College of Veterinary Medicine, Gansu Agricultural University, 1 Yingmencun, Lanzhou, 730070, PR China.
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 518 Ziyue Road, Minhang District, Shanghai, 200241, PR China.
Microb Pathog. 2021 Jun;155:104851. doi: 10.1016/j.micpath.2021.104851. Epub 2021 Mar 29.
Mycoplasma synoviae (MS) is an important pathogen which causes huge economic losses to the poultry industry worldwide, and research on MS can provide the foundation for diagnosis, prevention, and treatment of MS infection. In this study, primers designed based on the sequences of pyruvate dehydrogenase complex (PDC) E1 alpha and beta subunit genes (pdhA and pdhB, respectively) of MS 53 strain(AE017245.1) in GenBank were used to amplify the pdhA and pdhB genes of MS WVU1853 strain through PCR. Subsequently, the prokaryotic expression vectors pET-28a(+)-pdhA and pET-28a(+)-pdhB were constructed and expressed in Escherichia coli BL21(DE3) cells. The recombinant proteins rMSPDHA and rMSPDHB were purified, and anti-rMSPDHA and anti-rMSPDHB sera were prepared by immunizing rabbits, respectively. Subcellular localization of PDHA and PDHB in MS cells, binding activity of rMSPDHA and rMSPDHB to chicken plasminogen (Plg) and human fibronectin (Fn), complement-dependent mycoplasmacidal assays, and adherence and adherence inhibition assays were accomplished. The results showed that PDHA and PDHB were distributed both on the surface membrane and within soluble cytosolic fractions of MS cells. The rMSPDHA and rMSPDHB presented binding activity with chicken Plg and human Fn. The rabbit anti-rMSPDHA and anti-rMSPDHB sera had distinct mycoplasmacidal efficacy in the presence of guinea pig complement, and the adherence of MS to DF-1 cells pretreated with Plg was effectively inhibited by treatment with anti-rMSPDHA or anti-rMSPDHB sera. These findings indicated that surface-associated MSPDHA and MSPDHB were adhesion-related factors of MS and that the binding between MSPDHA/MSPDHB and Plg/Fn contributed to MS adhesion to DF-1 cells.
滑液支原体（MS）是一种重要的病原体，它给全球家禽养殖业造成了巨大的经济损失，对 MS 的研究可以为 MS 感染的诊断、预防和治疗提供基础。在本研究中，根据 MS 53 株（AE017245.1）在 GenBank 中的丙酮酸脱氢酶复合物（PDC）E1α和β亚基基因（pdhA 和 pdhB）序列设计引物，通过 PCR 扩增 MS WVU1853 株的 pdhA 和 pdhB 基因。随后，构建了原核表达载体 pET-28a(+)-pdhA 和 pET-28a(+)-pdhB，并在大肠杆菌 BL21(DE3)细胞中表达。纯化了重组蛋白 rMSPDHA 和 rMSPDHB，并分别用它们免疫家兔制备抗 rMSPDHA 和抗 rMSPDHB 血清。完成了 PDHA 和 PDHB 在 MS 细胞中的亚细胞定位、rMSPDHA 和 rMSPDHB 与鸡血浆纤溶酶原（Plg）和人纤维连接蛋白（Fn）的结合活性、补体依赖性杀支原体试验以及黏附和黏附抑制试验。结果表明，PDHA 和 PDHB 分布在 MS 细胞的表面膜和可溶胞质部分。rMSPDHA 和 rMSPDHB 与鸡 Plg 和人 Fn 具有结合活性。兔抗 rMSPDHA 和抗 rMSPDHB 血清在豚鼠补体存在时具有明显的杀支原体作用，用抗 rMSPDHA 或抗 rMSPDHB 血清处理可有效抑制 Plg 预处理的 MS 对 DF-1 细胞的黏附。这些发现表明，表面相关的 MSPDHA 和 MSPDHB 是 MS 的黏附相关因子，MSPDHA/MSPDHB 与 Plg/Fn 的结合有助于 MS 对 DF-1 细胞的黏附。
