金黄色葡萄球菌肠毒素SEC3抑制剂的筛选及活性鉴定

发布时间：2018-04-09 11:56

本文选题：金黄色葡萄球菌　切入点：肠毒素　出处：《中国病原生物学杂志》2014年04期

【摘要】：目的从噬菌体随机12肽库中筛选出金黄色葡萄球菌SEC3抑制剂并鉴定其活性。方法用纯化的重组的金黄色葡萄球菌SEC3包被酶标板,按吸附-洗脱-扩增的淘洗过程对噬菌体随机12肽库进行3轮筛选;用竞争ELISA法观察单个噬菌体克隆的竞争抑制效应,评价其活性。结果噬菌体3轮筛选的投入产出比逐轮升高,回收率从4.5×10-6升高至6.3×10-4,升高140倍,提示具有良好的富集效果;与阴性对照(未加噬菌体)相比,随机挑选的8个噬菌体克隆(A1~A8)竞争抑制率为10.6%~38.3%,差异有统计学意义(t值为9.0~23.5,P0.05),其中A1平均竞争抑制率最高,为(30.5±7.4)%,A5的平均竞争抑制率最小,为(16.4±4.7)%。以噬菌体滴度对数值为x轴,以克隆噬菌体各滴度对应的平均竞争抑制率为y轴,绘制竞争抑制曲线,其回归方程为Y=2.7943X-4.7733,相关系数r=0.935,决定系数R2=0.8727。在噬菌体滴度在108~1012pfu范围内,随着噬菌体滴度的增高,其抑制率也增高,且克隆噬菌体滴度在1011pf或1012pfu的竞争抑制率最高。结论用纯化的SEC3从噬菌体随机12肽库中筛选的SEC3抑制剂具有一定的竞争抑制活性,而且竞争抑制活性随着噬菌体滴度的增高而增强。
[Abstract]:Objective to screen Staphylococcus aureus SEC3 inhibitor from phage random 12 peptide library and to identify its activity.Methods the phage phage random 12 peptide library was screened with purified recombinant Staphylococcus aureus SEC3 coated with enzyme label, and the competitive inhibitory effect of single phage clone was observed by competitive ELISA method, according to the process of adsorption, elution and amplification of phage random 12 peptide library.Its activity was evaluated.Results the input-output of bacteriophage screening increased from 4.5 脳 10 ~ (-6) to 6.3 脳 10 ~ (-4) from 4.5 脳 10 ~ (-6) to 6.3 脳 10 ~ (-4).The logarithm of phage titer was taken as x axis and the average competitive inhibition rate of clone phage titer was y axis. The regression equation was Y _ (2.7943) X-4.7733, the correlation coefficient was 0.935, and the determining coefficient was R _ (2) O _ (2) O _ (0.8727).In the range of 108~1012pfu, the inhibition rate of bacteriophage titer increased with the increase of phage titer, and the competitive inhibition rate of clone phage titer was the highest in 1011pf or 1012pfu.Conclusion the SEC3 inhibitor screened by purified SEC3 from phage random 12 peptide library has a certain competitive inhibitory activity, and the competitive inhibitory activity increases with the increase of phage titer.
【作者单位】：中南大学湘雅二医院检验科;
【基金】：湖南省科技计划项目(No.2013SK3048) 湖南省卫生厅科研基金项目(No.132012024)
【分类号】：R378

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