4种抗原定量方法观察牛支原体培养特征

Observation of the Growth Trends of Mycoplasma bovis with Four Different Antigen Quantitation Methods

为探索在疫苗研制过程中牛支原体抗原收获时间及抗原定量替代方法,将牛支原体08M株接种于含10%马血清的Thiaucourt’s培养基,在110h内同时监测其颜色变化单位（color change units,CCU）、菌落形成单位（colony forming units,CFU）、菌体蛋白浓度和核酸含量的变化,绘制相应曲线。活菌计数结果（CCU和CFU）显示,牛支原体生长可分为明显的4期,10h进入对数期,30h进入稳定期,活菌数最高可达1.0×108 CCU/mL和7.7×107 CFU/mL,75h进入衰亡期;蛋白浓度从15h开始迅速增长,至35h蛋白浓度最高,为72.06μg/mL,此后维持在58.38～70.65μg/mL;核酸含量从15h开始增长,至25h后Ct值维持在15.32～17.84。结果表明,牛支原体蛋白含量在稳定期初期与活菌数具有良好的相关性。因此,在牛支原体灭活疫苗生产中,稳定期初期是最佳抗原收获时间,可用蛋白浓度法代替活菌计数法进行抗原定量。

To explore the antigen harvest time of Mycoplasma bovis（M.bovis）and the antigen quantitation alternative method,M.bovis 08 Mstrain was inoculated in the Thiaucourt＇s medium.Four growth curve plottings were made by measuring color change units（CCU）,colony forming units（CFU）,protein concentration and nucleic acid levels.Both the results of CCU and CFU counts showed that the growth of M.bovis was divided into four phases,the logarithmic phase appeared after being cultrured 10 h,the stationary phase appeared at 30 hwith the highest number of viable cells up to 1.0×108 CCU/mL and 7.7×107 CFU/mL,and the decline phase started at 75 h.The protein concentration of M.bovis increased rapidly from 15 to 35h,reached 72.06μg/mL at 35 h,then maintained at 58.38 to 70.65μg/mL.The nucleic acid levels of M.bovis increased rapidly from 15 h,and the cycle threshold（Ct）values were maintained between 15.32 to 17.84 after 25h.These results indicated that there was a good correlation between the protein concentration and viable count of M.bovis at the early stationary phase,which was the best time period to harvest antigen.The protein concentration determination could be an alternative method to quantify antigen contents of M.bovis when preparing inactivated M.bovis vaccine.