CAJ | 학술논문

目的通过体外试验探讨furin切割核心蛋白的可能性,并通过重组载体表达,研究其假定产物在HepG2细胞内的分布特点. 方法重组HBV核心蛋白,并将其在不同温度和时间条件下(4℃消化16 h、30℃消化1h和30℃消化3h)接受furin切割,产物采用Western blot分析.构建HBV核心蛋白furin切割假定产物的真核表达载体,并以完整核心蛋白表达载体作为对照,转染HepG2细胞,获得稳定表达细胞株.使用核心蛋白与假定产物共同区的单克隆抗体进行间接免疫荧光染色,用共聚焦显微镜观察其细胞内分布. 结果 HBV核心蛋白在多种条件下均被furin切断,其主要产物相对分子质量约为15 000,与预期相符,且30℃切割1 h的效果最好.真核表达的核心抗原假定切割产物能与核心蛋白单克隆抗体结合,且在细胞内的分布与完整核心蛋白类似,主要以颗粒形式分布在细胞质. 结论 HBV核心蛋白在体外能被furin切割,其主要产物与完整核心蛋白有类似的免疫原性和相同的细胞内分布,提示furin或其家族成员参与了HBV复制调节,其切割产物对机体抗病毒免疫可能存在深远影响,值得深入研究.
목적 통과체외시험탐토furin절할핵심단백적가능성,병통과중조재체표체,연구기가정산물재HepG2세포내적분포특점. 방법 중조HBV핵심단백,병장기재불동온도화시간조건하(4℃소화16 h、30℃소화1h화30℃소화3h)접수furin절할,산물채용Western blot분석.구건HBV핵심단백furin절할가정산물적진핵표체재체,병이완정핵심단백표체재체작위대조,전염HepG2세포,획득은정표체세포주.사용핵심단백여가정산물공동구적단극륭항체진행간접면역형광염색,용공취초현미경관찰기세포내분포. 결과 HBV핵심단백재다충조건하균피furin절단,기주요산물상대분자질량약위15 000,여예기상부,차30℃절할1 h적효과최호.진핵표체적핵심항원가정절할산물능여핵심단백단극륭항체결합,차재세포내적분포여완정핵심단백유사,주요이과립형식분포재세포질. 결론 HBV핵심단백재체외능피furin절할,기주요산물여완정핵심단백유유사적면역원성화상동적세포내분포,제시furin혹기가족성원삼여료HBV복제조절,기절할산물대궤체항병독면역가능존재심원영향,치득심입연구.
Objective To investigate the cleavage of HBV core protein in vivo by proprotein convertase furin or its family members and observe the intracellular localization of the putative cleaved product.Methods Recombinant HBV core protein was incubated with furin under different conditions in vitro, and the reaction was checked with Western blotting. The recombinant vectors expressed the putative cleaved fragment and intact core protein (serves as control) were constructed. The stable expression cell lines were established by transfecting constructs into HepG2 cell line, for which indirect immunofluorescence staining was used by monoclonal anti-HBc against the region shared by core protein and its cleaved product .The confocal microscopy was carried out to observe the intracellular distribution. Results HBV core protein was cleaved by furin in vitro under different tested conditions. The molecular weight of the major cleaved product just about 15 000 was in concordance with the expectation. The expressed cleaved fragment could react to the monoclonal antibody against core protein, and mainly located in cytosol in particle style just like the intact core protein. Conclusion HBV core protein can be cleaved by furin in vitro. The major cleaved product has similar antigenicity and subcellular distribution to core protein. These data suggest that proprotein convertase furin or its family members play important roles in HBV replication regulation, and the cleaved product may be involved in antiviral immunity of HBV infection. Further investigations are imperative.