稳定表达犬信号淋巴细胞激活因子基因细胞株的建立与鉴定

Construction and Identification of a Vero Cell Line Stably Expressing Canine SLAM Gene

[目的]构建稳定表达犬瘟热病毒细胞受体———犬信号淋巴细胞激活因子(SLAM)的非洲绿猴肾细胞株(Vero)。[方法]采用RT-PCR方法从犬外周血淋巴细胞中扩增出SLAM基因,将其克隆到哺乳动物真核表达载体pcDNA3.1(+)中,构建重组质粒pcDNA3.1/SLAM。采用脂质体将pcDNA3.1/SLAM转染到Vero细胞中,利用G418加压筛选和纯化培养获得稳定表达SLAM的重组Vero细胞株。应用RT-PCR和间接免疫荧光试验检测SLAM的表达。[结果]重组蛋白SLAM在Vero细胞中获得表达,并且在不同代次的阳性细胞株中均能稳定表达目的蛋白。[结论]该研究建立了稳定表达犬SLAM的细胞株Vero/SLAM,为犬瘟热病毒的分离和生物学特性研究提供了平台。

[Objective] Theaim was to construction a Vero cell line stably expressing SIAM. [Method] SLAM gene was amplified by RT-PCR from canine peripheral blood Lymphocytes and inserted into the eukaryotic expression vector pcDNA3.1 （ ＋ ） to construct a recombinant plasmid pcDNA3.1 （ ＋ ）/SLAM. The pcDNA3.1 （ ＋ ）/SLAM was then transfected into the Vero cells by Lipofectamine 2000. The Vero cell stably ex- pressing the SLAM was screened and purified under the drug selection of G418. The expression of SLAM was confirmed by RT-PCR and indirect immunofluorescence assay. [ Result ] The recombinant SLAM was correctly and effectively expressed in Vero cells. [ Conclusion ]The study estab- lishes SLAM-expressing cell line,which orovides a platform for studying the isolation and biological functions of canine distemper virus.