Determination of OHC protein motor

据估计，外毛细胞（OHC）响应电刺激的运动性的来源是细胞侧壁中的运动蛋白的构象变化。然而，这一机制尚未得到澄清。因此，本研究首先从豚鼠体内取出Corti器，构建Corti器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马达蛋白。结果，发现了两个可能在耳蜗中起重要作用的推定的新基因，然而，P.Dallos等人鉴定了编码运动蛋白的基因，并将其命名为“普雷斯廷”（Nature 405：149-155，2000）。因此，之后，基于公开到公共网站的普雷斯廷的碱基序列，尝试通过聚合酶链反应（PCR）克隆沙鼠普雷斯廷cDNA。结果如下：1。由于普雷斯廷cDNA全长编码序列较长，难以从沙土鼠耳蜗cDNA中直接获得，因此首先获得了4个普雷斯廷的片段。通过PCR扩增，获得了普雷斯廷cDNA的全长编码序列.组合方法对于通过PCR克隆长cDNA是有用的。在该方法中，为了排除组合区域中的错误，重要的是考虑组合方法的反应条件，例如，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.