基于可见光测定的山羊支原体山羊肺炎亚种抗原定量

Quantification of antigen of Mycoplasma capricolum subsp. capripneumoniae by optical assay

摘要:

山羊支原体山羊肺炎亚种(Mycoplasma capricolum subsp. capripneumoniae, Mccp)是山羊传染性胸膜肺炎(contagious caprine pleuropneumonia, CCPP)的病原，可用灭活疫苗和荚膜多糖(capsular polysaccharide, CPS)间接血凝试剂进行预防和血清学检测，但高昂的培养成本和复杂的抗原定量一直困扰着生产人员。为解决生产实际中出现的这些问题，本研究基于Mccp代谢组学的前期理论基础，通过改变初始pH值的方法，初步筛选出初始pH值为7.8的可以同时提高2种抗原产量的糖发酵培养基。利用紫外可吸收光谱可识别酚红，以及十六烷基三甲基溴化铵(cetyltrimethylammonium bromide, CTAB)可与阴离子荚膜多糖结合的理论依据，建立了利用紫外光谱分析Mccp达到的培养阶段，以及利用CTAB沉淀法相对定量发酵液荚膜多糖抗原产量的方法。通过紫外图谱观察的方法可对应Mccp生长曲线进行指导生产，大大节省传统颜色变化单位(color change unit, CCU)法的监测时间，提高了原肉眼观察方法的精确度。建立的CTAB沉淀法可在5 h内完成对CPS含量的监测，与传统的差值法相比大大缩短了时间，并且其准确度得到苯酚-硫酸法的验证。本研究优化的一种培养基和建立的两种相关性比较方法，可有效降低Mccp生产成本，提高生产效率，这些方法已在本实验室的研究阶段得到应用，为进一步改进CCPP灭活疫苗和荚膜多糖的生产工艺以及快速定量提供了实验数据。

Abstract:

Mycoplasma capricolum subsp. capripneumoniae (Mccp) is the cause of contagious caprine pleuropneumonia (CCPP) in goats. Inactivated vaccines and capsular polysaccharide (CPS) indirect hemagglutination reagents are available for prevention and serological detection, but high culture costs and complex antigen quantification have been plagued by production staff. In order to solve these problems in production practice, a sugar fermentation medium with an initial pH value of 7.8, which could improve the production of two antigens simultaneously, was screened out by changing the initial pH value based on previous Mccp metabolomics analysis. Since phenol red can be identified by UV absorption spectrum and cetyltrimethylammonium bromide (CTAB) can bind to anionic capsular polysaccharide, a UV spectrum measurement method for analyzing the culture stage reached by Mccp and a CTAB precipitation test for relative quantification of capsular polysaccharide antigen content in the fermentation broth were established. The UV spectrum observation method can guide the production of Mccp according to the growth curve of Mccp, which greatly reduces the monitoring time of the traditional CCU method and improves the accuracy of the original eye-observation method. The established CTAB precipitation test can complete the monitoring of CPS content within 5 hours, which greatly reduces the time required compared with the traditional differential technique, and its accuracy was verified by the phenol-sulfuric acid method. The optimized culture medium and the two correlation comparison methods established in this study can effectively reduce the production cost of Mccp and improve the production efficiency. The two assays have been used in the research at our laboratory, which provides experimental data for further improvement of the production process of CCPP inactivated vaccine and capsular polysaccharide as well as rapid quantification.