利用基因重组技术构建MRSA流行株假体关节感染模型的实验研究

构建一种基于MRSA临床流行株的假体关节感染模型是研究难治性耐药金黄色葡萄球菌假体感染分子机制和检测各种预防和治疗措施的重要工具。本项目针对目前假体感染体内模型中缺乏临床流行株工具耐药金葡、无法在体实时定量检测感染以及体内材料表面生物膜内活菌检测困难等缺陷，在前期分子流行病学筛选基础上，利用同源重组技术改造细菌，构建含有密码子优化Lux基因生物发光标记MRSA流行株及密码子优化eGFP荧光标记的MRSA流行株。建立具有长效荧光和生物发光稳定性的耐药金葡假体感染体内研究模型，并运用新建模型研究流行株srtA基因敲除对MRSA体内致病机制的影响和注银纳米颗粒钛合金作为关节假体内植物对MRSA感染预防的体内有效性。本研究不仅提供了流行株MRSA假体感染体内分子机制研究的基本平台，同时也解决了目前骨科生物材料体内抗菌性能研究的关键技术问题。

Constructing an animal model of prosthetic joint infection using clinical epidemic MRSA strains is a key tool of molecular mechanism study in vivo for refractory resistant Staphylococcus aureus prosthetictic joint infection and an important tool of preventive and therapeutic measures testing. Models in vivo without clinical epidemic strains, lack of real-time quantitative testing for infection in vivo and difficult detection of viable cells embedded into biofilm on the surface of biomaterial are main faults of current prosthetic joint infection animal models. The clinical epidemic strain was pre-screened with molecular epidemiological study and will be transformed into a labeled MRSA with codon-optimal eGFP fluorescence or codon-optimal Lux bioluminescence through DNA homologous recombination. PJI animal models of MRSA recombinant with long term stability of fluorescence or bioluminescence will be established. The new models will be used to investigate the pathogenesis involved srtA gene of epidemic strain MRSA on PJI and to test in vivo antimicrobial effectiveness of silver nanoparticles plasma immersion titanium alloy as joint implant against MRSA. The project not only provides the basic platform for in vivo molecular mechanisms studies of epidemic MRSA PJI, but also solves current key technical issue of in vivo studies for antimicrobial properties of orthopedic biomaterials.