Determination of OHC protein motor

据估计，外毛细胞(OHC)对电刺激反应的运动性起源于细胞侧壁中运动蛋白的构象变化。然而，这一机制目前还没有得到澄清。因此，在本研究中，首先从豚鼠中取出Corti器官，构建了Corti器官的cDNA文库。然后，在对每个克隆进行测序后，试图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发挥重要作用的新基因。然而，P.Dalls等人。确定了编码马达蛋白的基因，并将其命名为“Prestin”(自然405：149-155,2000)。因此，在此基础上，利用已公开的prestin基因的碱基序列，利用聚合酶链式反应(PCR)技术克隆了沙土鼠prestin基因。主要研究结果如下：1.鉴于prestin基因全长编码序列较长，很难直接从沙土鼠耳蜗组织中获得，本研究首先获得了prestin基因的四个片段。然后将这些片段进行聚合酶链式反应，获得了prestin基因的全长编码序列。在这种方法中，为了排除合并区域中的错误，必须考虑合并方法的反应条件，如温度、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.