中文摘要：

目的构建携带ccrC和ccrAB重组酶基因的载体质粒,并导入耐甲氧西林金黄色葡萄球菌（MRSA）中,验证ccrC重组酶具有把Ⅴ型SCCmec基因岛从细菌染色体上特异性剪切下来的功能。方法以携带Ⅴ型SCCmec基因岛的菌株81/0342的染色体DNA为模板,应用PCR法扩增ccrC基因,再以携带Ⅱ型SCCmec基因岛的菌株N315的染色体DNA为模板,应用PCR法扩增ccrAB基因,分别克隆到温度敏感质粒pYT3上构建重组质粒pSR5C和pSR2AB,电穿孔技术交叉导入原野生菌株81/0342和N315中构建6株细菌转化株,在适宜温度培育下充分表达质粒,应用PCR法检测SCCmec基因岛从染色体上脱落,并连续10d传代培养细菌,应用影印实验检查细菌的药物敏感性变化。结果ccrC重组酶基因的PCR扩增产物为1.9kb,小于Ⅱ型SCCmec基因岛上携带的ccrAB重组酶基因。经鉴定证实成功构建了重组质粒pSR5C和pSR2AB,当菌株81/0342中导入重组质粒pSR5C和pSR2AB后,81/0342所携带的Ⅴ型SCCmec基因岛能从染色体上剪切下来,并以环状DNA结构存在于细菌中,而只有当N315中导入重组质粒pSR2AB时,N315所携带的Ⅱ型SCCmec基因岛才能脱落下来,SCCmec基因岛脱落的细菌成为对妥布霉素和头孢唑肟药物敏感的菌株。结论ccrC重组酶与ccrAB一样具有特异性剪切酶的功能,可单独介导Ⅴ型SCCmec基因岛的脱落,从而为社区MRSA所携带的Ⅴ型SCCmec基因岛在金黄色葡萄球菌之间的广泛传播提供了实验依据。

英文摘要：

Objective To construct 2 recombinant plasmids carrying cassette chromosome recombinase C（ccrC） and ccrAB respectively and introduce the plasmids into methicillin-resistant Staphylococcus aureus （MRSA）strain 81/0432,and to observe the precise excision of Staphylococcal cassette chromosome mec （SCCmec）island from bacterial chromosome. Methods ccrC and ccrAB genes were amplified with chromosomal DNAs isolated from MRSA strains 81/0342 and N315 as PCR templates. We constructed recombinant plasmids pSR5C and pSR2AB by cloning ccrC and ccrAB genes into temperature-sensitive plasmid pYT3,after introducing them into MRSA strains 81/0432 and N315 by electroporation. PCR was performed to identify SCCmec excision from the bacterial chromosome. The transformants were serial passaged for 10 days,and then the drug resistance of these transformants was detected by replica experiment. Results The fragment length of ccrC gene was 1.9 kb,smaller than the fragment length of ccrAB from N315. The recombinant plasmids of pSR5C and pSR2AB were successfully constructed. After these 2 recombinant plasmids were introduced into MRSA strain 81/0342,type-Ⅴ SCCmec island was excised from the chromosome and formed a closed circular structure in the bacteria. However,type-Ⅱ SCCmec island could be excised only in N315 strain after the expression of pSR2AB. Replica experiment verified that transformed strains of 0342 （pSR2AB）,0342 （pSR5C）,and N315 （pSR2AB）were mostly susceptible to ceftizoxim or tobramycin after the excision of SCCmec island. Conclusion ccrC could serve as a recombinase as ccrAB,which could mediate precise excision of SCCmec island from the chromosome of type-Ⅴ MRSA strain. This study shows that type-Ⅴ SCCmec island is widely disseminated between Staphylococcus aureus strains in community setting.