马立克氏病病毒pp38基因上游的一个双向启动子研究  

A Bi-directional Promoter at The Upstream of pp38 Gene from Marek's Disease Virus

作　　者：丁家波[1] 崔治中[1] 孙淑红[1] 姜世金[1] 

机构地区：[1]山东农业大学动物科技学院,泰安271018

出　　处：《微生物学报》2004年第2期162-166,共5页Acta Microbiologica Sinica

基　　金：国家自然科学基金 ( 3 0 0 70 5 44 )~~

摘　　要：马立克氏病病毒 (MDV)pp38基因上游是病毒基因组DNA复制原点。在其两侧均含有启动子TATA box、CAAT box等特征性的保守基元 ,推测是一个天然的双向启动子。为了在体外验证其双向启动活性 ,本研究以MD Vpp38为报告基因 ,并将其ORF插入到pUC18中 ,构建了pUC pp38质粒。将包含该启动子完整区域的 789bp序列分别以正反两个方向克隆进pUC pp38质粒中pp38报告基因的上游 ,获得的重组质粒pProfpp38和pProrpp38。将所获得的重组质粒分别转染鸡胚成纤维细胞 (CEF) ,通过间接免疫荧光试验检测pp38基因的表达以验证该启动子的双向启动活性。结果表明 ,马立克氏病病毒复制原点区的启动子无论以何种方向插入pUC pp38质粒中 ,在转染细胞2 4h内能检测到pp38基因的表达 ,4 8h后能获得高效和持续的表达。逐渐缩小该启动子的范围 ,最终在 32 0bp时 ,仍能检测到两个方向较强的启动活性。Marek's disease virus(MDV)'s replicating origin is at the upstream of pp38 gene. On both sides of the region, there are several conserved promoter motifs such as TATA-box, CAAT-box, etc, which is regarded as a bi-directional promoter and enhancer. In order to validate the divergent promoting activity in vivtro, we cloned MDV pp38 gene open reading frame(ORF) into pUC18 vector, and constructed pUC-pp38 as a basic plasmid. The 789bp PCR fragment which contains the complete sequences of MDV's replicating origin was cloned at the upstream of pp38 gene in pUC-pp38 at two different directions. The positive clones named as pPro fpp38 and pPro rpp38 were transfected into chicken embryo fibroblast(CEF) cells. 24 hours after the transfection, green fluorescence can been seen on the cytoplasm of CEF in immunofluorecent assay(IFA). 48 hours and on after the transfection, the IFA positive cells will be up to 50% and the expression level can be maintained for a few days. The results show that this region has bi-directional promoting activity. 320bp was confirmed as the core sequence of this promoter with PCR technique.

关 键 词：马立克氏病病毒 PP38基因 双向启动子 重组质粒 鸡胚成纤维细胞 MDV 

分 类 号：S852.65[农业科学—基础兽医学]