摘要： 为研究口蹄疫病毒RNA聚合酶3D基因的分子生物学特性及定位,构建了3D基因真核表达载体pEGFP-3D,观察了3D在BHK-21细胞中的瞬时表达情况。从重组质粒pMD18-T-3D扩增到3D基因,与pGEM-T easy载体连接,将鉴定为阳性的重组质粒pGEM-3D与表达载体pEGFP-N1分别经BamHⅠ/HindⅢ酶切,进行定向亚克隆;重组表达质粒pEGFP-3D经PCR、酶切鉴定,阳性质粒进行测序。利用脂质体介导阳性pEGFP-3D质粒转染BHK-21细胞,免疫组化检测转染细胞中3D基因表达情况和3D基因在细胞中的定位,Western blotting验证pEGFP-3D融合基因的表达。结果表明,成功构建了重组表达质粒pEGFP-3D,并在BHK-21细胞中进行了表达。免疫组化分析表明,3D蛋白主要定位于细胞核;Western blotting证实pEGFP-3D融合蛋白在80 kDa处出现阳性条带,说明表达的外源蛋白具有免疫活性。pEGFP-3D表达载体转染细胞后很快就能通过观察荧光蛋白而检测出基因的瞬时表达情况,为基因转染研究中确定转染效率、3D的表达情况及蛋白定位等提供了直观的靶标,有望应用于FMDV聚合酶分子的生物学特性研究。

关键词: 口蹄疫病毒, pEGFP-3D表达载体, 定位, 免疫活性

Abstract: To construct pEGFP -3D expression vector and to detect its transient expression and its localization in transfected BHK -211Depending on tow steps of cleavage with BamH I and Hind FMDV 3Dgene can be cleaved from pGEM -T -3D and inserted into the plasmid pEGFP -N11The eukaryotic expression vector pEGFP -3D was constructed, which can express 3D fusion protein. The recombinant plasmid pEGFP -3D was transfected into BHK -21 cells1The pEGFP -3D plasmid was identified by restircted enzyme analysis and DNA sequencing and it was showed it contained. the full length of 3D gene. Immunohistochemistry analysis showed that 3D -GFP fusion protein mainly localized in BHCK -21 cell nucleus1The 3D -GFP fusion protein expressed could react with FMDV polyclonal antibodies inwestern blot analysis, demonstrating that 3D -GFP fusion protein having an immunological activity structure. The pEGFP -3D expression vector makes it possible to study the expression of 3D -GFP fusion protein and the protein localization in transfected cells1Moreover, as a useful tool to study the bio logical function of 3D protein will to necessary.

Key words: Foo- t and -mouth disease virus, pEGFP -3D transient expression vector, Immunohistochemistry analysis, Immunological activity

中图分类号: 

• S432.4+