在基于BAC的EB病毒基因组中引入突变

Gene modification in the genome of Epstein-Barr virus cloned as a bacterial artificial chromosome

为了在Epstein-Barr病毒(EBV)172kb的基因组中引入突变以研究基因功能,建立了一种简单有效的基因操作方法。在载体pcDNA3.1(+)上操作,将两端含有重组蛋白FLP识别位点(FRT)的卡那霉素筛选标记基因(kan)与鼻咽癌(NPC)来源的、包含LMP1基因全长ORF的gDNA"无缝"连接(无外源序列插入)。连接后的kan-LMP1线性DNA片段经转化、由λ噬菌体中redαβγ系统介导在E.coli中发生同源重组(ET克隆),用kan-LMP1替代了BAC-EBV(p2089)中相应的LMP1基因区域,然后经过重组蛋白FLP对FRT-kan-FRT特异性的识别,切除了引入的kan基因,留下一个69bp的FRT"疤痕"。通过抗性筛选和对菌液进行PCR扩增可以鉴定突变子。这种经改进并程序化的方法,也适应于引入其它突变或在其它BAC-疱疹病毒基因组中引入突变。

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 pKD 13 and inserted into the vector of pcDNA3.1 （＋）. Through the introduction of restriction endonuclease BsmB Ⅰ in PCR primers, NPC-derived LMPI 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 redαβγ system from bacteriophage λ. 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.