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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。
Objective To screen the S. aureus SEC3 inhibitor from phage random 12-peptide library and identify its activity. Methods Purified recombinant Staphylococcus aureus SEC3 was used to coat the plate and the phage random 12-peptide library was screened by the elution-elution-amplification process. The competitive ELISA was used to observe the competition of single phage clones Inhibit the effect, evaluate its activity. Results The results showed that the input-output of three rounds of phage screening was higher than that of one round. The recovery increased from 4.5 × 10-6 to 6.3 × 10-4, which was 140-fold higher than that of the negative control (A1 ~ A8) were randomly selected from 10.6% to 38.3%, the difference was statistically significant (t = 9.0-23.5, P <0.05), in which A1 average competition The highest inhibitory rate was (30.5 ± 7.4)%, while the average inhibitory rate of A5 was (16.4 ± 4.7)%. The phage titer was x-axis, and the average competitive inhibition rate of each phage titer of phage clones was y-axis. The competitive inhibition curve was drawn. The regression equation was Y = 2.7943X-4.7733, correlation coefficient r = 0.935, R2 = 0.8727. The phage titer was in the range of 108-1012 pfu, with the phage titer increasing, the inhibition rate also increased, and the competition inhibition rate of clonal phage titer at 1011pf or 1012pfu was the highest. Conclusions SEC3 inhibitors screened from the phage random 12-peptide library using purified SEC3 have competitive inhibitory activity and competitive inhibitory activity increases with increasing phage titer.