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Cx31突变可以导致常染色体显性听力下降、常染色体隐性听力下降、周围神经疾病伴听力丧失,以及变性红皮肤病角化病,其导致不同疾病的机理一直是研究的重点.利用定点突变技术(site-directed mutagenesis,SDM)构建connexin31显性听力下降相关突变体Cx31R180X,Cx31E183K并插入到真核表达载体pEGFP-N1,转染HeLa细胞,转染Cx31R180X-pEGFP-N1和Cx31E183K-pEGFP-N1Cx31突变体质粒的HeLa细胞质膜上没有出现斑块状染色和聚集现象,分别用内质网染料conA和高尔基体染料WGA进行免疫荧光染色,结果显示Cx31显性听力下降相关突变体蛋白主要分布在细胞质内,且大部分定位在内质网和高尔基体上.同时分别用抗Cx31和抗GFP抗体进行蛋白质印迹检测,证实Cx31R180X,Cx31E183K在转染的HeLa细胞中都有表达.研究发现Connexin31显性耳聋相关突变体Cx31R180X,Cx31E183K不能正常地形成间隙连接通道,这与connexin31 EKV(变性红皮肤病角化病)相关突变体能够运输到细胞膜上形成间隙连接通道的报道不相同,提示connexin31不同部位突变导致不同疾病的致病机理可能不一样,从而为解释“one connexin two diseases”提供分子水平的依据.
Cx31 mutations can lead to autosomal dominant hearing loss, autosomal recessive hearing loss, peripheral neuropathy with hearing loss, and degenerative erythrocytic keratosis, which led to different mechanisms of disease has been the focus of research. The use of site-directed mutagenesis (Cx31R180X, Cx31E183K) were constructed by site-directed mutagenesis (SDM) and inserted into eukaryotic expression vector pEGFP-N1. HeLa cells were transfected with Cx31R180X-pEGFP-N1 and Cx31E183K-pEGFP-N1Cx31 Plaque-like staining and aggregation were not observed on the plasma membrane of HeLa cells. Immunofluorescent staining with endoplasmic reticulum dye conA and Golgi dye WGA showed that the Cx31 dominant hearing loss-associated mutant proteins mainly distributed in the cytoplasm , And most of them were located on the endoplasmic reticulum and the Golgi.At the same time, Western blotting with anti-Cx31 and anti-GFP antibody respectively confirmed that both Cx31R180X and Cx31E183K were expressed in transfected HeLa cells.The study found that Connexin31 dominant deafness Mutants Cx31R180X, Cx31E183K do not normally form gap junctional channels, which is associated with connexin31 EKV (Denatured Red Skin Disease keratosis) can be transported to the cell membrane to form gap junction channels are not the same report, suggesting that mutations in different parts of connexin31 lead to different diseases may be different pathogenesis, in order to explain the “one connexin two diseases” to provide molecular level The basis.