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首页> 外文期刊>Journal of neurovirology >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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Enhanced green fluorescent protein expression may be used to monitor murine coronavirus spread in vitro and in the mouse central nervous system.

机译：增强的绿色荧光蛋白表达可用于监测鼠冠状病毒在体外和小鼠中枢神经系统中的传播。

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摘要

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 MHV-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.
机译：靶向重组被用于选择稳定和有效表达编码增强型绿色荧光蛋白（EGFP）的基因的小鼠肝炎病毒分离株。将EGFP基因插入鼠源性冠状病毒基因组中，代替非必需基因4。这些病毒从电泳迁移率比mRNA 4稍慢的mRNA中表达EGFP基因。在感染细胞裂解物和EGFP的Western印迹中检测到EGFP蛋白。通过显微镜在感染的细胞和病毒斑块中观察到活性（荧光）。在组织培养物中和小鼠中枢神经系统急性感染过程中，EGFP的表达通过至少六次传代保持稳定。这些病毒在组织培养和小鼠中枢神经系统（CNS）中均以与野生型病毒相似的动力学和最终程度复制。它们以野生型病毒的形式引起了动物的脑炎和脱髓鞘。但是，它们的毒力有所减弱。比较了仅在穗基因上不同并且表达高神经毒性MHV-4株或神经毒性较弱MHV-A59株的穗基因的等基因表达EGFP的病毒;病毒抗原的毒力和传播方式的差异反映了表达每个这些刺突基因的亲本病毒之间的差异。因此，表达EGFP的病毒将可用于小鼠鼠冠状病毒发病机理的研究。

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