Development of new method for construction of adenovirus vector using gene exchange reaction

建立一种利用Cre重组介导的基因交换反应构建腺病毒载体的方法。我们检测了野生型loxP独立识别的突变体loxP和Cre的真实靶序列，并鉴定了loxP V为突变型loxP，具有很高的敏感性和特异性。我们检测了野生型和突变型loxP基因交换反应的效率，效率不低于10%。构建EL缺失型腺病毒的受体病毒设计为能够通过Cre介导的位于Ψ序列两侧的一对loxP位点之间的重组来切除病毒包装信号(Ψ)。我们将含有Ψ序列和目的基因的受体病毒和供体质粒分别导入表达Cre的293细胞，通过基因交换反应获得了表达目的基因的腺病毒载体。纯目的病毒载体经293细胞有限稀释后可以很容易地分离出来，为了构建一个目的DNA取代所有病毒基因的“空载体”，我们制备了一个新的受体病毒作为辅助病毒。我们还构建了四个含有8碱基识别酶切割位点的供体粘粒盒作为克隆位点，以插入目的基因和大尺寸的填充DNA。

To develop a method for constructing adenovirus vector using gene exchange reaction mediated by Cre recombination. We assayed mutant loxPs recognized independently of wild-type loxP, authentic target sequences of Cre, and identified loxP V as a mutant loxP with high sensitivity and specificity. We examined efficiency of gene exchange reaction using both wild-type loxP and mutant loxP V and showed that the efficiency was no less than 10 %.The recipient virus for constructing recombinant adenovirus of El -deleted type was designed so that the viral packaging signal (Ψ) was able to be excised by Cre-mediated recombination between a pair of loxP sites located at the both sides of Ψ sequences. We introduced to Cre-expressing 293 cells both a recipient virus and a donor plasmid containing Ψ sequences and a purpose gene flanked with loxP and V. After five cycles of infection of Cre-293 cells with crude viral stock obtained, an adenovirus vector expressing the purpose gene was generated through gene exchange reaction. Pure purpose virus vector was able to be easily isolated after limited dilution using 293 cells.To construct "gutted vector" whose all viral genes were replaced by a purpose DNA, we prepared a new recipient virus that serves as a helper virus. We also constructed four different donor cosmid cassettes containing cleavage sites of 8-base recognition enzyme as a cloning site in order to insert a purpose gene and a stuffer DNA of large size.