绵羊肺炎支原体荚膜多糖的提取纯化及反应原性分析

Extraction,purification and reactivity analysis of capsular polysaccharide of Mycoplasma ovipneumoniae

本研究旨在提取绵羊肺炎支原体(Mycoplasma ovipneumoniae,Mo)GH3-3的荚膜多糖,并验证其反应原性。用无水乙醇沉淀得到粗荚膜多糖,再通过苯酚抽提、酶处理去除核酸和蛋白质,进而纯化得到荚膜多糖,利用免疫荧光试验证明该荚膜多糖的反应原性,利用刚果红试验检测其是否具有三螺旋结构。从1 L培养基上清中初步提取得到Mo GH3-3粗荚膜多糖12.24 mg,经过纯化得到9.8 mg的精制荚膜多糖,其纯化得率约为80%。免疫荧光试验结果表明Mo GH3-3荚膜多糖具有良好的反应原性,刚果红试验首次证明绵羊肺炎支原体荚膜多糖具有三螺旋结构。经过乙醇沉淀、苯酚抽提、酶处理、超滤浓缩后,得到高纯度的Mo GH3-3荚膜多糖。该提取纯化方法简便、成本低,便于生产实践中应用。荚膜多糖具有反应原性和三螺旋结构,证明其生物活性良好,为相关诊断试剂、疫苗、佐剂等的研发提供了可参考的多糖制备方法。

This study aimed to extract the capsular polysaccharides from GH3-3 strain of Mycoplasma ovipneumoniae and verify its reactivity.Crude capsular polysaccharide was obtained by absolute ethanol precipitation method,then nucleic acid and protein were removed by phenol extraction and enzyme treatment to obtain purified capsular polysaccharide.Immunofluorescence test was used to prove the reactivity of the capsular polysaccharide,and Congo red test was carried out to verify the triple helix structure of the capsular polysaccharides.12.24 mg of crude capsular polysaccharides derived from Mo GH3-3 was initially extracted from the supernatant of 1 L medium,and 9.8 mg of refined capsular polysaccharide was obtained after purification.The purified yield was about 80%.The results of immunofluorescence experiment showed that Mo GH3-3 capsule polysaccharides had good reactivity.The Congo red assay confirmed that the capsular polysaccharides of Mo has a triple helical structure.The high purity capsular polysaccharides of Mo GH3-3 strain were obtained using a combination of technical methods,comprising alcohol precipitation,phenol extraction,enzyme hydrolysis,ultrafiltration and concentration.This extraction and purification assay for Mo capsular polysaccharides is not only simple but also cost-effective,making it is suitable for application in production practice.The capsular polysaccharides exhibit good reactivity and have a triple helix structure,indicating that its biological activity is well-preserved.This work provides a reference method for the preparation of polysaccharide for the research and development of relevant diagnostic reagents,vaccines and adjuvants.