修饰的痘苗病毒安卡拉株（MVA）基因组中高频的同源重组

痘苗病毒由于其外源基因容量大，表达产物后加工完善等优势而广泛用于基因工程的研究以及基因治疗，痘苗病毒基因组的同源重组现象为其基因操作带来了方便，也被用于很多痘苗病毒基因结构和功能的研究，痘苗病毒安卡拉株（MVA）是一种修饰的复制限制的痘苗病毒，由于极高的安全性，正在实验室和临床应用的很多领域取代普通的痘苗病毒，为提高重组MVA系统的安全性以及筛选重组MVA的效率，发展了一种暂时选择系统，此系统利用分子内2段同向的相同序列发生同源重组去除选择标记k1l基因，从而消除选择标记对宿主可能的危害。利用此暂时表达系统构建了4个携带编码不同长度外源多蛋白质序列的重组MVA，并估算了每次传代的重组频率，结果显示，MVA同源重组频率虽然比其他痘苗病毒株要低，但仍然是较斋的，将带有k1l基因的重组MVA经3-4次盲传（blind passage)，即可获得完全去除选择标记的重组MVA。进一步证明上述利用暂时选择标记k1l基因构建重组MVA的系统具有十分可靠的安全性，适合作为人体活疫苗开发和基因治疗的载体，而且，通过盲传进行筛选，能大大提高去除选择标记的效率，降低鸺建重组MVA的成本。

High Frequency of Homologous Recombination in the Genome of Modified Vaccinia Virus Ankara Strain (MVA)

MVA is a genetically modified vaccinia virus strain, which is replication defective and extremely safe for research and clinical use. To further improve the safety of MVA and achieve more efficient selection of recombinant constructs, a transient selection marker system has been developed, which contains vaccinia virus k1l gene flanked by two identical fragments. In this report, four recombinant MVAs were constructed with this system, and homologous recombination of recombinant MVAs was studied during the construction procedure. The results showed that the recombination frequency of these rMVAs was significantly high, though lower than that observed in other vaccinia virus strains. The k1l free rMVAs could be conveniently obtained after 3 or 4 blind passages. Our data indicated that recombinant MVA with a transient selection marker system was safe for use as live vaccine and gene therapy vector for human. In addition, blind passages could enhance the efficiency in isolation of k1l free recombinants.