Novel Biomarkers of TBI Identified Using Phage Display

使用噬菌体展示鉴定 TBI 的新型生物标志物

• 批准号: 8795230
• 负责人: James W. Geddes
• 金额: $ 18.79万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-01 至 2017-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8795230
• 关键词: Address; Amino Acids; Animal Model; Antibodies; Bacteriophage M13; Bacteriophages; Binding; Binding Proteins; Biochemical Markers; Biological Markers; Biomechanics; Brain Concussion; Brain Injuries; Capsid Proteins; Cell surface; Clinical; Clinical Research; DNA; DNA Sequence; Data; Diagnosis; Diagnostic; Discrimination; Disease; Dizziness; Engineering; Evaluation; Genes; Head; Headache; Health; Image; Incubated; Individual; Inflammatory; Injury; Inovirus; Lateral; Libraries; Link; Liquid substance; Magnetism; Mass Spectrum Analysis; Modeling; Mus; Nausea; Nervous System Physiology; Outcome; Peptide Phage Display Library; Peptides; Percussion; Phage Display; Phase; Post-Concussion Syndrome; Property; Proteins; Protocols documentation; Radiology Specialty; Rattus; Recombinants; Recovery; Sensitivity and Specificity; Serum; Severities; Symptoms; Techniques; Technology; Time; Translating; Translations; Traumatic Brain Injury; Treatment Efficacy; Unconscious State; base; diagnosis evaluation; fluid percussion injury; injured; instrument; interest; mild traumatic brain injury; mouse model; novel; research study; tool

DESCRIPTION (provided by applicant): Traumatic brain injury (TBI) can be frustratingly difficult to diagnose, particularly for mild cases, which can result in inadequate or improper treatment. As a result, there has been considerable interest in identifying biochemical markers of TBI to aid in diagnosis and evaluation of treatment efficacy. CSF and serum alterations in several proteins have been investigated in both animal models and clinical studies, but these appear to be best suited for severe TBI cases. Urgently needed are biomarkers for mild TBI, which represents the majority of the cases and which are the most difficult to diagnose. To identify novel serum and CSF biomarkers for mild and moderate TBI, we propose an unbiased approach utilizing phage display. Phage display is a powerful tool for selecting peptides, proteins or antibodies with specific binding properties. It uses bacteriophages in which DNA encoding a peptide or protein is inserted into the gene encoding a coat protein of a filamentous phage such as M13 phage. The encoded protein or peptide is expressed on the cell surface of the phage and used to attract and bind proteins of interest. We propose to use engineered phage display peptide libraries in which >2 million independent clones express random sequences of 7 or 12 amino acid peptides. In Aim 1, three different phage libraries will be screened to identify phages that bind to the serum and CSF obtained from rats with mild TBI, produced using the lateral fluid percussion model. The biofluids will be collected at 6h and 24h after injury. We will utilize a subtractive panning technique in which phage libraries are first bound to the biofluid (CSF or serum) from uninjured mice to remove non- specific phages, then incubated with injury biofluids to identify injury-selective phages. Preliminary data demonstrate the feasibility of this approach using serum from a mouse TBI model at 6h postinjury. Aim 2 will use sequence and identify the proteins which bind to the phages selective for the injury biofluids. Together, the above results will identify novel biomarkers of mild TBI to assist in the diagnosis of TBI, determination of injury severity, evaluation of recovery and therapeutic efficacy, and prediction of outcomes.

描述（由申请人提供）： 创伤性脑损伤 (TBI) 的诊断非常困难，尤其是对于轻度病例，这可能会导致治疗不充分或不正确。因此，人们对鉴定 TBI 的生化标志物以帮助诊断和评估治疗效果产生了很大的兴趣。脑脊液和血清中几种蛋白质的改变已在动物模型和临床研究中进行了研究，但这些似乎最适合严重的 TBI 病例。轻度 TBI 占大多数病例，也是最难诊断的，迫切需要生物标志物。为了识别轻度和中度 TBI 的新型血清和脑脊液生物标志物，我们提出了一种利用噬菌体展示的公正方法。噬菌体展示是选择具有特定结合特性的肽、蛋白质或抗体的强大工具。它使用噬菌体，其中编码肽或蛋白质的DNA被插入到编码丝状噬菌体（例如M13噬菌体）的外壳蛋白的基因中。编码的蛋白质或肽在噬菌体的细胞表面表达并用于吸引和结合感兴趣的蛋白质。我们建议使用工程噬菌体展示肽文库，其中超过 200 万个独立克隆表达 7 或 12 个氨基酸肽的随机序列。在目标 1 中，将筛选三个不同的噬菌体文库，以鉴定与从患有轻度 TBI 的大鼠获得的血清和脑脊液结合的噬菌体，这些血清和脑脊液是使用横向流体冲击模型产生的。损伤后6小时和24小时收集生物液。我们将利用消减淘选技术，其中噬菌体文库首先与未受伤小鼠的生物液（脑脊液或血清）结合以去除非特异性噬菌体，然后与损伤生物液一起孵育以识别损伤选择性噬菌体。初步数据证明了该方法使用损伤后 6 小时小鼠 TBI 模型血清的可行性。目标 2 将使用序列并鉴定与对损伤生物液具有选择性的噬菌体结合的蛋白质。总之，上述结果将确定轻度 TBI 的新生物标志物，以协助 TBI 的诊断、损伤严重程度的确定、恢复和治疗效果的评估以及结果的预测。