牛支原体生物被膜形成相关基因的鉴定和功能研究

The epidemic of Mycoplasma bovis (M. bovis) infection in cattle in recent years has caused important economic losses to the cattle industry. Today, to combat M. bovis, antimicrobial therapy is the major measure available, but often fails. The antibiotic resistance and chronic infections of M. bovis has become the focus of researcher’s attention. There is direct relationship between biofilm formation and chronic infection with antibiotic resistance of M. bovis. However, genes involved in biofilm formation by M. bovis and their functions have not been investigated. In this study, transposon mutant library of M. bovis strain LZ01 will be constructed using the transposon Tn4001mod. The defective biofilm-forming mutants will be phenotypic screened, and then the method of genome walking, Southern hybridization and sequence analysis will be applied to identify genes related to biofilm formation. On this basis, the biofilm-related gene deletion mutants and their complementary strains will be constructed, and the correlation between biofilm formation, biological characteristics, antibiotic resistance and pathogenicity of M. bovis will be studied in vitro and in vivo. This study will help to reveal the mechanism of biofilm formation and molecular pathogenisis of M. bovis, further to clarify chronic infection mechanism of this important pathogen and has great scientific significance.

近年来牛支原体病的流行给养牛业造成巨大的经济损失，目前主要依赖抗菌素治疗，但效果却不理想。牛支原体耐药现象和慢性感染成为研究关注焦点。牛支原体生物被膜的形成与其耐药性的变迁和造成的慢性感染有直接关系。关于牛支原体生物被膜形成的相关基因及功能目前尚未阐明。本项目拟通过转座子Tn4001mod诱变，构建牛支原体LZ01株突变体库，通过表型筛选出生物被膜形成缺陷突变株，通过基因组步移法、Southern杂交和序列分析，筛选鉴定生物被膜形成相关基因。构建生物被膜相关基因缺失株与互补株，通过体内试验和体外试验，研究生物被膜形成相关基因与其生物学特性、耐药性和致病性的关系。本研究对揭示牛支原体生物被膜形成机制及致病机理，阐明致病菌抗生素耐受性和慢性感染机制具有重要的科学意义。

牛支原体病的流行给全世界养牛业造成重大经济损失，目前依赖的抗生素治疗效果却不理想。牛支原体生物被膜的形成可增加耐药性和造成慢性感染。目前关于牛支原体生物被膜形成相关基因及其功能尚不清楚。本研究通过转座子Tn4001mod，构建了牛支原体突变体库，共筛选阳性突变体1120个；通过表型筛选，已鉴定出生物被膜缺陷突变体11个，并进行PCR和Southern鉴定；对生物被膜和浮游生长状态下的牛支原体蛋白组学和免疫蛋白组学进行比较分析，系统分析出生物被膜形成相关基因及其功能，鉴定出牛支原体中有102个基因编码的蛋白与生物被膜形成密切相关，并利用生物信息学进行了生物功能的预测；本研究有利于揭示牛支原体生物被膜形成机制及致病机理，同时为牛支原体感染防控奠定了基础。