Novel Biomarkers of TBI Identified Using Phage Display

描述（由申请人提供）：创伤性脑损伤（TBI）可能难以诊断，特别是对于轻度病例，这可能导致治疗不足或不当。因此，人们对鉴定TBI的生化标志物以帮助诊断和评价治疗效果产生了相当大的兴趣。在动物模型和临床研究中已经研究了几种蛋白质的CSF和血清改变，但这些似乎最适合于严重的TBI病例。迫切需要的是轻度TBI的生物标志物，这代表了大多数病例，也是最难诊断的。为了确定新的血清和CSF生物标志物的轻度和中度TBI，我们提出了一个公正的方法，利用噬菌体展示。噬菌体展示是筛选具有特异性结合特性的多肽、蛋白质或抗体的有力工具。它使用噬菌体，其中编码肽或蛋白质的DNA插入编码丝状噬菌体（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上表达，并用于吸引和结合感兴趣的蛋白质。我们建议使用工程化的噬菌体展示肽文库，其中> 200万个独立克隆表达7或12个氨基酸肽的随机序列。在目的1中，将筛选三种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CSF结合的噬菌体，所述血清和CSF使用侧向流体撞击模型产生。分别于伤后6h和24h采集生物液。我们将利用消减淘选技术，其中噬菌体文库首先与来自未受伤小鼠的生物流体（CSF或血清）结合以去除非特异性噬菌体，然后与损伤生物流体孵育以鉴定损伤选择性噬菌体。初步数据表明，这种方法的可行性，使用血清从小鼠TBI模型在伤后6小时。目的2将使用序列分析和鉴定与损伤生物液选择性结合的蛋白质。总之，上述结果将确定轻度TBI的新生物标志物，以帮助诊断TBI，确定损伤严重程度，评估恢复和治疗效果以及预测结果。

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.