Enhanced green fluorescent protein expression may be used to monitor murine coronavirus spread in vitro and in the mouse central nervous system
Enhanced green fluorescent protein expression may be used to monitor murine coronavirus spread in vitro and in the mouse central nervous system
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DOI：
10.1080/13550280290100815
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发表时间：
2002-10-01
影响因子：
3.2
通讯作者：
Weiss, SR
中科院分区：
文献类型：
--
作者：
Das Sarma, J;Scheen, E;Weiss, SR
利用靶向重组技术筛选稳定高效表达增强型绿色荧光蛋白(EGFP)基因的小鼠肝炎病毒分离株。EGFP基因被插入到小鼠冠状病毒基因组中，取代了非必需基因4。这些病毒从比mRNA4稍慢的mRNA中表达EGFP基因。在感染细胞裂解产物的Western印迹上检测到EGFP蛋白，并在显微镜下观察到感染细胞和病毒斑块中的EGFP活性(荧光)。在组织培养中和在小鼠中枢神经系统急性感染期间，EGFP的表达在至少六代中保持稳定。这些病毒在组织培养和小鼠中枢神经系统(CNS)中复制的速度和最终程度与野生型病毒相似。它们作为野生型病毒在动物身上引起脑炎和脱髓鞘；然而，它们的毒力有所减弱。比较了表达强神经毒力的MHV-4毒株和神经毒力较弱的NMV-A59毒株的表达尖峰基因的同基因EGFP病毒；毒力和病毒抗原传播模式的差异反映了表达这些尖峰基因的亲本病毒之间的差异。因此，表达EGFP的病毒将在小鼠冠状病毒致病机制的研究中发挥重要作用。
Targeted recombination was used to select mouse hepatitis virus isolates with stable and efficient expression of the gene encoding the enhanced green fluorescent protein (EGFP). The EGFP gene was inserted into the murine coronavirus genome in place of the nonessential gene 4. These viruses expressed the EGFP gene from an mRNA of slightly slower electrophoretic mobility than mRNA 4. EGFP protein was detected on a Western blot of infected cell lysates and EGFP activity (fluorescence) was visualized by microscopy in infected cells and in viral plaques. Expression of EGFP remained stable through at least six passages in tissue culture and during acute infection in the mouse central nervous system. These viruses replicated with similar kinetics and to similar final extents as wild-type virus both in tissue culture and in the mouse central nervous system (CNS). They caused encephalitis and demyelination in animals as wild-type virus; however, they were somewhat attenuated in virulence. Isogenic EGFP-expressing viruses that differ only in the spike gene and express either the spike gene of the highly neurovirulent MHV-4 strain or the more weakly neurovirulent NMV-A59 strain were compared; the difference in virulence and patterns of spread of viral antigen reflected the differences between parental viruses expressing each of these spike genes. Thus, EGFP-expressing viruses will be useful in the studies of murine coronavirus pathogenesis in mice.