Spacer区的缺失突变体可导致Huh7胞内CAT值显著下降,而其他各类TSR'r'缺失突变体共转染后胞内CAT值无变化.结论 HBV 458 nt-1308 nt剪接特异性蛋白抑制Huh7细胞对IFN-α仅的反应性,其活性与N端的TP及Spacer区有关.
目的 研究乙型肝炎病毒(HBV)458 nt～1308nt剪接特異性蛋白TSR'r'(源于HBVDNA聚閤酶讀碼框,T代錶TP區,S為Spacer區,R'為截短的RT區,r'為截短的RNaseH區)抗α-榦擾素(IFN-α)作用併分析其功能區域.方法 PCR擴增穫得HBV 458 nt～1308 nt剪接變異體剪接特異性基因TSR'r'及其缺失突變體,併剋隆于pcDNA3.1/HisC.重組載體以FuGENE6轉染Huh7肝細胞,通過融閤錶達的多肽錶位抗體,Western blot檢測目的蛋白錶達.TSR'r'及其缺失突變體重組載體分彆與IFN-α反應報告載體p6-16CAT共轉染Huh7肝細胞,轉染後48h給予終濃度為100 IU/ml的IFN-α2a刺激,作用24 h後裂解細胞,檢測胞內氯黴素乙酰基轉移酶(CAT)含量.所有實驗數據採用單因素方差分析法進行統計分析.結果 構建TSR'r'及其缺失突變體重組真覈錶達載體,Westernblot顯示各基因片段在Huh7細胞中均錶達相應蛋白.p6-16CAT共轉染結果顯示,隨著TSR'r'重組錶達載體轉染量的遞增,Huh7胞內CAT值逐漸降低.此外,轉染TP+Spacer區的缺失突變體可導緻Huh7胞內CAT值顯著下降,而其他各類TSR'r'缺失突變體共轉染後胞內CAT值無變化.結論 HBV 458 nt-1308 nt剪接特異性蛋白抑製Huh7細胞對IFN-α僅的反應性,其活性與N耑的TP及Spacer區有關.
목적 연구을형간염병독(HBV)458 nt～1308nt전접특이성단백TSR'r'(원우HBVDNA취합매독마광,T대표TP구,S위Spacer구,R'위절단적RT구,r'위절단적RNaseH구)항α-간우소(IFN-α)작용병분석기공능구역.방법 PCR확증획득HBV 458 nt～1308 nt전접변이체전접특이성기인TSR'r'급기결실돌변체,병극륭우pcDNA3.1/HisC.중조재체이FuGENE6전염Huh7간세포,통과융합표체적다태표위항체,Western blot검측목적단백표체.TSR'r'급기결실돌변체중조재체분별여IFN-α반응보고재체p6-16CAT공전염Huh7간세포,전염후48h급여종농도위100 IU/ml적IFN-α2a자격,작용24 h후렬해세포,검측포내록매소을선기전이매(CAT)함량.소유실험수거채용단인소방차분석법진행통계분석.결과 구건TSR'r'급기결실돌변체중조진핵표체재체,Westernblot현시각기인편단재Huh7세포중균표체상응단백.p6-16CAT공전염결과현시,수착TSR'r'중조표체재체전염량적체증,Huh7포내CAT치축점강저.차외,전염TP+Spacer구적결실돌변체가도치Huh7포내CAT치현저하강,이기타각류TSR'r'결실돌변체공전염후포내CAT치무변화.결론 HBV 458 nt-1308 nt전접특이성단백억제Huh7세포대IFN-α부적반응성,기활성여N단적TP급Spacer구유관.
Objective To investigate the anti-IFN-α effects of the novel protein TSR'r' encodedby the 458 nt-1308 nt spliced variant of hepatitis B virus genome,and to determine its functional domaias.Methods the TSR'r' gene(originated from open reading frame of HBV DNA polymerase,T represents terminal protein region,S represents the Spacer region,R'represents the truncated reverse transcriptase region,and r'represents the truncated RNaseH region)of the 458 nt-1308 nt spliced variant of HBV genome and its deletants were amplified by PCR and were cloned into the pcDNA3.1/HisC vector.The recombinant vector was transfected into Huh7 hepatocytes individually by FuGENE6 transfection reagent,and the expression of the fusion protein was detected by Western blot.Huh7 hepatocytes were co-transfected with p6 16CAT and the recombinant vector harboring either TSR'r'or the related deletant,and treated with IFN-α 2a 48 h post transfection.After 24 h stimulating.the cells were lysed and the intracellular CAT value was calculated.All data were processed with One-way analysis of variance(ANOVA).Resuits Recombinant vectors harboring either the TSR'r'gene or related deletant were constructed successfully,and the fusion proteins were expressed well in Huh7 cells.When Huh7 hepatocytes were co-transfected with p6-16CAT and TSR'r' recombinant.the intracellular CAT values reduced gradually as paralleled with the increasing amount of TSR'r'recombinant.Furthermore,as compared with the empty vector,intracellular CAT values also decreased significantly when the Huh7 cells co-transfected with recombinant harboring TP plus Spacer regions,while any of the other deletants(harboring either TP or Spacer region or neither)showed no significant difference.Conclusion The novel protein encoded by the 458 nt-1308 nt spliced variant of hepatitis B virns genome suppressed the response of Huh7 hepatocytes to IFN-α.and the N-terminal TP plus Spacer region was the functional domain of the protein for anti-IFN-α effects.
