稳定表达貉犬瘟热病毒细胞受体SLAM的Vero细胞系的建立及应用

Establishment and application of a Vero cell line stably expressing raccoon dog SLAM，the cellular receptor of canine distemper virus

摘要:

摘要:【目的】信号淋巴激活分子(SLAM 又称CD150)为犬瘟热病毒(Canine distemper virus，CDV)的细胞受体。本研究目的在于建立稳定表达犬瘟热易感动物貉SLAM基因的Vero细胞系，用于犬瘟热诊断及CDV强毒株的快速分离。【方法】从重组质粒pMD-18-T-rSLAM扩增rSLAM 基因编码区，通过分子克隆技术构建真核表达质粒pIRES2-EGFP-rSLAMhis。将该质粒转染Vero细胞后，经EGFP荧光观察、G418抗性压力和单细胞克隆化及RT-PCR筛选表达rSLAM阳性细胞系。应用获得细胞系对临床犬瘟热病例进行病毒分离，对分离得到CDV进行RT-PCR鉴定及动物回归试验。【结果】经过RT-PCR和免疫组化试验证实，本实验筛选获得一株能稳定表达rSLAM的Vero细胞系———Vero-rSLAM。该细胞系接种CDV阳性样品36－48h即可产生典型CDV致细胞融合病变，而其亲本Vero细胞接种至6d均无可见细胞病变出现。应用Vero-rSLAM细胞系从狐狸、貉犬瘟热阳性病料中分离获得了3株犬瘟热病毒。其中犬瘟热病毒LN(10)f1株对易感狐狸、貉均可产生致死性感染。【结论】成功建立了稳定表达rSLAM的Vero细胞系，用它分离的CDV对本动物保 持较强的毒力。

Abstract:

Abstract:［Objective］ The signaling lymphocyte activation molecule (SLAM，also known as CD150)，is used as a cellular receptor by canine distemper virus (CDV). Wild-type strains of CDVs can be isolated and propagated efficiently in non-lymphoid cells expressing this protein. Our aim is to establish a Vero cells expressing raccoon dog SLAM(rSLAM) to efficiently isolate CDV from pathological samples.［Methods］ A eukaryotic expression plasmid，pIRES2-EGFPrSLAMhis，containing rSLAM gene fused with six histidine-coding sequence，EGFP gene，and neomycin resistance gene was constructed. After transfection with the plasmid，a stable cell line，Vero-rSLAM，was screened from Vero cells with the identification of EGFP reporter and G418 resistance． Three CD positive specimens from infected foxes and raccoon dogs were inoculated to Vero-rSLAM cells for CDV isolation． Foxes and raccoon dogs were inoculated subcutaneously LN (10) f1 strain with 4×102. 39 TCID50 dose to evaluate pathogenicity of CDV isolations.［Results］The rSLAMh fused gene was shown to transcript and express stably in Vero-rSLAM cells by RT-PCR and Immunohistochemistry assay．Three CDV strains were isolated successfully in Vero-rSLAM cells 36－48 hours after inoculation with spleen or lung specimens from foxes and raccoon dogs with distemper． By contrast，no CDV was recovered from those CD positive specimens when Vero cells were used for virus isolation．Infected foxes and raccoon dogs with LN(10) f1 strain all showed typical CD symptoms and high mortality (2/3 for foxes and 3/3 for raccoon dogs) in 22 days post challenge.［Conclusion］Our results indicate that Vero-rSLAM cells stably expressing raccoon dog SLAM are highly sensitive to CDV in clinical specimens and the CDV isolation can maintain high virulence to its host animals.