金黄色葡萄球菌肠毒素SEC3抑制剂的筛选及活性鉴定

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,P〈0.05）,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。

Screening and analysis of the activity of a recombinant Staphylococcus aureus enterotoxin C3 inhibitor

Objective To screen a recombinant Staphylococcus aureus enterotoxin C3（SEC3）inhibitor against a random peptide phage display library and preliminarily assess its activity. Methods A random 12-peptide phage display library was immunoscreened with purified recombinant S.aureus enterotoxin C3via binding,elution,and amplification.Phage clones were obtained after 3rounds of biopanning,competitive inhibition of phage binding was determined using competitive ELISA,and the activity of these clones was investigated. Results After 3rounds of screening,the ratio of output to input increased 140-fold and the titer increased from 4.5×10-6 to 6.3×10-4,indicating satisfactory enrichment.Compared to the negative control,8phage clones（A1-A8）randomly selected from the 3rd round of screening were found to compete with SEC3polyclonal antibody.The percent inhibition increased from 10.6%to 38.3%,indicating a significant difference（t=9.0-23.5,P0.05）.A1had the highest percent competitive inhibition（30.5±7.4%）on average while A5had the lowest（16.4±4.7%）.A competitive inhibition curve was created with the logarithm of the phage titer serving as the x axis and the average percent inhibition serving as the y axis.The linear regression equation was y= 2.7943X﹣4.7733,the correlation coefficient r was 0.935,and the coefficient of determination R2 was 0.8727.The percent inhibition increased with an increase in the phage titer from 108 pfu-1012 pfu,and the greatest percent inhibition was obtained with a phage titer of 1011 pfu or 1012 pfu. Conclusion An SEC3inhibitor was screened against a random 12-peptide phage display library.The SEC3inhibitor had a certain level of competitive inhibition and this inhibition activity increased as the phage titer increased.