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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。
Objective To construct four mini SCC recombinant plasmid vectors and introduce them into the recipient strain N315ex to validate and compare the function of ccrC and ccrAB recombinase in recognizing specific DNA fragments to integrate the Ⅴ or SC SCCmec island into the bacterial chromosome . Methods The DNA fragments attⅤ and attⅡ were amplified by PCR using the chromosomal DNA extracted from the transformants 0342 (pSR5C) and N315 (pSR2AB) as templates. The DNA fragments were ligated to pYT3 vector to construct pYTattⅤ and pYTattⅡ. The genes ccrC0342 and ccrAB315 were cloned into pYTattⅤ and pYTattⅡ, respectively. The recombinant plasmids were constructed and introduced into N315ex recipient bacteria. The plasmids were fully expressed under proper temperature. The number of colonies on the drug-containing medium under different temperature and PCR method , Detecting the frequency of plasmid integration in each transformant and verifying the direction of plasmid insertion. Results Four mini-SCC recombinant plasmids were successfully constructed. The mini SCC plasmids of N315ex (pSR5attⅤ), N315ex (pSR5attⅡ) and N315ex (pSR2attⅡ) were integrated into the bacterial chromosomes at different frequencies and specific orientations with the integration efficiencies of 8.600%, 0.005% and 7.200%, respectively. No integration occurred in N315ex (pSR2attV). Conclusion The ccrC recombinase can recognize att Ⅴ of SCCmec of V type and att Ⅱ of SCCmec of type Ⅱ as recombination and integration, compared with that of cc Ⅱ, which can only recognize Ⅱ type SCCmec island.