牛支原体新型毒力相关基因的鉴定和功能研究

Mycoplasma bovis (M.bovis) is causing a great loss in cattle industry by leading to cattle pneumonia and mastitis. Since 2008, the M.bovis pneumonia of beef cattle has been epidemic in feedlots in China. The morbidity is 80% and case fatality is 10% on average,but likely as high as 60%.In addition to M.bovis mastitis, the outbreak of M.bovis pneumonia was reported in dairy cattle either. .The fact that pathogenesis of this pathogen is not clear impedes the development of effective control measures. Therefore, this project is aimed to identify the virulence-related genes of M.bovis and thereby enhance the understanding of its pathogenesis..The following tasks are mainly included: The virulent and attenuated strains previously obtained by the applicants will be used in this project. Firstly the genomes of three attenuated strains with various virulence will be sequenced and bioinformatics analysis performed to discover the differential genes. Then the transcription of differential genes will be further determined by transcriptome sequencing and real time RT-PCR. The differential genes are cloned and expressed. The antibodies to the recombinant differential proteins will be produced and the detection methods will be developed. The protein expression of these genes in M.bovis were subsequently confirmed in vivo by using the sera from animals with natural infection and in vitro by using the antiserum to the recomibant proteins. Meanwhile the genetic tools for M.bovis site-directed mutation are studied and developed.After taking the data altogether, the candidates from the differential genes will be selected as the targets to be mutated. The resultant mutants with mutation of the specific genes are going to be evaluated for their biological properties in vitro. Further artificial infection experiment in cattle with these mutants will be performed to evaluate their virulence and immune response. .Finally the main genes and their function related to the virulence of M.bovis will be determined.

2008年来我国肉牛流行牛支原体肺炎，发病率80%以上，病死率平均10%，可达60%。同时牛支原体也导致奶牛乳腺炎和肺炎，给养牛业造成重大损失。该病原致病机理不明阻碍了防控手段的有效研发。本项目旨在鉴定该病原的毒力相关基因，为阐明其分子致病机理提供理论依据。主要内容包括：首先完成牛支原体弱毒力菌株的基因组测序，比较分析强弱毒力菌株的基因组序列，筛选差异基因；通过转录组测序和实时荧光定量RT-PCR等进行差异基因的转录分析；建立牛支原体定点突变技术；克隆表达差异基因，制备抗体，建立差异蛋白检测方法；检测差异蛋白质的体内、外表达状态，确定强弱毒力菌株间的主要差异基因；构建相关基因的突变株；体外评价特定基因对牛支原体生物学特性的影响；进一步在牛体模型上进行菌株特征分析和毒力检测，并评价各突变菌株诱导的免疫反应，证实差异基因对细菌毒力和免疫原性的影响。最终确定牛支原体主要毒力相关基因及其功能。

牛支原体致牛肺炎、乳腺炎和关节炎等，是危害世界养牛业的重要病原体。虽然牛支原体自1961年首次在美国发现至今已有55年，但该病原体的致病机理仍然不清楚，阻碍了防控手段的研发，导致该病在临床上难治难防。因此，本项目旨在研究和发现牛支原体毒力相关因子（基因和蛋白），为阐明其致病机制、研发高效防控措施提供理论依据。.本项目立项前期已获得强毒力临床分离株和传代致弱的系列弱毒力株。本项目主要内容包括：完成了3株弱毒力菌株(P115, P150, P180)的基因组测序、比较基因组和比较蛋白组分析；确定了强弱毒力株之间的差异基因和差异蛋白；发掘了一批毒力相关和免疫相关的功能基因；利用细胞感染模型，系统研究了一些功能基因或蛋白的毒力相关功能。此外，还对临床分离株的耐药性和分子耐药机制进行了研究。.项目主要取得如下结果：发现致弱株与亲代株HB0801有一个14.2 kb大片段缺失和一些SNP，代次越高，SNP发生频率越高；通过免疫蛋白质组学技术发现了39个免疫原性蛋白和19个差异表达的免疫原性蛋白，确定其中Mbov_0579和Mbov_0730编码蛋白可以作为诊断抗原靶标；建立了高通过筛选毒力相关基因/蛋白的细胞模型（牛肺上皮细胞和牛肺泡巨噬细胞感染模型），比较了牛支原体强弱菌株黏附、侵袭和凋亡等生物学过程；成功构建了牛支原体随机突变体库（含2285个突变体），确定了600个突变体的突变基因。利用细胞感染模型和突变体库，筛选了一批在胞内存活、细胞黏附、侵袭、凋亡等毒力相关功能上具有缺陷的突变体；利用细胞模型和H2O2产生能力进一步评价了强弱毒力株7个差异基因的突变体，对4个毒力相关蛋白即1个差异蛋白PDHA、3个重要功能蛋白（牛支原体核酸酶、NADH氧化酶、CdpP）进行了细胞水平和分子水平的系统研究。通过牛体试验确定了弱毒力株P150的免疫保护作用，建立了牛体免疫保护评价指标体系，并探讨了其免疫保护机制。此外，对40株临床分株进行了基因组测序。通过比较基因组学探索了牛支原体多重耐药性的机制。项目实施期间发表学术论文5篇，其中SCI收录4篇；主编出版专著2部；获国家发明专利授权1个，申报国家发明专利3个；制定地方标准2个；培养研究生13名；疫苗也转让给了两家疫苗生产企业。相关成果对于阐明牛支原体分子致病机制及研发特异性防控技术和产品奠定了重要基础。