Determination of OHC protein motor

据估计，外毛细胞（OHC）响应电刺激的运动性的起源是细胞外侧壁运动蛋白的构象变化。然而，这一机制尚未得到澄清。因此，本研究首先从豚鼠身上取下Corti器官，构建Corti器官cDNA文库。然后，在每个克隆测序后，尝试通过分析序列和组织表达来鉴定运动蛋白。结果发现了两个可能在耳蜗中起重要作用的新基因。然而，P.Dallos等人发现了编码运动蛋白的基因，并将其命名为“Prestin”（Nature 405: 149-155, 2000）。因此，在此之后，我们以公开的沙鼠prestin碱基序列为基础，尝试用聚合酶链反应（PCR）克隆沙鼠prestin cDNA。研究结果如下：1。由于prestin cDNA全长编码序列较长，难以直接从沙鼠耳蜗cDNA中获得，因此首先获得了prestin的4个片段。然后用PCR方法将这些片段组合起来。结果获得了prestin cDNA的全长编码序列。该组合方法可用于PCR克隆长cDNA。在该方法中，为了排除组合区域中的误差，需要考虑组合方法的反应条件，如退火温度、时间等。

It is estimated that the origin of the motility of the outer hair cell (OHC) in response to electrical stimulation is the conformational change of the motor protein in the lateral wall of the cell. However, this mechanism has not been clarified yet. Therefore, in this study, firstly, the organ of Corti was removed from the guinea pig and the organ of Corti cDNA library was constructed. Then, after each clone was sequenced, an attempt was made to identify the motor protein by analyzing the sequence and tissue expression. As a result, two putative novel genes which might play an important role in the cochlea were found. However, P.Dallos et al. identified the gene that codes for the motor protein and designated it "Prestin" (Nature 405 : 149-155, 2000). Therefore, after that, based on the base sequences of prestin opened to the public web site, an attempt was made to clone gerbil prestin cDNA by polymerase chain reaction (PCR). The results are as follows :1. As the full length coding sequence of prestin cDNA is quite long and it is difficult to obtain it directly from the gerbil cochlear cDNA, firstly, four fragments of prestin were obtained. Then, these fragments were combined by PCR.As a result, the full length coding sequence of prestin cDNA is obtained.2. The combining method is useful to clone long cDNA by PCR.In this method, in order to exclude an error in the combined region, it is important to consider the reaction condition of combining method, e.g., the annealing temperature, time, and so on.