Bio-mimicry of paracellular surface on implant neck surface by immobilizing functional claudin peptide to establish an epithelial seal

通过固定功能性紧密蛋白肽来模拟种植体颈部表面的细胞旁表面以建立上皮密封

• 批准号: 22K17077
• 负责人: DILIPKUMAR・MOOTHA ARCHANA
• 金额: $ 2.91万
• 依托单位: Hiroshima University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Early-Career Scientists
• 财政年份: 2022
• 资助国家: 日本
• 起止时间: 2022-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-22K17077/
• 关键词: Claudins; membrane protein; protein expression; cell attachment; inclusion body; Claudin; Peri-implant; Epithelial seal

The aim of this research project is to immobilize recombinantly synthesized claudin membrane protein for epithelial cell attachment on an inert titanium surface to achieve soft tissue attachment around dental implant collar. Claudins DNA sequence was amplified from the human cDNA library using claudin specific primers. Subsequently, the amplified DNA sequence was ligated to pET-22 vector and inserted into Escherichia coli (E.coli) bacteria for cloning and transfected to BL-21 bacterial cells for protein synthesis. Claudin protein expression was attempted at several temperatures and variable durations to determine the best case scenario. Protein was usually expressed as inclusion bodies (IBs), and there were challenges despite several attempts using various solubilizing agents to solubilize these IBs. However, such hurdles were previously expected by the researcher since claudins are membrane proteins and consists of several hydrophobic amino acids, causing IB formation.Currently, to overcome these challenges the researcher will use non-bacterial protein synthesis such as cell-free systems for claudin protein synthesis to avoid IB formation and dependency on bacterial cells for protein expression.In the meantime, commercially claudin protein was immobilized on titanium discs with bio-functional groups and linkers for gingival epithelial cells adhesion to surface modified titanium discs. The preliminary tasks for protein expression using cell-free protein expression systems have already begun as of now.

本课题的研究目的是将重组合成的用于上皮细胞附着的克audin膜蛋白固定在惰性钛表面上，实现种植牙环周围软组织的附着。利用claudin特异性引物从人cDNA文库中扩增出claudin DNA序列。随后，将扩增的DNA序列连接到pET-22载体上，插入大肠杆菌（E.coli）进行克隆，转染到BL-21细菌细胞中进行蛋白质合成。在不同温度和不同时间下尝试Claudin蛋白表达，以确定最佳情况。蛋白质通常以包涵体（IBs）的形式表达，尽管多次尝试使用各种增溶剂对这些包涵体进行增溶，但仍存在挑战。然而，由于claudin是膜蛋白，由几种疏水氨基酸组成，导致IB形成，因此研究人员之前就预料到了这些障碍。目前，为了克服这些挑战，研究人员将使用非细菌蛋白质合成，例如无细胞系统来合成claudin蛋白，以避免IB形成和依赖细菌细胞来表达蛋白质。同时，利用生物功能基团和连接体将市售克audin蛋白固定在钛盘上，使牙龈上皮细胞粘附于表面修饰的钛盘上。目前，利用无细胞蛋白表达系统进行蛋白表达的初步工作已经开始。