摘要
滑液支原体(Mycoplasma synoviae,MS)是一种重要的禽病病原,严重危害我国养禽业.本研究旨在分析MS烯醇化酶(enolase,Eno)参与病原致病和诱导感染宿主免疫相关的功能.对MS的Eno蛋白进行种内和种间同源性和进化树分析,并预测Eno的蛋白结合位点和B细胞表位.MS Eno在不同MS菌株间同源性较高,且有别于其他物种.B细胞表位广泛分布在Eno蛋白的各个部分,且大都与预测的蛋白结合位点有重合.进行Eno原核表达和纯化,制备兔抗Eno多克隆抗血清.利用Western blot分析Eno在MS感染鸡或灭活苗免疫鸡中的反应原性.结果显示,MS Eno与兔抗Eno多克隆抗血清、兔抗 MS多克隆抗血清、灭活疫苗(HN01株或YBF-MS1株)高免鸡血清、HN01强毒攻毒后阳性鸡血清、3个不同地方来源的临床阳性鸡血清均能发生反应,说明其在疫苗免疫和病原感染鸡中均表现有反应原性.利用菌落印迹和双荧光检测分析并证实Eno为外膜蛋白.利用间接免疫荧光和ELISA板结合试验分析Eno对鸡滑膜鞘细胞(synovial sheath cells,SSCs)是否具有黏附素作用.试验证实Eno对SSCs具有黏附作用,且此黏附作用具有Eno浓度依赖性.以上证明MS Eno在病原感染和灭活苗免疫鸡中具有反应原性,并作为外膜锚定的细胞黏附素参与对关键宿主细胞SSCs的黏附,为后续深入研究Eno的致病和免疫相关功能提供了重要参考.
Abstract
Mycoplasma synoviae(MS)is an important avian disease pathogen that seriously harms the poultry industry in China.The aim of this paper was to analyze the enolase(Eno)of MS for its involvement in pathogenic and immune-related functions in host.Intraspecific and interspecific ho-mology and evolutionary tree analyses were performed for the Eno proteins.The protein binding sites and B-cell epitopes of Eno were also predicted.The results showed that the homology of MS Eno was high among different MS strains and was distinct from other species.B-cell epitopes were widely distributed in all parts of the Eno protein and mostly overlapped with the predicted protein binding sites.Eno recombinant expression and purification were performed.Rabbit anti-Eno poly-clonal antiserum was prepared.The reactogenicity of Eno in chickens infected with MS or immu-nized with inactivated vaccine was analyzed using Western blot.The result showed that MS Eno re-acted with rabbit anti-Eno polyclonal antiserum,rabbit anti-MS polyclonal antiserum,highly im-mune chicken serum of inactivated vaccine(HN01 strain),highly immune chicken serum of inacti-vated vaccine(YBF-MS1 strain),positive chicken serum after the chicken infected with virulent strain HN0l,and clinically positive serum from three different local sources,indicating that Eno showed reactogenicity in both vaccine immunization and pathogenic infection.Eno was analyzed and showed as an outer membrane protein using colony blotting and dual fluorescence assay.Indirect immunofluorescence and ELISA plate binding assays were used to analyze whether Eno has an ad-hesin effect on chicken synovial sheath cells(SSCs).The results showed that Eno had an adhesive effect on SSCs and this adhesive effect was Eno concentration-dependent.This study demonstrates that Eno of MS has reactogenicity in MS-infected or inactivated vaccine-immunized chickens and participates in adhesion to key host cell SSCs as an outer membrane-anchored cellular adhesion,which provides important reference information for subsequent in-depth studies on the pathogenic and immune-related functions of Eno.
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