诱生型一氧化氮合酶特异性抑制肽的制备

目的：通过噬菌体展示技术筛选iNOS特异性抑制肽。方法：将iNOSFAD结合区及其附近序列的基因片段装入pET-28A( )，在大肠杆菌BL21中表达，His.bind^TM亲和层析柱纯化目的蛋白，使用纯化蛋白筛选Ph.D.-12^TM噬菌体库，筛选iNOS活性抑制作用较高的噬菌体克隆，测序并合成其中具有一致序列的短肽。结果：得到具有较高表达量的目的蛋白，经His.Bind^TM柱亲和层析纯化后纯度大于95％，以纯化蛋白筛选Ph.D.-12^TM噬菌体库，经4轮筛选获得10株iNOS活性抑制作用较高的噬菌体克隆，测序发现其中5株序列完全相同，合成该12肽，初步研究表明其对iNOS表现为高浓度抑制，低浓度兴奋的作用，而对nNOS及eNOS则没有影响。结论：以iNOSFAD片段蛋白为靶蛋白筛选得到的克隆对iNOS活性具有特异性影响，可根据这些特征设计合成小分子前导药物，创造新的活性药物。

Preparation of the Specified Inhibitory Peptide of iNOS.

AIM The purpose is to prepare the specific inhibitory peptide of iNOS by Phage display. METHODS A fragment of iNOS(703-881AA)was inserted in pET-28a(+)plasmid the combinant construct was transformed into BL 21 E.coli. The expressed protein was purified by His.Bind TM -Sepharose column. The phage surface displayed Ph.D.-12 TM library was screened with the purified protein; the clone having inhibitory effect on iNOS activity was selected and sequenced. The peptide having consentaneous sequence was synthesized and the effect of it on iNOS activity was investigated. RESULTS A high level expression of the target protein was found after IPTG induction and fine purified target protein was obtained by His. Bind+{TM}-Sepharose column. 10 phages clone having a higher inhibitory effect on iNOS activity was selected after 4 rounds screening to Ph.D.-12 TM library, five of them have the same sequence. The peptide was synthesized. NOS activity study revealed that it inhibited iNOS activity at high concentration, but excited at low concentration, and it did not cross-react with nNOS and eNOS. CONCLUTION Screening the phage library with iNOSFAD fragment can get the phage containing the peptide which combines to iNOS specifically and have no effect on nNOS and eNOS, and the new inhibitory drug of iNOS can be designed according to the character of the peptide.