乙型肝炎病毒458nt-1308nt剪接特异性蛋白抗α-干扰素作用

The anti-IFN-a effects of the novel protein encoded by the 458 nt-1308 nt spliced variant of hepatitis B virus genome

目的研究乙型肝炎病毒（HBV）458nt-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HBV458nt-1308t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100IU／ml的IFN-α2a刺激，作用24h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，Western blot显示各基因片段在Huh7细胞中均表达相应蛋白。06-16CAT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HBV458nt-1308nt剪接特异性蛋白抑制Huh7细胞对IFN-α的反应性，其活性与N端的TP及Spacer区有关。

Objective To investigate the anti-IFN-α effects of the novel protein TSR′r′ encoded by the 458 nt-1308 nt spliced variant of hepatitis B virus genome, and to determine its functional domains. 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 ceils were lysed and the intracellular CAT value was calculated. All data were processed with One-way analysis of variance（ANOVA）. Results 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 virus 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.