重组铜绿假单胞菌外毒素A含量SDS-PAGE检测方法的建立及验证

Development and verification of a SDS-PAGE method for determination of content of recombinant exotoxin of Pseudomonas aeruginosa

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 develop and verify a SDS-PAGE method for determination of recombinant exotoxin of Pseudomonas aeruginosa（r EPA）. Methods SDS-PAGE gel was stained with Coomassie brilliant blue R-250 and analyzed for optical density with Image lab software, based on which the content of target protein in sample was calculated. The devel-oped method was verified for specificity, accuracy and reproducibility, determined for detection range, and used for deter-mination of fermentation yield of r EPA in wet host bacteria as well as r EPA content in shock liquid of host bacteria purified by column chromatography. Results The r EPA content at a range of 100 - 600 μg / ml showed good relationship to the optical density of the corresponding band, with a r2 value of more than 0. 97. Both the natural ingredients of E. coli and common additives during purification process showed no significant effect on the determination of r EPA content, indi-cating high specificity of the developed method. The recovery rates of 500, 300 and 150 μg / ml r EPA in ten tests by the developed method were（102. 32 ± 4. 18）%,（102. 77 ± 4. 94）% and（95. 04 ± 16. 41）%, with CVs of 4. 08%, 4. 81%and 17. 27%, respectively. The fermentation yields of r EPA of various batches were 250 - 500 mg / L, while the total recovery rate after three steps of chromatography was about 50%. Conclusion A SDS-PAGE method for determination of r EPA content was developed, which was specific, accurate and reproducible. It laid a foundation of optimization of pro-duction procedure.