目的:构建ERK-2基因重组腺病毒载体,检测构建的腺病毒感染原代大鼠生长板软骨细胞的效率以及目的基因的表达。方法:将ERK-2 cDNA亚克隆到腺病毒穿梭载体pAdTrack-CMV中,线性化后与腺病毒骨架质粒pAdEasy-1共同转染E.Col.i B J5183,将筛选、鉴定的重组腺病毒质粒线性化后转染HEK293细胞进行病毒颗粒的包装;流式细胞术检测不同感染复数(MO I)ERK-2重组腺病毒感染原代培养的大鼠肋生长板软骨细胞的效率,W estern b lot检测腺病毒感染的生长板软骨细胞中ERK-2蛋白的表达。结果:成功构建ERK-2重组腺病毒,MO I 50的腺病毒感染原代生长板软骨细胞的效率大于90%,感染的生长板软骨细胞中ERK-2表达显著增加。结论:构建的重组腺病毒可介导ERK-2基因在原代大鼠生长板软骨细胞中高表达。
Severe acute respiratory syndrome (SARS) is the first new epidemic of the twenty-first century. A novel coronavirus (SARS-CoV) has been identified as the causative agent of SARS. The genome of SARS-CoV has 29,727 nucleatides in length. The genome organization, with 11 open reading frames, is similar to that of coronaviruses. Phylogenetic analyses and sequence comparisons showed that SARS-CoV is not closely related to any of the known coronaviruses, indicating neither a mutant nor recombinant of well-characterized coronaviruses. It is a complete new coronavirus from nonhuman host. Pathological studies show that severe immune response, associated to cytokine dysregulation, may be related to the lung damage of fatal SRAS. Recombination of genomes of wild-type strains with vaccine coronavirus is a potential risk associated with the application of living attenuated coronavirus vaccines. The proteinases, controlling the activities of the SARS-CoV replication, and spike protein, involved in viral entry and pathogenesis, represent attractive targets of anti-SARS drug development. Comparative full-length genome sequence analysis of 14 SARS coronavirus isolates suggests a remarkable genetic conservation of the virus. Anti-SARS vaccine and drug development will benefit from this genetic conservation. SARS-CoV is not likely to change rapidly and thus may not readily mutate to a benign infection. The progress in anti-SARS research has been impressive. However, one of the most effective tools in the control of the SARS is quickly tracing and isolating the contacts of stricken patients before they spread the virus further.
