肠炎沙门氏菌SEF14菌毛相关功能的挖掘研究

Salmonellae enteritidis is an important food-borne zoonotic pathogens in humans and animals. Unique SEF14 fimbriae are only found in Salmonellae enteritidis and closely related serovar, which encoded by sef operon gene in chromosomal pathogenicity island may affect serovar-specific virulence and pathogenity traits. Based on the identification and characterization of adhesin and adhesin receptor binding domain to construct the recombinant E.coli with expressing plasmid, which could functionally surface-display the adhesin receptor binding domain on the cell surface, with the capability of adhering to the host cell well in vitro. The single and double mutants of SEF14 fimbriae from S. enteritidis strains with different subunit deletion will be constructed, the engineered SEF14 mutants will be tested for invasion, immune response and survival in infection in susceptible host cells, target tissues and organs to further study interaction between chicken host and Salmonellae enteritidi infection, compared with the wild type strains, mutants and their complementary recombinants. This project will target the molecular mechanism of the adhesion, colonization, infection, invasion, transmision, distribution, and long-term contaminating and sheding which mediated by the SEF14 fimbriae. And put dada together we expect to carry out, finally we will scientificly analyze and evaluate the key parameters of susceptibility and resistance in our major native chicken breeds against S. enteritidis infection and provide new advanced strategy for prophylactic and control S. enteritidis infection.

肠炎沙门氏菌是一种重要的食源性人畜共患病病原菌，其特异性菌毛黏附素SEF14与肠炎沙门氏菌种特异性毒力、致病性特性相关，由染色体毒力岛上sef操纵子全基因编码和表达。基于其黏附素和黏附素受体结合域的鉴定和特征化，利用DNA重组技术和细菌分子遗传学相关研究手段和技术，构建表面分泌表达功能性黏附素受体结合域的重组质粒表达菌，完成宿主靶细胞与特异性黏附素受体结合性能的体外检测；构建不同致病性肠炎沙门氏菌黏附素SEF14突变株，通过野生病原菌、黏附素突变株和突变株互补重组菌与易感宿主靶细胞、组织和器官的入侵、应答和抗性能力测试比较，建立和深入研究鸡和感染肠炎沙门氏菌相互作用，旨在阐明SEF14介导的病原菌在易感动物体内黏附定植、感染侵袭、扩散和长期排毒分子机理，并能针对性地对我国主要地方鸡种的肠炎沙门氏菌感染抗病和易感性进行综合评价，使我国能够站在高起点上开展家禽肠炎沙门氏菌感染控制新策略研究。

肠炎沙门氏菌是一种重要的食源性人畜共患病病原菌，其特异性菌毛黏附素SEF14与肠炎沙门氏菌种特异性毒力、致病性特性相关，由染色体毒力岛上sef操纵子全基因编码和表达。基于肠炎沙门氏菌特异性菌毛SEF14黏附素亚单位和黏附素操纵子完整基因的功能、特征化，表明肠炎沙门氏菌SEF14菌毛仅特异性表达在家禽病原菌相关的D群沙门氏菌肠炎沙门氏菌中，分析比较D群沙门氏菌染色体基因序列，发现SEF14菌毛操纵子亚单位基因sefD以及调节基因sefR在鸡白痢沙门氏菌中存在变异，且并不能表达，推测此变异可能是SEF14菌毛限制性表达的原因。基于重组表达的主要亚单位蛋白rSEFA介导的间接ELISA试剂盒的完善将为家禽种群肠炎沙门氏菌净化工作以及日常血清筛查提供了极大的便利。利用DNA重组技术和细菌分子遗传学相关研究手段和技术，构建表面分泌表达功能性黏附素受体结合域的重组质粒表达菌，完成宿主靶细胞与特异性黏附素受体结合性能的体外检测；构建不同致病性肠炎沙门氏菌黏附素SEF14突变株，通过野生病原菌、黏附素突变株和突变株互补重组菌与易感宿主靶细胞、组织和器官的入侵、应答和抗性能力测试比较，建立和深入研究鸡和感染肠炎沙门氏菌相互作用。试验结果表明SEF14菌毛一定程度上降低了小鼠腹腔和鸡巨噬细胞对肠炎沙门氏菌的吞噬作用，以及增强了肠炎沙门氏菌在巨噬细胞中存活率。借助SEF14菌毛的作用，肠炎沙门氏菌能更好的作用于巨噬细胞并在巨噬细胞中存活，利于其隐性感染和长期排毒，这和上述易感动物感染试验结果一致。本研究旨在阐明SEF14介导的病原菌在易感动物体内黏附定植、感染侵袭、扩散和长期排毒分子机理，并能针对性地对我国主要地方鸡种的肠炎沙门氏菌感染抗病和易感性进行综合评价，使我国能够站在高起点上开展家禽肠炎沙门氏菌感染控制新策略研究。