Novel Biomarkers of TBI Identified Using Phage Display

描述(由申请人提供)：创伤性脑损伤(TBI)很难诊断，特别是对于轻微的病例，这可能会导致治疗不充分或不适当。因此，人们对确定脑损伤的生化标志物以帮助诊断和评估治疗效果有相当大的兴趣。已经在动物模型和临床研究中研究了脑脊液和血清中几种蛋白质的变化，但这些似乎最适合于严重的脑外伤病例。迫切需要用于轻度脑外伤的生物标记物，这代表了大多数病例，也是最难诊断的。为了确定轻中度脑损伤的新的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无偏见的方法。噬菌体展示是筛选具有特定结合特性的多肽、蛋白质或抗体的有力工具。它使用噬菌体，将编码多肽或蛋白质的DNA插入到编码丝状噬菌体外壳蛋白的基因中，如M13噬菌体。编码的蛋白质或肽在噬菌体的细胞表面表达，用于吸引和结合感兴趣的蛋白质。我们建议使用工程化噬菌体展示肽库，其中200万个独立克隆表达7或12个氨基酸的随机序列。在目标1中，将筛选三个不同的噬菌体文库，以鉴定与轻微脑外伤大鼠的血清和脑脊液结合的噬菌体。分别于伤后6小时和24小时采集体液。我们将利用消减平移技术，首先将噬菌体文库结合到未受伤小鼠的生物液(脑脊液或血清)上，以去除非特定噬菌体，然后与受伤的生物液孵育，以识别损伤选择性噬菌体。初步数据表明，使用创伤后6h的小鼠脑损伤模型的血清，这种方法是可行的。目的2将利用测序和鉴定与噬菌体结合的蛋白来选择性地识别受伤的生物液。综上所述，上述结果将确定新的轻度脑损伤生物标志物，以辅助脑损伤的诊断、损伤严重程度的确定、恢复和治疗效果的评估以及预后的预测。

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.