CAJ | 학술논문

目的构建真核表达载体pDsRed-Monomer-N1-开放读码框(ORF)3,并在BHK-21细胞观察ORF3的表达及定位. 方法用RT-PCR扩增HEV ORF3基因,用Hind Ⅲ/EcoR Ⅰ酶切ORF3基因及pDsRed-Monomer-N1载体,并构建成重组真核表达载体pDsRed-Monomer-N1-ORF3,将鉴定为阳性的质粒经脂质体转染BHK-21细胞,用RT-PCR检测ORF3基因的过表达,用Wsternblot和免疫组织化学法检测ORF3的表达及定位.结果 pDsRed-Monomer-N1-ORF3重组质粒构建成功,转染BHK-21细胞后ORF3基因正常转录,免疫组织化学结果显示ORF3主要分布在BHK-21细胞的细胞质中,ORF3能与特异性抗体结合.结论在BHK-21中表达的融合蛋白主要定位于细胞质中,为进一步研究该蛋白的生理功能奠定了基础.
목적 구건진핵표체재체pDsRed-Monomer-N1-개방독마광(ORF)3,병재BHK-21세포관찰ORF3적표체급정위. 방법 용RT-PCR확증HEV ORF3기인,용Hind Ⅲ/EcoR Ⅰ매절ORF3기인급pDsRed-Monomer-N1재체,병구건성중조진핵표체재체pDsRed-Monomer-N1-ORF3,장감정위양성적질립경지질체전염BHK-21세포,용RT-PCR검측ORF3기인적과표체,용Wsternblot화면역조직화학법검측ORF3적표체급정위.결과 pDsRed-Monomer-N1-ORF3중조질립구건성공,전염BHK-21세포후ORF3기인정상전록,면역조직화학결과현시ORF3주요분포재BHK-21세포적세포질중,ORF3능여특이성항체결합.결론 재BHK-21중표체적융합단백주요정위우세포질중,위진일보연구해단백적생리공능전정료기출.
Objective To construct a eukaryotic expression vector to express the hepatitis E virus protein open reading frame 3 (ORF3) and investigate the intracellular location of the expressed protein using the baby hamster kidney (BHK-21) fibroblast cell line.Methods The ORF3 gene was amplified by RT-PCR,cloned into the HindⅢ and EcoRI sites in the multicloning site of the pDsRed-Monomer-N1mammalian expression vector that encodes a red fluorescent protein (DsRed),and confirmed by restriction enzyme digestion and sequencing.The recombinant plasmid was then transfected into BHK-21 cells via the Lipofectamine 2000 reagent; the subsequent ORF3 gene overexpression was confirmed by RT-PCR and the protein expression and location was detected by Western blotting and immunofluorescence assay.Results TThe pDsRed-Monomer-N1-ORF3 recombinant plasmid was successfully constructed.After transfection into BHK-21 ceils,the ORF3 gene was transcribed and expressed,and the ORF3 protein was mainly located in the cytoplasm,where it could react with a specific antibody.Conclusion The ORF3-DsRed fusion protein was mainly located in the cytoplasm of BHK-21 fibroblasts,and may represent a useful tool for research on the role of this protein in HEV infection.