7肽和12肽两种M13噬菌体展示库筛选肿瘤坏死因子alpha拮抗肽的比较

doi:10.13523/j.cb.20170501

中国生物工程杂志

2017, Vol. 37

Issue (5): 1-8 DOI: 10.13523/j.cb.20170501

研究报告

7肽和12肽两种M13噬菌体展示库筛选肿瘤坏死因子alpha拮抗肽的比较

胡昌武, 谢君, 朱乃硕

复旦大学生命科学学院遗传工程国家重点实验室微生物与分子免疫学实验室上海 200438

Comparison of the Inhibitory Efficiency of M13 Based 7-mer and 12-mer Phage Display Libraries Derived Peptides as Tumor Necrosis Factor Alpha Antagonist

HU Chang-wu, XIE Jun, ZHU Nai-shuo

State Key Lab of Genetic Engineering, Lad of Molecular Immunology, School of Life Science, Fudan University, Shanghai 200438, China

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摘要：

肿瘤坏死因子alpha的拮抗剂是治疗多种炎症性自身免疫疾病的首选，但抗体类拮抗物因副作用明显而使用受限，尤其是机体内抗抗体的产生，严重影响治疗效果和药物代谢。而肽类物除免疫原性低之外，和小分子相比也有更低的毒性和更强的靶标特异性。使用7肽和12肽两种M13噬菌体展示库筛选TNFα拮抗肽，以分析7肽和12肽分别作为TNFα拮抗肽的亲和性与功能性。经过3~4轮的筛选和验证，得到2条7肽序列和2条12肽序列。利用ELISA方法检测合成肽与TNFα结合的亲和性，编号632的7肽亲和性最强，Kd=138nmol/L；编号636的12肽亲和性稍差，Kd=8.59μmol/L。Insight Ⅱ软件分别分析632肽和636肽与TNFα二聚体结合，发现632肽与TNFα二聚体结合更加稳定，并且在细胞水平上632肽拮抗TNFα活性功能比636肽更强，有632肽存在的条件下TNFα诱导的L929细胞生存率上升了3倍，而636肽的作用只有2倍。7肽比12肽更适合作为TNFα拮抗肽。

关键词： 拮抗肽; 噬菌体展示库; 肿瘤坏死因子

Abstract:

The antagonist of tumor necrosis factor alpha is the first choice for the treatment of multiple inflammatory autoimmune diseases. Treatment with TNFα antibodies is restricted by their side effects, particularly, the production of anti-antibodies, which seriously affect the treatment efficacy and drug metabolism. Short peptides have low immunogenicity, and compared to small molecules, they also have lower toxicity and stronger target specificity. Using M13 based 7-mer and 12-mer peptides phage display libraries to screen TNFα binding peptide ligands, and analyzed the affinity and functionality of the selected TNFα antagonist. After 3-4 rounds of screening, two 7-mer and two 12-mer peptide sequences were obtained. Binding affinity of the synthetic peptides for TNFα was determined by ELISA. 7-mer peptide with number 632 showed affinity of Kd=138nmol/L, while the 12-mer peptide with number 636 showed lower affinity of Kd=8.59μmol/L. Insight II software was used to carry out Zdock with TNFα dimer, and it was found that both the 7-mer peptide could bind to TNFα with a more stable state than the 12-mer peptide. At the cellular level, the peptide 632 were more resistant to the activity of TNFα than peptide 636. In the presence of peptide 632, the survival rate of L929 cells induced by TNFα was 3 times higher, but the 636 peptides were only 2 times. Altogether, the 7-mer peptides were more suitable than 12-mer peptide as TNFα antagonist.

Key words: Phage display library Antagonistic peptide Tumor necrosis factor

收稿日期: 2016-11-01 出版日期: 2017-05-25

ZTFLH:

Q939.91

基金资助:

国家自然科学基金（30901315,30970144）、国家“863”计划重大课题（2011AA02A114）、上海市科委支撑项目（13431900602）资助项目

通讯作者: 朱乃硕 E-mail: nzhu@fudan.edu.cn

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引用本文:

胡昌武, 谢君, 朱乃硕. 7肽和12肽两种M13噬菌体展示库筛选肿瘤坏死因子alpha拮抗肽的比较[J]. 中国生物工程杂志, 2017, 37(5): 1-8.

HU Chang-wu, XIE Jun, ZHU Nai-shuo. Comparison of the Inhibitory Efficiency of M13 Based 7-mer and 12-mer Phage Display Libraries Derived Peptides as Tumor Necrosis Factor Alpha Antagonist. China Biotechnology, 2017, 37(5): 1-8.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20170501 或 https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I5/1

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