Efficient construction of a recombinant adenovirus vector by an improved in vitro ligation method
Efficient construction of a recombinant adenovirus vector by an improved in vitro ligation method
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DOI：
10.1089/hum.1998.9.17-2577
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发表时间：
1998-11-20
影响因子：
4.2
通讯作者：
Kay, MA
中科院分区：
文献类型：
--
作者：
Mizuguchi, H;Kay, MA
建立了一种基于体外连接的重组腺病毒载体的构建方法。为了将外源基因插入腺病毒DNA中，我们将三个独特的限制性酶切位点I-CeuI、SwaI和PI-SceI引入载体质粒的El缺失位点，该载体质粒含有完整的El，E3缺失的5型腺病毒基因组。I-CeuI和PI-SceI是内含子编码的核酸内切酶，分别具有至少9-10和11 bp的序列特异性。构建了穿梭质粒pHM 3，其在I-Cen I和PI-Sce I位点之间含有多个克隆位点。将目的基因插入该穿梭质粒后，通过使用I-CeuI和PI-SceI位点的体外连接，可以容易地制备用于El缺失的腺病毒载体的质粒。连接产物的SwaI消化阻止了含有亲本腺病毒基因组的质粒（空载体）的产生。转化E. coli中，90%以上的转化子具有正确的插入片段。为了制备载体，将PacI消化的线性化质粒转染到293细胞中，产生重组病毒的同质群体。独特限制性位点的大量和战略位置不仅将提高用于基因转移的新的第一代载体的生产速度，而且将允许载体DNA骨架的快速进一步改进。
An efficient method for constructing a recombinant adenovirus (Ad) vector, based on an in vitro ligation, has been developed. To insert the foreign gene into an adenoviral DNA, we introduced three unique restriction sites, I-CeuI, SwaI, and PI-SceI, into the El deletion site of the vector plasmid, which contains a complete El, E3-deleted adenovirus type 5 genome. I-CeuI and PI-SceI are intron-encoded endonucleases with a sequence specificity of at least 9-10 and 11 bp, respectively. A shuttle plasmid, pHM3, containing multiple cloning sites between the I-CenI and PI-SceI sites, was constructed. After the gene of interest was inserted into this shuttle plasmid, the plasmid for El-deleted adenovirus vector could be easily prepared by in vitro ligation using the I-CeuI and PI-SceI sites. SwaI digestion of the ligation products prevented the production of a plasmid containing a parental adenovirus genome (null vector). After transformation into E. coli, more than 90% of the transformants had the correct insert. To make the vector, a PacI-digested, linearized plasmid was transfected into 293 cells, resulting in a homogeneous population of recombinant virus. The large number and strategic location of the unique restriction sites will not only increase the rapidity of production of new first-generation vectors for gene transfer but will allow for rapid further improvements in the vector DNA backbone.