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.
复制标题
增强的绿色荧光蛋白表达可用于监测体外和小鼠中枢神经系统中鼠冠状病毒的扩散。
DOI：
10.1080/13550280260422686
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发表时间：
2002-10
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;Seo SH;Koval M;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。增强型绿色荧光蛋白基因被插入鼠冠状病毒基因组中，取代非必需基因4。这些病毒从电泳迁移率比mRNA 4稍慢的mRNA表达EGFP基因。在感染的细胞裂解物的蛋白质印迹上检测EGFP蛋白，并且通过显微镜观察感染的细胞和病毒噬斑中的EGFP活性（荧光）。EGFP的表达在组织培养中通过至少六次传代以及在小鼠中枢神经系统的急性感染期间保持稳定。这些病毒复制与野生型病毒在组织培养和小鼠中枢神经系统（CNS）中具有相似的动力学和相似的最终程度。它们作为野生型病毒在动物中引起脑炎和脱髓鞘;然而，它们的毒力有所减弱。比较了仅在刺突基因上不同并表达高神经毒性MHV-4株或更弱神经毒性MHV-A59株刺突基因的同基因EGFP表达病毒;病毒抗原的毒力和传播模式的差异反映了表达这些刺突基因的亲本病毒之间的差异。因此，表达EGFP的病毒将有助于研究小鼠冠状病毒的发病机制。
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.
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影响因子：
9.2
作者：
Haas, J;Park, EC;Seed, B
通讯作者：
Seed, B
影响因子：
3.5
作者：
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5.4
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9.9
作者：
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3.8
作者：
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通讯作者：
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