%0 Journal Article
%T Gene modification in the genome of Epstein-Barr virus cloned as a bacterial artificial chromosome<br>在基于BAC的EB病毒基因组中引入突变
%A Jianhong Lu
%A Yunlian Tang
%A Ming Zhou
%A Minghua Wu
%A Jue Ouyang
%A Jianming Gao
%A Liming Zhang
%A Dan Li
%A Qiong Chen
%A Wei Xiong
%A Xiaoling Li
%A Ke Tang
%A Guiyuan Li
%A <br>卢建红
%A 唐运莲
%A 周鸣
%A 武明花
%A 欧阳珏
%A 高建明
%A 张荔茗
%A 李丹
%A 陈琼
%A 熊炜
%A 李小玲
%A 唐珂
%A 李桂源
%J 微生物学报
%D 2008
%I 
%X Epstein-Barr virus (EBV) is an oncogenic herpesvirus associated with a variety of malignancies, including Burkitt's lymphoma and nasopharyngeal carcinoma (NPC). Functions of most EBV genes have not been determined. The use of bacterial artificial chromosome (BAC) to clone and modify the genome of EBV has enhanced the gene function study in the context of genome. Infectious clones of EBV were previously established by using EBV-BAC plasmid p2089. In order to further investigate EBV mutant biology, an easy and efficient method for gene modification in EBV-BAC was developed and detailed. The kanamycin gene (kan) flanked by recombinase FLP recognition targets (FRTs) was amplified from plasmid pKD13 and inserted into the vector of pcDNA3.1(+). Through the introduction of restriction endonuclease BsmB I in PCR primers, NPC-derived LMP1 gDNA containing the full-length ORF was then precisely ligated with kan on pcDNA3.1(+). The linear DNA segment of kan-LMP1 was transformed into E. coli DH10B cells containing p2089 and plasmid pKD46, homologous recombination was subsequently mediated by redalphabetagamma system from bacteriophage lambda. By this linear transformation and ET cloning, the full-length LMP1 in EBV-BAC (p2089) was replaced by the kan-LMP1. The introduced kan gene in EBV-BAC genome was eliminated specifically by the recombinase FLP when transformed by plasmid pCP20, leaving an FRT scar of 69 bp. The mutant could be identified by antibiotic screening and PCR amplification on bacteria medium. This method allows the gene of interest to be easily modified alone and then to be introduced into EBV-BAC genome. Following this example of gene substitution, other mutations such as deletion, insertion and point mutation become convenient work, and this improved method can be a potential use of gene modification in other BAC-based herpesvirus genome.
%K Epstein-Barr virus
%K mutation
%K homologous recombination
%K linear transformation<br>EB病毒
%K 突变
%K 同源重组
%K 线性转化
%K 病毒
%K 基因组
%K 突变
%K chromosome
%K artificial
%K bacterial
%K virus
%K genome
%K modification
%K 疱疹
%K 适应
%K 方法
%K 程序化
%K 改进
%K 菌液
%K 抗性筛选
%K 疤痕
%K 切除
%K 区域
%K 替代
%U http://www.alljournals.cn/get_abstract_url.aspx?pcid=90BA3D13E7F3BC869AC96FB3DA594E3FE34FBF7B8BC0E591&jid=A3C6BA55AB623B90FA9104CFFC826F3C&aid=B193A9B3BF82632D41843C9BDDB92339&yid=67289AFF6305E306&vid=B6DA1AC076E37400&iid=38B194292C032A66&sid=44FDB9366EDDFA2B&eid=A22854835F81B3F8&journal_id=0001-6209&journal_name=微生物学报&referenced_num=0&reference_num=17