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

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。

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

Objective] The aim was to construction a Vero cell line stably expressing SLAM.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 expressing 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 establishes SLAM-expressing cell line,which provides a platform for studying the isolation and biological functions of canine distemper virus.