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  

The Construction of a Novel Recombinant Virus Δ67R-RGV and Preliminary Analyses the Function of the 67R Gene

作　　者：黄星[1,2] 裴超[1,2] 何利波[1,2] 张奇亚[1,2] 

机构地区：[1]中国科学院水生生物研究所、淡水生态与生物技术国家重点实验室,武汉430072 [2]中国科学院研究生院,北京100049

出　　处：《病毒学报》2014年第5期495-501,共7页Chinese Journal of Virology

基　　金：国家自然科学基金(31430091)

摘　　要：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。The Rana grylio virus （RGV） is a member of the genus Ranavirus. It belongs to the family Iri- doviridae, and contains the gene 67R encoding dUTPase. In order to investigate the function of 67R in the replication and infection of RGV, we constructed △67R-RGV, a recombinant virus with deletion of 67R. First, we constructed the plasmid pGL3-67RL-p50-EGFP-67RR which carried an enhanced green fluores- cence gene （EGFP） as a selectable marker. After homologous recombination between pGL3-67RL-p50-EG- FP-67RR and the RGV genome, Epithelioma papulosurn cyprini （EPC） cells were infected with the resul- ting mixture. Through ten successive rounds of plaque isolation via EGFP selection, all plaques emitted green fluorescence, and finally △67R-RGV was generated. Total DNA of △67R-RGV infected cells was ex- tracted for PCR analyses. Simulateously, mock infected and wild-type RGV （wt-RGV） infected cells were used as a comparison. Results showed that 67R could be detected in wt-RGV infected cells, but that only the EGFP gene was detected in △67R-RGV infected cells. Furthermore, one-step growth curves of wt- RGV and △67R-RGV were similar. Therefore, 67R and its encoding product dUTPase might not be essen- tial for the growth of RGV. These results suggest that, homologous recombination and recombinant rana- virus could be used to study the gene function of viruses in aquatic animals.

关 键 词：重组虹彩病毒 沼泽绿牛蛙病毒(RGV) 尿嘧啶脱氧核糖核苷三磷酸酶(dUTPase) 基因缺失 一步生长曲线 

分 类 号：S852.65[农业科学—基础兽医学]