变异链球菌蛋白质提取方法研究

Study on protein extraction methods for Streptococcus mutans

目的建立稳定、有效的提取变异链球菌蛋白质方法。方法以裂解液为提取介质,分别采用反复冻融超声、超声、热裂解和热裂解超声4种方法提取细菌蛋白质,通过分析细菌破碎程度、蛋白质SDS-PAGE电泳及蛋白质质量浓度,比较各提取方法的效果。结果除热裂解法外,其他3种方法均可有效裂解细菌,其中超声起主要作用,同时这3种方法提取蛋白质的SDS-PAGE条带分布基本相同,但反复冻融超声法提取蛋白质条带丰度最高。蛋白质质量浓度测定显示,反复冻融超声法提取蛋白质质量浓度最高。4种方法均具有较好的稳定性和重复性。结论反复冻融超声法可以有效提取变异链球菌蛋白质,有较好的稳定性和重复性,且操作简单。

Objective To establish an efficient and stable method for protein extraction of Streptococcus mutans. Methods The collected bacteria were treated by freeze-thaw and ultrasonic （method 1）, ultrasonic （method 2）, boiling （method 3）, boiling and ultrasonic （method 4）, respectively. The index such as state of bacteria broken, concentration of extracted protein and SDS-PAGE of protein were employed to evaluate the effects of above four methods. Results Beside the method 3, the other three methods could break the bacteria effectively, of which ultrasonic was the key factor. The pattern of SDS-PAGE which treated by method 1, method 2 and method 4 was almost same, but method 1 resulted in the best abundance. There was significantly difference among the four protein concentration extracted by four methods （P〈0.05）. All methods exhibited good stability and reproducibility. Conclusion Method of freeze-thaw and ultrasonic resulted in an efficient proteins extraction of Streptococcus mutans which demonstrated good stability and reproducibility and easy to handle.