摘要
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.
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.
出处
《中华肝脏病杂志》
CAS
CSCD
北大核心
2014年第7期499-503,共5页
Chinese Journal of Hepatology
基金
国家自然科学基金（81260326）
兰州大学第一医院青年基金（Ldyyynqn201206）
