以重组TACE胞外功能域从噬菌体随机肽库中筛选TACE的抑制肽  

Screening of TACE Peptide Inhibitors from a Phage Display Random 15-Peptide Library by Recombinant TACE Ecotodomain

作　　者：黄巍[1] 李凌波[1] 韩玲[1] 张慧[1] 杨渝珍[1] 

机构地区：[1]华中科技大学同济医学院生物化学与分子生物学系,武汉430030

出　　处：《生物工程学报》2005年第1期30-35,共6页Chinese Journal of Biotechnology

基　　金：国家自然科学基金资助项目 (No .3 0 0 70 72 2 )。~~

摘　　要：肿瘤坏死因子转换酶 (TACE)是加工裂解TNF α前体的关键酶 ,参与了许多炎症的发生发展过程。为通过肽库筛选得到TACE的抑制肽 ,首先制备筛选靶分子 ,用RT PCR从人外周血单核细胞中分别扩增出TACE的催化区 (T80 0 )和整个胞外区 (T130 0 ) ,然后分别克隆至pET 2 8a和pET 2 8c中 ,转化大肠杆菌BL2 1(DE3) ,经IPTG诱导表达出带有His tag的目的蛋白 ,两者均为包涵体 ,变性复性后过Ni2 + NTA亲和层析柱得到纯度达 90 %的重组蛋白。以纯化的重组T80 0和T130 0分别筛选噬菌体展示随机 15肽库 ,对筛选克隆进行ELISA检测、竞争抑制实验和序列分析。从两个独立的筛选过程中得到一个相同的阳性克隆序列“TRWLVYFSRPYLVAT” ,固相Fmoc法合成该短肽 ,观察其在LPS诱导人单核细胞产生sTNF α中的作用。结果表明 ,筛选到的短肽可显著抑制TACE的活性 ,减少TNF α的分泌 ,抑制率可达 6 0 3% ,为抗炎小分子药物的设计和改造提供线索和依据。Tumour necrosis factor α converting enzyme(TACE) is the major protease responsible for processing proTNF from membrane anchored precursor into secreted TNF α. It was validated that TACE is involved in many diseases such as arthritis, multiple sclerosis and Alzheimers, therefore it represents a novel and significant target for therapeutic intervention in a variety of inflammatory and neuroimmunological diseases. To obtain the recombinant TACE ectodomain and use it as a selective molecule for the screening of TACE peptide inhibitors, the cDNA coded for catalytic domain(T800) and full length ectodomain(T1300) of TACE were amplified by RT PCR, the expression plasmid was constructed by inserting T800/T1300 into plasmid pET 28a/pET 28c and transformed into E.coli BL21(DE3). SDS PAGE and Western blotting analysis revealed that T800/T1300 was highly expressed in the form of inclusion body being induced by IPTG. After Ni 2+ NTA resin affinity chromatography, the purity of the recombinant T800/T1300 protein was more than 90%. T800 and T1300 protein were used in the screening of TACE binding peptides from the phage display random 15 peptide library. After four rounds of biopanning, the positive phage clones were analyzed by ELISA,competitive inhibition assay and DNA sequencing. A common amino acid sequence TRWLVYFSRPYLVAT was found and synthesized. The synthetic peptide was shown to bind to TACE and inhibit the TNF α release from LPS stimulated human peripheral blood mononuclear cells(PBMC) up to 60 3%. FACS analysis revealed that the peptide mediated the accumulation of TNF α on LPS stimulated PBMC surface. These results demonstrate that the TACE binding peptide is an effective antagonist of TACE and the deduced motif might be applied to molecular design of anti inflammation drugs.

关 键 词：肿瘤坏死因子转换酶 胞外功能域 噬菌体展示 抑制肽 

分 类 号：R346[医药卫生—基础医学]