摘要
由于目前用于小反刍兽疫病毒(PPRV)相关研究的羊源细胞均为原代细胞,其对PPRV的敏感性较差,而信号淋巴细胞激活分子(SLAM)作为PPRV的重要受体可以促进病毒的感染与复制。因此,为构建表达山羊SLAM(gSLAM)的重组腺病毒,为羊源原代细胞提供SLAM受体,本研究将gSLAM基因克隆至腺病毒穿梭载体pShuttle-CMV中,转化至含有5型腺病毒骨架的BJ5183感受态细胞中,获得重组腺病毒载体pAd-gSLAM,转染293A细胞,经PCR鉴定表明获得了重组腺病毒rAd-gSLAM。以MOI 3 rAd-gSLAM感染Vero细胞后,采用western blot检测SLAM的表达。结果显示,rAd-gSLAM能够感染Vero细胞并表达SLAM蛋白。将PPRV疫苗株rPPRV/GFP和中国流行株PPRV/CN/HLJ/13分别以MOI 0.1感染rAd-gSLAM预先感染后的Vero(Vero/rAd-gSLAM)细胞,24 h经荧光显微镜观察,并采用荧光定量PCR检测两株病毒在感染后不同时间点的拷贝数,绘制病毒的生长曲线。荧光显微镜观察可见,rPPRV/GFP感染的Vero/rAd-gSLAM细胞出现较大量绿色荧光且形成明显的细胞融合;PPRV/CN/HLJ/13感染的Vero/rAd-gSLAM细胞形成明显的细胞融合且出现红色荧光。病毒的生长曲线结果显示,rPPRV/GFP在Vero/rAdgSLAM细胞中的拷贝数与其在Vero和Vero/Ad对照细胞中的拷贝数在感染后各时间点差异却均不显著;PPRV/CN/HLJ/13的拷贝数在感染Vero/rAd-gSLAM细胞后24 h即达到峰值,且其在感染该细胞后72 h的拷贝数约为其感染Vero及Vero/Ad细胞在该时间点的48倍。将rAd-gSLAM(MOI 3)感染原代羊源睾丸(LT)细胞后,再以MOI 0.1分别感染rPPRV/GFP和PPRV/CN/HLJ/13,72 h后经荧光显微镜观察可见,前者感染的LT细胞出现绿色荧光,且有明显的细胞融合;后者感染的LT细胞出现明显的细胞融合和CPE。上述结果表明,本研究构建了表达gSLAM的重组腺病毒rAd-gSLAM,其感染Vero细胞系和原代LT细胞后均能够表达gSLAM,增强了PPRV对Vero细胞系和原代LT细胞的感染与复制。本研究为PP
Due to sheep cells currently used for PPRV related research are all primary cells,which had low sensitivity with PPRV infection,where as,signaling lymphocyte activation molecule(SLAM)is an important receptor for PPRV which can promote viral infection and replication.Therefore,in order to construct a recombinant adenovirus expressing goat SLAM(gSLAM)gene and provide SLAM receptors to sheep or goat primary cells,the gSLAM gene was cloned into the adenovirus shuttle vector pShuttle-CMV,and transformed into BJ5183 competent cells containing the gene backbone of the adenovirus type 5 to get plasmid pAd-gSLAM.The recombinant adenovirus plasmid pAd-gSLAM was constructed and transfected into 293A cells and PCR identification showed that the recombinant adenovirus expressing gSLAM(rAd-gSLAM)was obtained.After infection of Vero cells with rAd-gSLAM at a MOI of 3,the expression of foreign protein was detected by western blot test.The western blot results showed that rAd-gSLAM could infect cells and express SLAM protein.Vero cells infected with rAd-gSLAM at a MOI of 3 first,then infected with 0.1 MOI rPPRV/GFP or PPRV/CN/HLJ/13,the cells was observed by fluorescence microscope at 24 hpi,fluorescence quantitative PCR was used to detect the copy number of different viruses at different time points after infection,and the growth curve of the virus was drawn.Fluorescence microscopy showed that Vero/rAd-gSLAM cells infected with rPPRV/GFP showed more green fluorescence and formed obvious cell fusion.Vero/rAd-gSLAM cells infected with PPRV/CN/HLJ/13 formed obvious cell fusion and showed red fluorescence.The virus growth curve results showed that the copy number of rPPRV/GFP in Vero/rAd-gSLAM cells was not significantly different from that in Vero and Vero/Ad cells at each time point after infection.The copy number of PPRV/CN/HLJ/13 infected Vero/rAd-gSLAM cells reached its peak 24 hours after infection,and the copy number of PPRV/HLJ/13 infected Vero/rAd-gSLAM cells at 72 hours after infection was about 48 times that of Vero and V
作者
陈合凤
张帅
王喜军
王金良
温志远
葛金英
陈伟业
步志高
CHEN He-feng;ZHANG Shuai;WANG Xi-jun;WANG Jin-liang;WEN Zhi-yuan;GE Jin-ying;CHEN Wei-ye;BU Zhi-gao(State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Reasearch Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou University,Yangzhou 225009,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2020年第12期1207-1213,共7页
Chinese Journal of Preventive Veterinary Medicine
基金
国家重点研发计划(2016YFD0500108)
国家国际合作专项(2015DFA31300)。
关键词
小反刍兽疫
SLAM
重组腺病毒
VERO
羊源细胞
peste des petitsruminants
SLAM
recombinant adenovirus
Vero
sheep or goat derived cells
