CAJ | 학술논문

目的：构建表达双链 RNA 依赖性蛋白激酶（PKR）融合绿色增强荧光蛋白（pEGFP-PKR）真核表达质粒，并进一步研究 PKR 蛋白在体外抗乙型肝炎病毒（HBV）活性。方法以 pEGFP-N1为空载体，运用分子克隆技术构建重组质粒pEGFP-PKR，通过双酶切和直接测序两种方法验证重组质粒pEGFP-PKR 是否构建成功。以能分泌完整 HBV 病毒颗粒子的肝胚瘤细胞株 HepG2.2.15细胞为细胞模型，采用重组质粒转染方式处理 HepG2.2.15细胞，运用荧光显微镜观察融合蛋白 pEGFP-PKR 在细胞内的表达，以电化学发光方法和实时荧光定量 PCR 技术分析细胞上清 HBV 抗原表达和细胞病毒复制水平。结果酶切鉴定和序列分析证实成功构建重组质粒 pEGFP-PKR，转染 HepG2.2.15细胞后在荧光显微镜下可见融合蛋白 pEGFP-PKR 表达，同时细胞分泌的HBV 抗原与空载体组相比较明显下降（P ＜0.05），而细胞外HBV 复制水平未见明显变化。结论在体外肝细胞模型中，PKR 蛋白具有一定的抗 HBV 活性作用。
목적：구건표체쌍련 RNA 의뢰성단백격매（PKR）융합록색증강형광단백（pEGFP-PKR）진핵표체질립，병진일보연구 PKR 단백재체외항을형간염병독（HBV）활성。방법이 pEGFP-N1위공재체，운용분자극륭기술구건중조질립pEGFP-PKR，통과쌍매절화직접측서량충방법험증중조질립pEGFP-PKR 시부구건성공。이능분비완정 HBV 병독과입자적간배류세포주 HepG2.2.15세포위세포모형，채용중조질립전염방식처리 HepG2.2.15세포，운용형광현미경관찰융합단백 pEGFP-PKR 재세포내적표체，이전화학발광방법화실시형광정량 PCR 기술분석세포상청 HBV 항원표체화세포병독복제수평。결과매절감정화서렬분석증실성공구건중조질립 pEGFP-PKR，전염 HepG2.2.15세포후재형광현미경하가견융합단백 pEGFP-PKR 표체，동시세포분비적HBV 항원여공재체조상비교명현하강（P ＜0.05），이세포외HBV 복제수평미견명현변화。결론재체외간세포모형중，PKR 단백구유일정적항 HBV 활성작용。
Aim To construct and express the eukary-otic expression vector of double-stranded RNA-depend-ent protein kinase (PKR)fusion green fluorescent and analyse its antiviral activity of HBV in vitro.Methods The PKR gene was cloned into an empty expression vector pEGFP-N1 using molecular clone technology. After being confirmed by restriction enzyme digestion and sequencing methods,the recombinant plasmid was named as pEGFP-PKR that was subsequently transfect-ed into HepG2.2.15 cells using LipofectamineTM2000. The expression level of PKR in HepG2.2.15 cells was confirmed by using fluorescent microscopy. Mean-while,HBV DNA and HBsAg/HBeAg were detected by real-time PCR and electrochemiluminescence meth-od,respectively.Results Both restriction enyme di-gestion and sequencing assays showed that the recombi-nant vector pEGFP-PKR was successfully constructed in our study.Fluorescent microscopy observation indi-cated that the fusion protein pEGFP-PKR expressed ef-ficiently in HepG2.2.15 cells.Moreover,compared with the empty vector group,the expression of HBV antigen in supernatants was significantly decreased (P<0.05 ).However,the extracellular HBV DNA ex-pression was not inhibited significantly.Conclusion In vitro,PKR proteion has certain antiviral activity of HBV.