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Abstract：
Mycoplasma synoviae (M. synoviae) is one of the major poultry pathogens causing infectious synovitis, airsacculitis, a high incidence of shell breakage, and egg production loss. However, the pathogenesis of M. synoviae remains unclear. Adhesion of mycoplasmas to host cells is a crucial step in infection and colonization. The purpose of this study was to determine the adhesive function of a putative P80 family lipoprotein (LP78) and evaluate its application in the detection of antibodies against M. synoviae. Recombinant LP78 (rLP78) was expressed in the supernatant component of Escherichia coli and mouse anti-rLP78 serum was prepared. Bioinformatic analysis and western blotting results revealed that LP78 was conservative among M. synoviae strains. It was distributed not only in the cytoplasm but also on the membrane of M. synoviae through western blotting and indirect immunofluorescence (IFA). The adherence of M. synoviae to DF-1 cells was significantly inhibited by mouse anti-rLP78 serum (p < 0.01). IFA revealed that rLP78 adhered to DF-1 cells, and this adherence was prevented by mouse anti-rLP78 serum. Furthermore, rLP78 was found to bind to the DF-1 cells membrane proteins in a dose-dependent manner by enzyme-linked immunosorbent assay (ELISA). Screening of DF-1 cells membrane proteins by western blotting showed that proteins with molecular weight of 35-40 kDa and 55-70 kDa bound to rLP78. Moreover, rLP78 was identified to be a fibronectin/plasminogen binding protein. The sensitivity and specificity of rLP78-based iELISA were 85.7 and 94.1%, respectively. The maximum dilution of positive serum (HI titer, 1:128) detected via rLP78-based iELISA was 1:6,400, whereas that detected using a commercial ELISA kit was 1:12,800-1:25,600. Both rLP78-based iELISA and the commercial ELISA kit detected seroconversion after 7 days of challenge and immunization. No cross-reactivity with positive sera against other avian pathogens was observed in rLP78-based iELISA. Collectively, these results indicate that LP78 is a fibronectin/plasminogen-binding adhesion protein of M. synoviae and a potential diagnostic antigen. The present study will facilitate a better understanding of the pathogenesis of M. synoviae and the development of new diagnostic.
摘要：
滑膜支原体（M.滑膜)是引起传染性滑膜炎的主要家禽病原体之一，气囊炎,外壳破损的发生率很高，和鸡蛋产量损失。然而,滑膜分枝杆菌的发病机制尚不清楚。支原体与宿主细胞的粘附是感染和定植的关键步骤。这项研究的目的是确定推定的P80家族脂蛋白（LP78）的粘附功能，并评估其在检测抗滑膜分枝杆菌抗体中的应用。在大肠杆菌的上清液成分中表达重组LP78(rLP78),制备小鼠抗rLP78血清。生物信息学分析和免疫印迹结果表明，LP78在滑膜分枝杆菌菌株中是保守的。通过蛋白质印迹和间接免疫荧光（IFA），它不仅分布在细胞质中，而且分布在滑膜分枝杆菌上。小鼠抗rLP78血清显著抑制滑膜分枝杆菌对DF-1细胞的粘附(p<0.01)。IFA显示rLP78粘附于DF-1细胞，小鼠抗rLP78血清阻止了这种粘附。此外，通过酶联免疫吸附测定（ELISA），发现rLP78以剂量依赖性方式与DF-1细胞膜蛋白结合。通过蛋白质印迹法筛选DF-1细胞膜蛋白显示分子量为35-40kDa和55-70kDa的蛋白与rLP78结合。此外，rLP78被鉴定为纤连蛋白/纤溶酶原结合蛋白。基于rLP78的iELISA的敏感性和特异性分别为85.7％和94.1％，分别。阳性血清的最大稀释度（HI滴度，1：128）通过基于rLP78的iELISA检测到的是1：6,400，而使用商业ELISA试剂盒检测到的是1：12,800-1：25,600。基于rLP78的iELISA和商业ELISA试剂盒都在攻击和免疫7天后检测到血清转化。在基于rLP78的iELISA中未观察到与针对其他禽类病原体的阳性血清的交叉反应性。总的来说，这些结果表明LP78是滑膜分枝杆菌的纤连蛋白/纤溶酶原结合粘附蛋白和潜在的诊断抗原。本研究将有助于更好地理解滑膜分枝杆菌的发病机制和新的诊断方法的发展。
