诱生型一氧化氮合酶特异性抑制肽的制备  

Preparation of the Specified Inhibitory Peptide of iNOS.

作　　者：潘英[1] 朱敏生[1] 沈月[1] 许祥裕[1] 朱东亚[2] 朱有华[1] 

机构地区：[1]南京军区军事医学研究所,南京210002 [2]中国药科大学新药研究中心,南京210009

出　　处：《中国药科大学学报》2002年第1期66-69,共4页Journal of China Pharmaceutical University

基　　金：国家自然科学基金资助项目 ( 396 70 85 6 )

摘　　要：目的 :通过噬菌体展示技术筛选 i NOS特异性抑制肽。方法 :将 i NOS FAD结合区及其附近序列的基因片段装入 p ET-2 8a( + ) ,在大肠杆菌 BL2 1 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.-1 2 TM噬菌体库 ,筛选 i NOS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.-1 2 TM噬菌体库 ,经 4轮筛选获得 1 0株 i NOS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 1 2肽 ,初步研究表明其对 i NOS表现为高浓度抑制 ,低浓度兴奋的作用 ,而对 n NOS及 e NOS则没有影响。结论 :以 i NOSFAD片段蛋白为靶蛋白筛选得到的克隆对 i NOS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 。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.

关 键 词：iNOSFAD片段 噬菌体展示 iNOS抑制肽 制备 诱生型一氧化氮梧酶 特异性抑制肽 

分 类 号：Q591.2[生物学—生物化学]