DC-SIGN真核表达载体的构建及其稳定转染HeLa细胞系的建立

CONSTRUCTION OF EUKARYOTIC EXPRESSION VECTOR AND ESTABLISHMENT OF HELA CELL LINE WITH STABLE EXPRESSION OF DC- SIGN

为研究新城疫病毒(Newcastle disease virus,NDV)与树突状细胞特异性粘附分子-3-结合非整合素分子(dendritic cell specific intercellular adhesion molecule-3-grabbing non-integrin,DC-SIGN)的相互作用对NDV感染树突状细胞(dendritic cell,DC)的影响,本研究拟构建能够稳定表达DC-SIGN蛋白的细胞系,为研究DC-SIGN蛋白与病毒蛋白互作提供基础。以小鼠DCs提取的c DNA为模板,通过PCR方法获得DC-SIGN基因,连入真核表达载体,并命名为pcDNA-DCSIGN,利用LipofectamineTM 2000转染HeLa细胞,G418进行药物压力筛选,经RT-PCR和Western blot鉴定,获得了一株可以高效表达DC-SIGN蛋白的HeLa细胞,且经过多次传代后仍然可以稳定表达DC-SIGN,说明该细胞系构建成功。

To investigate the influence of the interaction between Newcastle diseases virus（NDV） protein with dendritic cells specific adhesion molecules- 3- grabbing non-integrin（DC-SIGN） on NDV infection of dendritic cell（DC）, we generated a Hela cell line stably expressing DC- SIGN protein for studying DC- SIGN protein and virus protein interactions. The cDNA was extracted from mouse DCs and used as template. The DC- SIGN gene was amplified in polymerase chain reaction（PCR） for construction of eukaryotic expression vector pcDNA-DC-SIGN, which was then transfected into He La cells using LipofectamineTM 2000. A HeLa cell line efficiently expressing DC- SIGN protein was identified through RT-PCR and Western blot, and the specific Hela cell line was stable after many passages.