中国医科大学学报
中國醫科大學學報
중국의과대학학보
JOURNAL OF CHINA MEDICAL UNIVERSITY
2010年
3期
161-164
,共4页
马笑雪%黄惠颖%孙丹丹%罗恩杰
馬笑雪%黃惠穎%孫丹丹%囉恩傑
마소설%황혜영%손단단%라은걸
金黄色葡萄球菌%SCCmec基因岛%ccrC基因%重组酶%质粒
金黃色葡萄毬菌%SCCmec基因島%ccrC基因%重組酶%質粒
금황색포도구균%SCCmec기인도%ccrC기인%중조매%질립
Staphylococcus aureus%staphylococcal cassette chromosome mec island%cassette chromosome recombinase C gene%recombinase%plasmid
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SCCmee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pSRSC和pSR2AB,电穿孔技术交又导入原野生菌株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10 d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1.9 kb,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.
目的 構建攜帶ccrC和ccrAB重組酶基因的載體質粒,併導入耐甲氧西林金黃色葡萄毬菌(MRSA)中,驗證ccrC重組酶具有把Ⅴ型SCCmec基因島從細菌染色體上特異性剪切下來的功能.方法 以攜帶Ⅴ型SCCmee基因島的菌株81/0342的染色體DNA為模闆,應用PCR法擴增ccrC基因,再以攜帶Ⅱ型SCCmec基因島的菌株N315的染色體DNA為模闆,應用PCR法擴增ccrAB基因,分彆剋隆到溫度敏感質粒pYT3上構建重組質粒pSRSC和pSR2AB,電穿孔技術交又導入原野生菌株81/0342和N315中構建6株細菌轉化株,在適宜溫度培育下充分錶達質粒,應用PCR法檢測SCCmec基因島從染色體上脫落,併連續10 d傳代培養細菌,應用影印實驗檢查細菌的藥物敏感性變化.結果 ccrC重組酶基因的PCR擴增產物為1.9 kb,小于Ⅱ型SCCmec基因島上攜帶的ccrAB重組酶基因.經鑒定證實成功構建瞭重組質粒pSR5C和pSR2AB,噹菌株81/0342中導入重組質粒pSR5C和pSR2AB後,81/0342所攜帶的Ⅴ型SCCmec基因島能從染色體上剪切下來,併以環狀DNA結構存在于細菌中,而隻有噹N315中導入重組質粒pSR2AB時,N315所攜帶的Ⅱ型SCCmec基因島纔能脫落下來,SCCmec基因島脫落的細菌成為對妥佈黴素和頭孢唑肟藥物敏感的菌株.結論 ccrC重組酶與ccrAB一樣具有特異性剪切酶的功能,可單獨介導Ⅴ型SCCmec基因島的脫落,從而為社區MRSA所攜帶的Ⅴ型SCCmec基因島在金黃色葡萄毬菌之間的廣汎傳播提供瞭實驗依據.
목적 구건휴대ccrC화ccrAB중조매기인적재체질립,병도입내갑양서림금황색포도구균(MRSA)중,험증ccrC중조매구유파Ⅴ형SCCmec기인도종세균염색체상특이성전절하래적공능.방법 이휴대Ⅴ형SCCmee기인도적균주81/0342적염색체DNA위모판,응용PCR법확증ccrC기인,재이휴대Ⅱ형SCCmec기인도적균주N315적염색체DNA위모판,응용PCR법확증ccrAB기인,분별극륭도온도민감질립pYT3상구건중조질립pSRSC화pSR2AB,전천공기술교우도입원야생균주81/0342화N315중구건6주세균전화주,재괄의온도배육하충분표체질립,응용PCR법검측SCCmec기인도종염색체상탈락,병련속10 d전대배양세균,응용영인실험검사세균적약물민감성변화.결과 ccrC중조매기인적PCR확증산물위1.9 kb,소우Ⅱ형SCCmec기인도상휴대적ccrAB중조매기인.경감정증실성공구건료중조질립pSR5C화pSR2AB,당균주81/0342중도입중조질립pSR5C화pSR2AB후,81/0342소휴대적Ⅴ형SCCmec기인도능종염색체상전절하래,병이배상DNA결구존재우세균중,이지유당N315중도입중조질립pSR2AB시,N315소휴대적Ⅱ형SCCmec기인도재능탈락하래,SCCmec기인도탈락적세균성위대타포매소화두포서우약물민감적균주.결론 ccrC중조매여ccrAB일양구유특이성전절매적공능,가단독개도Ⅴ형SCCmec기인도적탈락,종이위사구MRSA소휴대적Ⅴ형SCCmec기인도재금황색포도구균지간적엄범전파제공료실험의거.
Objective To construct 2 recombinant plasmids carrying cassette chromosome recombinase C(ccrC)and ccrAB respectively and introduce the plasmids into methicillin-resistant Staphlococcus 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 N31S as PCR templates.We constructed recombinant plasmids pSR5C and pSR2AB by cloning ccrC and ccrAB genes into temperature-sensitive plasmid pYI3,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 rransformants 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 N31S 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 cciC 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 aweus strains in community setting.
