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为建立一株稳定表达山羊淋巴细胞活化分子(signaling lym phocyte activation molecule,SLA M)的细胞系,给研究小反刍兽疫病毒(peste des petits ruminants virus,PPRV)感染机制、病毒的分离和疫苗生产等奠定基础,本研究采用PCR扩增了SLAM基因,并将其胞外区连接至pD isplay载体,构建了重组质粒pDisplayg SLAM;利用p Display载体的G 418抗性及抗SLAM单克隆抗体,筛选表达gSLAM的Vero-E6细胞系,对该细胞系进行PCR鉴定、间接免疫荧光试验和Western-blot鉴定。采用该细胞系分离PPRV,并与Vero-E6细胞同时吸附PPRV分离株和疫苗株病毒后进行比较。结果,成功筛选出表达g SLAM的Vero-E6细胞系gSLAM/Vero-E6,并通过PCR方法成功扩增出gSLAM基因。通过间接免疫荧光试验和Western-blot鉴定,gSLAM基因成功表达在细胞膜上,并分离出一株PPRV。gSLAM/Vero-E6细胞系和Vero-E6细胞同时吸附PPRV分离株和疫苗株后,gSLAM/Vero-E6细胞系较Vero-E6细胞产生更明显的细胞病变。上述研究结果表明,gSLAM/Vero-E6细胞系对PPRV是高度敏感,为PPRV的后续研究提供了试验材料。
To establish a cell line that stably expresses signaling lym phocyte activation molecule (SLA M), the mechanism of infection of peste des petits ruminants virus (PPRV), isolation of virus and vaccine production In this study, SLAM gene was amplified by PCR and its extracellular region was connected to pD isplay vector to construct recombinant plasmid pDisplayg SLAM. The expression of G 418 resistance and anti-SLAM monoclonal antibody of p Display vector was screened gSLAM Vero-E6 cell line, the cell line PCR identification, indirect immunofluorescence assay and Western-blot identification. PPRV was isolated using this cell line and compared with Vero-E6 cells after adsorbing PPRV isolate and vaccine strain virus. As a result, gSLAM / Vero-E6 cell line expressing g SLAM was successfully screened and the gSLAM gene was successfully amplified by PCR. By indirect immunofluorescence assay and Western-blot identification, gSLAM gene was successfully expressed on the cell membrane and a PPRV was isolated. After gSLAM / Vero-E6 cell line and Vero-E6 cell line adsorbed PPRV isolate and vaccine strain simultaneously, gSLAM / Vero-E6 cell line produced more obvious cytopathic effect than Vero-E6 cell line. The above results show that gSLAM / Vero-E6 cell line is highly sensitive to PPRV, providing experimental materials for the follow-up study of PPRV.