迷你SCC重组质粒的构建及ccr重组酶对SCCmec基因岛的整合功能研究

目的构建4组迷你SCC重组质粒载体,并导入受体菌N315ex中,验证并比较ccrC和ccrAB重组酶具有识别特异性DNA片段,进而把Ⅴ型或Ⅱ型SCCmec基因岛整合入细菌染色体上的功能。方法以转化株0342（pSR5C）和N315（pSR2AB）中抽提的染色体DNA为模板,PCR扩增DNA片段attⅤ和attⅡ,并连接到pYT3载体上,构建pYTattⅤ和pYTattⅡ;将PCR扩增的重组酶基因ccrC0342和ccrAB315分别交叉克隆到pYTattⅤ和pYTattⅡ上,构建重组质粒,并导入N315ex受体菌,在适宜温度培育下充分表达质粒,应用在不同温度培养下含药培养基上菌落数量变化及PCR方法,检测各转化株中质粒整合频率,验证质粒插入方向。结果成功构建4组迷你SCC重组质粒。转化株N315ex（pSR5attⅤ）、N315ex（pSR5attⅡ）和N315ex（pSR2attⅡ）中迷你SCC质粒以不同频度和特定方向整合入细菌染色体中,整合效率分别为8.600%、0.005%和7.200%。N315ex（pSR2attⅤ）中未见整合发生。结论与ccrAB仅可识别Ⅱ型SCCmec基因岛的attⅡ相比,ccrC重组酶可识别V型SCCmec的attⅤ和Ⅱ型SCCmec的attⅡ两组DNA片段,发挥其重组整合作用。

Construction of mini-SCC recombinant plasmids and functional research on ccr recombinase genes mediating SCCmec island integration

Objective To construct four recombinant plasmids designed as mini-SCCs and introduce them into recipient strain N315ex in order to observe the accurate integration of mini-SCCs into bacterial chromosomes mediated by ccr recombinase genes.Methods AttⅤ and attⅡ DNA fragments were amplified with chromosomal DNA isolated from 0342（pSR5C） and N315（pSR2AB） as PCR templates.AttⅤ and attⅡ fragments were cloned into temperature-sensitive plasmid pYT3 to construct pYTattⅤ and pYTattⅡ.Cloning the PCR-amplified ccrC0342 and ccrAB315 genes into pYTattⅤ and pYTattⅡ,respectively,yielded four recombinant plasmids,i.e.mini-SCC plasmids.Mini-SCC plasmids were introduced into N315ex by electroporation.The subsequent transformants were tested for plasmid integration frequency and integration direction by counting bacterial clones grown on medium at different temperatures using PCR.Results Accurate integration of mini-SCC plasmids into bacterial chromosomes was observed in transformants of N315ex（pSR5attⅤ）,N315ex（pSR5attⅡ）,and N315ex（pSR2attⅡ）with varying frequency.Integration of mini-SCCs in N315ex（pSR2attⅤ）was not observed.Conclusion Compared to ccrAB recombinase,which recognizes only the att DNA sequence in type Ⅱ SCCmec islands,ccrC recombinase recognized att DNAs in type Ⅴ SCCmec islands as well as in type Ⅱ SCCmec islands,thus acting as a true integration recombinase.