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。
Objective To identify the fimctional region of terminal protein （TP） of HBV DNA polymerase with anti-IFN-α effects. Methods The promoter region of IFN-α-inducible human 6-16 gene was amplified by PCR and cloned into pCAT-Basic vector to construct the IFN-α response reporter vector p6-16CAT. Then the vector was transfected into Huh7 hepatoeytes by FuGENE6 and the cells were treated with serial concentrations of IFN-0.2a. The intracellular CAT （ chloramphenicol acetyltransferase） value was calculated by ELISA to quantitatively evaluate the cellular responsiveness to IFN-α. The recombinant eukaryotic expression vector harboring the serial deletants of TP （including spacer region） was constructed and expression of the fusion protein in Huh7 hepatocytes was examined by Western blot. Huh7 hepatoeytes were co-transfeeted with p6-16CAT and the individual TP deletant expression vector. Forty-eight hours later, the cells were stimulated by IFN-α2a for 24 h. Then the cells were lysed and the intracellular CAT value was calculated to evaluate the anti-IFN-α effects of TP deletants. All data were calculated by one-way analysis of variance（ANOVA）. Results The promoter region of human 6-16 gene was obtained and the IFN-α response reporter vector p6-16CAT was constructed successfully. When Huh7 hepatocytes were transfected with p6-16CAT and treated with IFN-α2a, the expression of CAT increased significantly and peaked when the concentration of IFN-α2a reached 100 IU/ml. Western blot showed that TP （including Spacer region） ddetants expressed well in Huh7 cells. When Huh7 hepatocytes were co-transfected with p6-16CAT and coding for partial or full length of TP and treated with IFN-α2a, the intracellular CAT values showed no significant difference with empty vector group. In contract, the intracellular CAT values significantly decreased while the recombinant coding full length TP plus partial or full length of spacer region was used for co-transfection. Spacer region of DNA polymerase was closely correlated with the anti-IFN-α effects of HBV.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2007年第6期540-545,共6页
Chinese Journal of Microbiology and Immunology
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
