Novel Biomarkers of TBI Identified Using Phage Display

描述（由申请人提供）：创伤性脑损伤（TBI）的诊断非常困难，特别是对于轻微的病例，这可能导致治疗不足或不当。因此，人们对识别TBI的生化标记物以帮助诊断和评估治疗效果非常感兴趣。在动物模型和临床研究中已经研究了脑脊液和血清中几种蛋白质的改变，但这些似乎最适合于严重的脑外伤病例。目前迫切需要的是轻度创伤性脑损伤的生物标志物，这代表了大多数病例，也是最难诊断的。为了鉴定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、蛋白质或抗体的有力工具。它使用噬菌体，将编码肽或蛋白质的DNA插入编码丝状噬菌体（如M13噬菌体）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，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，其中有200万个独立克隆表达7或12个氨基酸肽的随机序列。在Aim 1中，将筛选三种不同的噬菌体文库，以鉴定与轻度脑损伤大鼠血清和脑脊液结合的噬菌体，这些噬菌体是使用侧流式冲击模型产生的。在伤后6h和24h采集生物体液。我们将利用减法筛选技术，首先将噬菌体文库与未受伤小鼠的生物液（CSF或血清）结合以去除非特异性噬菌体，然后与损伤生物液孵育以识别损伤选择性噬菌体。初步数据表明，该方法在小鼠TBI模型损伤后6小时的血清中是可行的。目标2将使用序列和识别与选择性损伤生物液体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，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.