不同方法提取和测定粪肠球菌生物膜总蛋白含量的比较

The Comparison of Different Methods on Extracting and Measuring the Total Protein of Biofilms Formed by Enterococcus faecalis

目的:以标准菌株ATCC33186为例,用超声破碎法提取粪肠球菌生物膜中总蛋白,结合3种不同方法测定总蛋白的含量,确定一种简单快速有效的检测细菌生物膜中总蛋白含量的方法。方法:将培养24h的生物膜加入PBS缓冲液清洗后,使用超声破碎法提取生物膜中总蛋白,选择作用时间、振幅Amp、超声和间隔时间等参数进行超声破碎,结合SDS-PAGE凝胶电泳分析蛋白电泳图谱确定最佳超声条件后,确定最佳超声条件后,使用BCA法、考马斯亮蓝Bradford法和Lowry法测定生物膜中总蛋白含量,对结果进行比较分析。结果:最佳超声破碎条件参数:作用时间2min,振幅Amp20%,超声2s间隔2s。用BCA法测得总蛋白含量每皿为2299.1μg,考马斯亮蓝Bradford法测得含量为每皿3793.8μg,Lowry法测定含量为每皿1858.0μg。结论:确定超声破碎提取粪肠球菌生物膜蛋白的最优化条件,是一种简单可行测定细菌生物膜中总蛋白含量的方法。

Objective： In order to measure the total protein of bacterial biofilm formed by Enterococcus faecalis ATCC33186,ultrasonication was used to extract the protein.After optimizing ultrasonic extraction conditions by different parameters,three methods commonly used to determine the protein concentration were taken.And we tried to confirm an accurate,rapid and simply method for quantifying protein in the biofilm.Methods： Ultrasonication was used to extract the protein from biofilm which was washed by PBS buffer after culturing 24 h.In order to determine the optimum ultrasonication condition,we chose different parameters as times,amplitudes,ultrasonic and interval times.After choosing the best ultrasonic treatment combined with SDS-PAGE electrophoresis,BCA,Bradford and Lowry methods were used to determine the total protein of biofilms.And we compared the data discrepancy of different methods.Results： The optimum condition of ultrasonication was ultrasonic time 2 min,Amp 20% and ultrasonic treatment for 2 s with interval of 2 s.The average concentration of total protein concentration of three times was 2299.1μg/dish by BCA,3793.8 μg/dish by Bradford,1858.0 μg/dish by Lowry.Conclusion： The optimum ultrasonic condition for protein extraction from Enterococcus faecalis biofilm was determined.The Bradford method was feasible and precise to determine the total protein concentration of bacterial biofilm.