| 应用T7噬菌体展示技术筛选与sLRIG1亲和结合蛋白 |
| 投稿时间：2018-01-15 修订日期：2018-01-17 点此下载全文 |
| 引用本文：朱锡群,易伟.应用T7噬菌体展示技术筛选与sLRIG1亲和结合蛋白[J].医学研究杂志,2018,47(11):49-51 |
| DOI：
10.11969/j.issn.1673-548X.2018.11.011 |
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| 基金项目:国家自然科学基金资助项目（30973073，81172402） |
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| 中文摘要:目的 用T7噬菌体展示技术（phage-displayed technique， PDT）筛选与可溶性多亮氨酸重复区免疫球蛋白样蛋白1（soluble leucine-rich repeats and immunoglobulin-like domains 1， sLRIG1）相互作用的蛋白，为进一步研究sLRIG1作用于人脑胶质瘤的机制找到突破点。方法 利用BacPAK6杆状病毒蛋白表达系统（baculovirus expression system， BEVS）获得人sLRIG1重组蛋白，以该蛋白为靶标，采用亲和淘洗方法经6轮筛选人肝细胞cDNA T7噬菌体展示文库；随机挑选96个筛选到的噬菌体行PCR扩增，琼脂糖凝胶电泳回收DNA片段并送测序。结果 经过6轮筛选，噬菌体投入和产出量比达1.85×106PFU，趋于稳定。回收的DNA片段多在250~750bp，纯化回收后得到35个克隆样本，送测序后得到35条有效表达序列标签，对其行同源性搜索及功能预测等生物信息学分析，最终获得12个可能与sLRIG1蛋白有相互作用的蛋白或多肽，这些蛋白大多与肿瘤相关。结论 通过PDT可获得与sLRIG1蛋白相互结合的蛋白，这些结果为下一步研究sLRIG1的生物功能及其对人脑胶质瘤的作用及机制奠定了基础。 |
| 中文关键词:T7噬菌体 可溶性多亮氨酸重复区免疫球蛋白样蛋白1 胶质瘤 相互作用蛋白 |
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| Screening of Proteins Interacting with Soluble Leucine-rich Repeats and Immunoglobulin-like Domains 1 from T7-phage Display Library |
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| Abstract:Objective To screen proteins who interact with the ectodomains of leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) (soluble LRIG1, sLRIG1) in human cells and to discover new cues for the mechanism researches on sLRIG1 and glioma cells. Methods Method of T7-phage display system was used to find out the proteins that have high affinity to the sLRIG1 protein. A recombination protein of sLRIG1 was used as the target protein, which was obtained from the baculovirus expression system (BEVS). Results After six rounds of T7-phage screening, the ratio of input and output reached 1.85×106 PFU,96 specific phages were obtained. Thirty-five of these phages were identified to be available by PCR amplification and DNA sequencing. Lengths of these DNA fragments were among 250-750bp. After homological, functional and other biological information analysis,12 proteins or peptides were finally selected that supposed to interact with sLRIG1. Conclusion T7-phage display system is an effective method to obtain proteins that interact with sLRIG1. Obtaining these proteins is important for further sLRIG1 researches on its biological functions and therapeutic effects in human glioma. |
| keywords:T7-phage Soluble leucine-rich repeats and immunoglobulin-like domains 1 Human glioma Interacting proteins |
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