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目的建立测定重组铜绿假单胞菌外毒素A(recombinant exotoxin of Pseudomonas aeruginosa,r EPA)含量的SDSPAGE法,并进行验证。方法采用SDS-PAGE,经考马斯亮蓝R-250染色后,利用Image Lab软件分析获得目标蛋白条带光密度值,计算目标蛋白在待测样品中的含量。对建立的方法进行专属性、准确性和重复性验证,确定检测范围,并用建立的方法检测r EPA宿主湿菌体中r EPA发酵产量及r EPA宿主菌休克液柱层析纯化样品中r EPA含量。结果 r EPA含量在100~600μg/ml范围内与其对应条带的光密度值具有良好的相关性,决定系数在0.97以上。大肠埃希菌固有组分及纯化过程中常用的添加物对r EPA含量检测均未产生明显影响,专属性较好;用建立的方法检测500、300、150μg/ml r EPA样品10次的回收率分别为(102.32±4.18)%、(102.77±4.94)%和(95.04±16.41)%,CV值分别为4.08%、4.81%和17.27%。各批r EPA发酵产量为250~500 mg/L,三步柱层析纯化后,r EPA总回收率约为50%。结论建立了测定r EPA含量的SDS-PAGE法,该方法专属性、准确性和重复性良好,为r EPA生产工艺的优化奠定了基础。
Objective To establish and validate the SDSPAGE method for the determination of recombinant exotoxin of Pseudomonas aeruginosa (r EPA). Methods SDS-PAGE, Coomassie Brilliant Blue R-250 stained, the use of Image Lab software to obtain the target protein optical density values, calculate the target protein in the sample content. Specificity, accuracy and repeatability of the established method were validated to determine the detection range and the established method was used to determine the r EPA fermentative production in the r EPA host wet cell and r EPA host bacterium. content. Results r EPA content in the range of 100 ~ 600μg / ml and its corresponding band optical density value has a good correlation, the determination coefficient of 0.97 or more. The inherent components of Escherichia coli and the additives commonly used in the purification process had no significant effect on the detection of r EPA content and the specificity was good. The recovery of 500, 300 and 150μg / ml r EPA samples was tested by the established method The rates were (102.32 ± 4.18)%, (102.77 ± 4.94)% and (95.04 ± 16.41)%, respectively. The CV values were 4.08%, 4.81% and 17.27%, respectively. Each batch of r EPA fermentation yield of 250 ~ 500 mg / L, three-column chromatography purification, r EPA total recovery of about 50%. Conclusion The SDS-PAGE method for the determination of r-EPA content was established. The specificity, accuracy and repeatability of the method were good, which laid the foundation for the optimization of r EPA production technology.