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沼泽绿牛蛙病毒(Rana grylio virus,RGV)属于虹彩病毒科(Iridoviridae),蛙病毒属(Ranavirus),其基因组中含编码尿嘧啶脱氧核糖核苷三磷酸酶(dUTPase)的基因67R。通过构建缺失67R的重组病毒Δ67R-RGV,探讨67R在RGV复制和感染过程中的功能。首先构建携带有作为标记的绿色荧光蛋白(Enhanced green fluorescence protein,EGFP)基因的质粒(pGL3-67RL-p50-EGFP-67RR),然后将该质粒与RGV基因组在67R位点进行同源重组(homologous recombination),并接种到培养的鲤鱼上皮瘤细胞(Epithelioma papulosum cyprinid,EPC)单层细胞中。利用荧光显微观察,判断并筛选有感染性的重组病毒(Δ67R-RGV)。经过十轮挑斑分离,直至在普通显微镜下观察到的细胞病变空斑与荧光显微镜观察到的绿色荧光空斑完全吻合,获得一株新的、纯化的重组蛙病毒Δ67R-RGV。再以野生型蛙病毒(wt-RGV)为对照,分别扩增并提取wt-RGV和Δ67R-RGV的基因组DNA,进行PCR检测。结果显示,在wt-RGV基因组中可检测到67R;但在Δ67R-RGV基因组中仅检测到EGFP,却检测不到67R,证实在构建重组病毒时,EGFP的确按预期插入67R位点。进一步分别测定了wt-RGV与Δ67R-RGV的一步生长曲线,结果无显著差别,表明67R及其编码产物dUTPase的缺失并不影响RGV的正常复制,67R为病毒非必需基因。
Abstract：The Rana grylio virus(RGV)is a member of the genus Ranavirus.It belongs to the family Iridoviridae,and contains the gene 67 Rencoding dUTPase.In order to investigate the function of 67 Rin the replication and infection of RGV,we constructedΔ67R-RGV,a recombinant virus with deletion of 67 R.First,we constructed the plasmid pGL3-67RL-p50-EGFP-67 RR which carried an enhanced green fluorescence gene(EGFP)as a selectable marker.After homologous recombination between pGL3-67RL-p50-EGFP-67 RR and the RGV genome,Epithelioma papulosum cyprini(EPC)cells were infected with the resulting mixture.Through ten successive rounds of plaque isolation via EGFP selection,all plaques emitted green fluorescence,and finallyΔ67R-RGV was generated.Total DNA ofΔ67R-RGV infected cells was extracted for PCR analyses.Simulateously,mock infected and wild-type RGV(wt-RGV)infected cells were used as a comparison.Results showed that 67 Rcould be detected in wt-RGV infected cells,but that only the EGFP gene was detected inΔ67R-RGV infected cells.Furthermore,one-step growth curves of wtRGV andΔ67R-RGV were similar.Therefore,67 Rand its encoding product dUTPase might not be essential for the growth of RGV.These results suggest that,homologous recombination and recombinant ranavirus could be used to study the gene function of viruses in aquatic animals.
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基本信息:
DOI：10.13242/j.cnki.bingduxuebao.002538
中图分类号:S941.41
引用信息:
[1]黄星,裴超,何利波,等.一株新的重组蛙病毒Δ67R-RGV的构建及基因67R的初步功能鉴定[J].病毒学报,2014,30(05):495-501.DOI:10.13242/j.cnki.bingduxuebao.002538.
基金信息:
国家自然科学基金(31430091)
2014-09-23
2014-09-23
2014-09-23