中华检验医学杂志
中華檢驗醫學雜誌
중화검험의학잡지
CHINESE JOURNAL OF LABORATORY MEDICINE
2009年
1期
76-79
,共4页
倪明%余冰%田德英%宋世会%谢琳卡%陈安群
倪明%餘冰%田德英%宋世會%謝琳卡%陳安群
예명%여빙%전덕영%송세회%사림잡%진안군
假单胞菌,铜绿%生物膜%藻酸盐%细菌蛋白质类%突变
假單胞菌,銅綠%生物膜%藻痠鹽%細菌蛋白質類%突變
가단포균,동록%생물막%조산염%세균단백질류%돌변
Pseudomonas aeruginosa%Biofilms%Alginates%Bacterial proteins%Mutation
目的 研究新型突变的mucA基因对黏液型铜绿假单胞菌PA17生物被膜形成过程和成熟生物被膜形态的影响.方法 将PAO1的mucA基因全长克隆到铜绿假单胞菌表达质粒pUCP20上,转化到含新型mucA基因突变的黏液型铜绿假单胞菌PA17,酶切、测序鉴定;异丙基-β-D-硫代半乳糖苷(IPTG)诱导重组质粒表达,半定最逆转录(RT)-PCR检测IPTG诱导前后藻酸盐合成关键基因algD的表达水平;改良平板培养法建立PA17、含重组质粒的PA17及PAO1的生物被膜模型,扫描电镜观察其生物被膜形成过程.结果 IPTG诱导后,表达重组质粒的PA17浮游状态时algD表达下降,证实PAO1的mucA基因转化PA17成功,其生物被膜形成速度居PAO1和PA17之间,8 h时即可出现不可逆性黏附,6 d形成成熟生物被膜,PA17、PAO1和含重组质粒的PA17所形成的成熟生物被膜形态相似.结论 PA17所含的新型mucA基因突变造成PA17生物被膜形成初始阶段的不可逆黏附过程延迟,但对成熟生物被膜形态无影响.
目的 研究新型突變的mucA基因對黏液型銅綠假單胞菌PA17生物被膜形成過程和成熟生物被膜形態的影響.方法 將PAO1的mucA基因全長剋隆到銅綠假單胞菌錶達質粒pUCP20上,轉化到含新型mucA基因突變的黏液型銅綠假單胞菌PA17,酶切、測序鑒定;異丙基-β-D-硫代半乳糖苷(IPTG)誘導重組質粒錶達,半定最逆轉錄(RT)-PCR檢測IPTG誘導前後藻痠鹽閤成關鍵基因algD的錶達水平;改良平闆培養法建立PA17、含重組質粒的PA17及PAO1的生物被膜模型,掃描電鏡觀察其生物被膜形成過程.結果 IPTG誘導後,錶達重組質粒的PA17浮遊狀態時algD錶達下降,證實PAO1的mucA基因轉化PA17成功,其生物被膜形成速度居PAO1和PA17之間,8 h時即可齣現不可逆性黏附,6 d形成成熟生物被膜,PA17、PAO1和含重組質粒的PA17所形成的成熟生物被膜形態相似.結論 PA17所含的新型mucA基因突變造成PA17生物被膜形成初始階段的不可逆黏附過程延遲,但對成熟生物被膜形態無影響.
목적 연구신형돌변적mucA기인대점액형동록가단포균PA17생물피막형성과정화성숙생물피막형태적영향.방법 장PAO1적mucA기인전장극륭도동록가단포균표체질립pUCP20상,전화도함신형mucA기인돌변적점액형동록가단포균PA17,매절、측서감정;이병기-β-D-류대반유당감(IPTG)유도중조질립표체,반정최역전록(RT)-PCR검측IPTG유도전후조산염합성관건기인algD적표체수평;개량평판배양법건립PA17、함중조질립적PA17급PAO1적생물피막모형,소묘전경관찰기생물피막형성과정.결과 IPTG유도후,표체중조질립적PA17부유상태시algD표체하강,증실PAO1적mucA기인전화PA17성공,기생물피막형성속도거PAO1화PA17지간,8 h시즉가출현불가역성점부,6 d형성성숙생물피막,PA17、PAO1화함중조질립적PA17소형성적성숙생물피막형태상사.결론 PA17소함적신형mucA기인돌변조성PA17생물피막형성초시계단적불가역점부과정연지,단대성숙생물피막형태무영향.
Objective To study the effect of a new mucA gene mutation on the biofilm formation process and the morphology of matured biofilm of Pseudomonas aeruginosa. Methods The mucA gene of PAO1 was cloned into the Pseudomonas aeruginosa expression plasmid pUCP20. The recombinant plasmid was transformed to mucoid PA17 which contained a new mucA mutation. Positive clones of the plasmids were identified by enzyme digestion and sequencing. The expression levels of algD in the positive clones were assessed by semi-quantitative RT-PCR. The modified plate culture method was used to establish the biofilm models of PA17, PA17 with recombinant plasmid and PAO1 in vitro. Results Transformation was identified by the decreased expression of algD in positive clones. The rate of biofilm formation of the positive clones was between those of PAO1 and PA17. The irreversible adhesion occurred after 8 h and the matured biofilm was observed on day 6. The morphologies of PA17, PAO1 and PA17 with recombinant plasmid were the same. Conclusion The mucA gene mutation of PA17 delays the formation of irreversible adhesion of PA17 biofilm, but it has no effect on the morphology of matured biofilm.
