CAJ | 학술논문

目的构建乙型肝炎病毒核心蛋白( HBc )突变体L60V重组质粒,并探讨L60V拮抗α干扰素( IFN-α)抗病毒活性的机制。方法构建HBc突变体融合表达载体pEGFP-L60V,转染人肝胚瘤细胞株 HepG2细胞,荧光显微镜及Western blot 法检测其在细胞中的表达；并以 RT-PCR 及Western blot法检测JAK-STAT信号转导途径分子及抗病毒蛋白表达。结果经酶切和测序分析,成功构建HBc突变体L60V重组质粒；将L60V和HBc野生株表达质粒( pEG-FP-WT)分别转染 HepG2细胞后,以1000 IU/ml IFN-α处理,转染L60V和HBc野生株表达质粒的细胞内抗黏液病毒A(MxA)及 JAK-STAT 信号转导途径分子 STAT1、STAT2、IRF9表达减少,其中在转染 pEGFP-L60V 细胞内的 MxA、STAT1、STAT2和IRF9表达显著减少。结论HBc突变体L60V可能通过 JAK-STAT 信号转导途径抑制抗病毒蛋白MxA的表达来拮抗IFN-α的抗病毒作用；其拮抗IFN-α抗病毒作用比HBc更显著。
목적구건을형간염병독핵심단백( HBc )돌변체L60V중조질립,병탐토L60V길항α간우소( IFN-α)항병독활성적궤제。방법구건HBc돌변체융합표체재체pEGFP-L60V,전염인간배류세포주 HepG2세포,형광현미경급Western blot 법검측기재세포중적표체；병이 RT-PCR 급Western blot법검측JAK-STAT신호전도도경분자급항병독단백표체。결과경매절화측서분석,성공구건HBc돌변체L60V중조질립；장L60V화HBc야생주표체질립( pEG-FP-WT)분별전염 HepG2세포후,이1000 IU/ml IFN-α처리,전염L60V화HBc야생주표체질립적세포내항점액병독A(MxA)급 JAK-STAT 신호전도도경분자 STAT1、STAT2、IRF9표체감소,기중재전염 pEGFP-L60V 세포내적 MxA、STAT1、STAT2화IRF9표체현저감소。결론HBc돌변체L60V가능통과 JAK-STAT 신호전도도경억제항병독단백MxA적표체래길항IFN-α적항병독작용；기길항IFN-α항병독작용비HBc경현저。
Objective To construct recombinant plasmid of L60V mutant core proteins of HBV, and explore its an-tagonistic action on the antiviral action of interferon-α( IFN-α) . Methods Built the fusion expression vector pEG-FP-L60V of HBc mutant,the expression of the proteins was tested by the fluorescent microscope and Western blot after transfection to HepG2 cells. Detect moleculars of JAK-STAT signal transduction pathway and the antiviral pro-tein expression by RT-PCR and Western blot. Results The recombinant plasmid of L60V mutant core proteins of HBV was established successfully according to the result of restriction endonuclease digestion and DNA sequencing;HepG2 cells were transfected with pEGFP-L60V and pEGFP-WT, the expression of the antiviral proteins MxA and JAK-STAT signal transduction pathway moleculars were decreased after treatment with 1 000 IU/ml IFN-α, and de-creased even more in cells transfected with pEGFP-L60V. Conclusion L60V mutant core protein of HBV has the mechanism to antagonize the IFN-α antiviral activity by lowering antiviral protein MxA expression through JAK-STAT signal transduction pathway, and its functions are more efficiently than that of HBc.