滑液支原体NADH氧化酶的酶学活性及亚细胞定位研究

Enzymatic activity and subcellular localization analyses of the NADH oxidase in Mycoplasma synoviae

摘要:

【背景】许多研究表明，支原体的NADH氧化酶(NADH oxidase，NOX)不仅在胞浆中发挥生物酶学功能，也存在于细胞膜上发挥黏附宿主细胞功能。【目的】对滑液支原体(Mycoplasma synoviae，MS)的NOX进行酶学活性及亚细胞定位研究，分析其在MS致病过程中的潜在作用。【方法】对MS的NOX蛋白进行原核表达、纯化，然后对重组MSNOX (rMSNOX)的酶学活性及影响酶活的条件进行研究，测定其酶比活力、米氏常数及最大反应速率，接着用MS阳性血清及制备的rMSNOX兔多克隆抗体，分别与rMSNOX蛋白及MS全菌、膜蛋白和胞浆蛋白进行Western blotting反应，鉴定rMSNOX的免疫原性及其在MS中的分布情况。【结果】在大肠杆菌BL21(DE3)中成功表达rMSNOX蛋白，相对分子质量约为53 kD，并获得纯化的rMSNOX蛋白；酶活测定显示rMSNOX蛋白的酶比活力为14.17 IU/mg，最适酶促温度为37 °C，最适pH为7.5，双倒数法求得rMSNOX的最大反应速率Vmax为21.8 μmol/(L·min)，米氏常数Km(NADH)为244.0 μmol/L；rMSNOX蛋白与MS阳性血清特异性结合，证明具有良好的免疫原性；亚细胞定位研究表明NOX蛋白主要存在于MS的胞浆中，细胞膜上有少量的分布。【结论】首次证实MS的NOX不仅具有NADH氧化酶活性，也是MS具有免疫原性的膜蛋白，为进一步探索NOX在MS致病过程中的作用提供了分子基础。

Abstract:

[Background] Studies have shown that the NADH oxidase (NOX) of Mycoplasma not only functions as an enzyme in cytoplasm, but also exists on the cell membrane as an adhesin. [Objective] To study the enzymatic activity and subcellular localization of the NOX of Mycoplasma synoviae (MS), analyze its potential role in the MS pathogenesis. [Methods] The recombinant MS NOX (rMSNOX) protein was prokaryotic expressed and purified. Then the enzymatic activity of purified rMSNOX and the factors affecting enzymatic activity were studied. In addition, the enzymatic specific activity, the maximum reaction rate (Vmax) and the Michaelis constant (Km) of the rMSNOX were determined. The MS positive chicken serum was used for Western blotting analysis of the immunogenicity of the rMSNOX. The Mycoplasma whole-cell, membrane and cytoplasmic fractions were prepared for Western blotting analysis to determine the subcellular localization of MSNOX using the rabbit anti-rMSNOX serum. [Results] The rMSNOX protein was successfully expressed in E. coli BL21(DE3) and purified. The relative molecular mass was about 53 kD. The enzymatic specific activity of the rMSNOX protein was determined as 14.17 IU/mg, the optimum enzymatic temperature was 37 °C, and the optimum pH was 7.5 by an enzyme activity analysis. The Km (NADH) of rMSNOX was determined as 244.0 μmol/L, and the Vmax as 21.8 μmol/(L·min) using the double-reciprocal method. The rMSNOX presented a specific binding to MS positive chicken serum, which proved that it had good immunogenicity. Subcellular localization analysis indicates that NOX protein distributed in MS cytoplasm and cell membrane. [Conclusion] This study demonstrates for the first time that MSNOX not only possesses NADH oxidase activity, but also is an immunogenic membrane protein, which provides a molecular basis for further exploring the role of NOX in the MS pathogenesis.