乙型肝炎病毒3022～1787核苷酸缺失突变体编码蛋白具有抗α干扰素作用

The anti-IFN-α effects of a novel protein encoded by the 3022-1787nt deletion mutant of hepatitis B virus

为证实乙型肝炎病毒(HBV)3022～1787核苷酸缺失突变体编码蛋白TS′X′(源于DNA聚合酶读码框架,T为TP区,S′为部分缺失的spacer区,X′为截短的X蛋白)具有抗α干扰素(IFN-α)作用并确定其功能区域,用聚合酶链反应(PCR)扩增获得TS′X′全长及片段TS′(含TP区及部分缺失的spacer区),并克隆于pcDNA3.1/HisC载体。重组质粒以FuGENE6转染Huh7肝细胞,48h后裂解细胞,以蛋白质印迹法(Western blot)证实目的蛋白可在Huh7肝细胞中表达。重组质粒及空载体pcDNA3.1/HisC分别与IFN-α反应报告质粒p6-16CAT按分子数5∶1、10∶1、15∶1、30∶1共转染Huh7肝细胞,转染后48h给予终浓度为100IU/ml的IFN-α2a刺激,作用24h后裂解细胞,用酶联免疫吸附试验(ELISA)检测胞内氯霉素乙酰基转移酶(CAT)含量。结果显示,与空白载体相比,随着TS′X′及TS′重组表达质粒转染量的递增,Huh7胞内CAT值逐渐降低(n=6,P<0.05),但TS′X′与TS′之间无显著差异(n=6,P>0.05)。本研究证实,HBV3022～1787核苷酸缺失突变体编码的TS′X′蛋白可抑制Huh7细胞对IFN-α的反应性,其活性与其N端TP及部分缺失的spacer区有关。

The purpose of the current study is to investigate the anti-IFN-α effects and to determine the functional region of the novel protein TS′X′ encoded by the 3022-1787nt deletion mutant of hepatitis B virus （HBV）. Regions coding for TS′X′ （ in frame with the opening reading frame of DNA polymerase,in which T stands for terminal protein, S′ for partially deleted spacer region, and X′ for truncated X protein ） and TS′ （ N-terminal of TS′ X′ containing terminal protein and partially deleted spacer region ） were amplified by PCR and cloned into the pcDNA3.1/HisC vector separately. The recombinant vector was transfected into Huh7 hepatocytes individually by FuGENE6 transfection reagent, and the expression of the fusion protein was verified by Western blot analysis. The recombinant or empty vector was co-transfected to Huh7 hepatocytes with IFN-α response reporter plasmid p6-16CAT at the molar ratio of 5： 1,10： 1,15：1 and 30：1 ,and cells were treated with IFN-α2a （100 IU/ml） 48 h post-transfection. After 24 h of stimulation, the cells were lysed and the intraeellular CAT value was calculated by ELISA assay. The results demonstrated that, as compared to the empty vector, the intracellular CAT values were gradually reduced in parallel with an increasing amount of TS′X′ or TS′ recombinant （n = 6, P 〈 0.05 ） , while no significant differences were observed between TS′X′ and TS′ recombinants （n = 6,P 〉 0. 05 ）. It was concluded that the novel protein TS′X′ encoded by the 3022-1787nt deletion mutant of HBV suppressed the response of Huh7 hepatocytes to IFN-α,and the N- terminal region was a functional domain for its anti-IFN-α effects.