重组工程是近几年发展的新型遗传工程技术. 以PCR扩增的线性低拷贝质粒pACYC184为载体,用Gap-repair方法从大肠杆菌DY330染色体上直接体内亚克隆了包括Red重组酶基因在内的长约6.7 kb的基因序列,构建了pYM-Red重组质粒. 在宿主菌W3110体内进行了染色体上galk基因的敲除,验证了Red重组酶的生物功能,并确定了影响pYM-Red重组效率的诱导时间和线性DNA 片段用量. 在42℃诱导10 min和线性DNA打靶分子浓度为300 ng时,pYM-Red的重组效率可达到大约每4 000个电转存活细胞中有1个重组阳性克隆,分别比pKD46和pBR322-Red系统高5~6倍.Recombineering is a new developed genetic engineering technology in the past few years. A new recombineering system named pYM-Red was constructed by gap-repair, that is a technology called in vivo cloning. Linear PCR fragments that amplified from low copy plasmid pACYC184 were used as gene targeting vector. The length of subcloned DNA sequence including Red gene and a series regulatory sequences are about 6.7 kb. The biology function of Red gene in pYM-Red was tested by gene replacement (galkkan) of W3110 chromosome. Factors that effect recombination efficiency were precisely confirmed. When induced 10 min at 42℃ and using 300 ng linear DNA fragment as targeting molecules, the efficiency of pYM-Red mediated recombination can reach one positive recombination clone per four thousands electroporation survived cells, it is 5~6 folds higher than pBR322-Red and pKD46 recombination system.军队“十五”医药卫生科学基金( 01MA 089).~
