乙型肝炎病毒DNA聚合酶末端蛋白抗α-干扰素功能区域分析

发布时间：2018-04-12 12:03

本文选题：乙型肝炎病毒 + α-干扰素　；参考：《中华微生物学和免疫学杂志》2007年06期

【摘要】：目的确定乙型肝炎病毒(HBV)DNA聚合酶末端蛋白(terminal protein,TP)抗α-干扰素(IFN-α)的功能区域。方法PCR扩增IFN-α诱导人类6-16基因启动子并克隆入pCAT-Basic中,构建IFN-α反应报告载体p6-16CAT。以FuGENE6转染该报告载体至Huh7细胞并给予不同浓度IFN-α2a刺激,ELISA法检测细胞内氯霉素乙酰基转移酶(CAT)的表达,建立Huh7细胞对IFN-α反应强弱的定量判定标准。构建TP(包括Spacer区)基因缺失突变体重组真核表达载体,通过融合表达的多肽表位抗体,Western blot法检测目的蛋白在Huh7细胞中的表达。重组表达载体分别与报告载体p6-16CAT共转染Huh7细胞,转染后48 h给予终浓度为100 IU/ml的IFN-α2a刺激,作用24 h后裂解细胞,通过检测胞内CAT含量高低评价各缺失突变体抗IFN-α作用,并据此确定TP(包括Spacer区)抗IFN-α作用的功能区域。结果获得6-16基因启动子并构建IFN-α反应报告载体p6-16CAT。转染p6-16CAT的Huh7细胞在IFN-α2a刺激下CAT表达显著增强,且在IFN-α2a终浓度为100 IU/ml时达到最大。West- ern blot显示TP(包括Spacer区)各基因缺失突变体在Huh7细胞中表达相应蛋白。共转染p6-16CAT及部分/全长TP的Huh7细胞在IFN-α2a刺激下,胞内CAT值和对照组相比无显著差别,相反,全长TP+部分/全长Spacer使细胞内CAT值显著下降。结论DNA聚合酶Spacer区与HBV抗IFN-α作用密切相关。
[Abstract]:Objective to determine the functional region of hepatitis B virus (HBV) terminal terminal protein (TPTP) against interferon 伪 (IFN- 伪).Methods IFN- 伪 -induced human 6-16 gene promoter was amplified by PCR and cloned into pCAT-Basic. The IFN- 伪 response report vector p6-16CAT was constructed.The expression of chloramphenicol acetyltransferase (FuGENE6) was detected by FuGENE6 transfection into Huh7 cells with different concentrations of IFN- 伪 2a. The quantitative criteria for the response of Huh7 cells to IFN- 伪 were established.The eukaryotic expression vector of body weight group with deletion mutation of TP- (including Spacer) gene was constructed, and the expression of the target protein in Huh7 cells was detected by Western blot assay with fusion expressed polypeptide epitope antibody.The recombinant expression vector co-transfected Huh7 cells with the report vector p6-16CAT. After 48 h of transfection, the cells were stimulated with 100 IU/ml IFN- 伪 2a at the final concentration. After 24 h, the cells were lysed. The effect of the deletion mutants against IFN- 伪 was evaluated by detecting the CAT content in the cells.The functional regions of TP- (including Spacer region) against IFN- 伪 were determined.Results the 6-16 gene promoter was obtained and the IFN- 伪 response report vector p6-16 CAT was constructed.The expression of CAT was significantly increased in p6-16CAT transfected Huh7 cells stimulated by IFN- 伪 2a, and reached the maximum when the final concentration of IFN- 伪 2a was 100 IU/ml. West- blot showed that TP- 伪 2a gene deletion mutants (including Spacer region) expressed the corresponding protein in Huh7 cells.Under the stimulation of IFN- 伪 2a, the intracellular CAT values of p6-16CAT and partial / full length Huh7 cells were not significantly different from those of the control group. On the contrary, the full length TP partial / full length Spacer significantly decreased the intracellular CAT value.Conclusion the Spacer region of DNA polymerase is closely related to the anti-IFN- 伪 effect of HBV.
【作者单位】：福建医科大学分子医学研究中心;
【基金】：国家自然科学基金(30300015) 福建省重大科技基金(2002F005) 福建省高校创新团队培育计划基金
【分类号】：R373

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