人连接蛋白Connexin26基因的克隆、表达及初步鉴定

目的研究含人连接蛋白Connexin26（Cx26）基因的真核表达载体构建及其在非洲猴肾细胞（COS-7）中获得稳定、高效表达的方法。方法提取人外周血淋巴细胞RNA，经逆转录制备成cDNA，设计特异性引物，用PCR方法从cDNA中扩增Cx26基因，将其定向插入真核表达载体pCI-neo中获得pCI-Cx26重组子-采用酶切法和测序法鉴定。用脂质体将pCI-Cx26转染COS-7细胞，经G418筛选，获得阳性克隆。用RT-PCR和SDS-PAGE检测对表达产物进行检测。结果从人外周血RNA制备而成的cDNA中可扩增出预期大小的Cx26基因片段，pCI-Cx26经双酶切测序证实构建成功。RT-PCR和SDS-PAGE检测到pCI-Cx26在COS-7细胞中获得稳定、高效表达。结论本研究成功构建了人连接蛋白Cx26基因重组真核表达载体pCI-Cx26：pCI-Cx26脂质体转染COS-7细胞，可见Cx26基因在COS-7细胞中获得稳定、高效的表达。该结果为今后进行pCI-Cx26基因治疗Cx26基因突变引起的遗传性耳聋的研究奠定了实验基础。

Expression of Human Connexin26 in COS-7 Cells

Objective To construct the eukaryotic expression plasmid for the expression of human Connexin26 in COS-7 cells. Methods Total RNA was isolated from human peripheral blood lymphocytes and used as template for the PCR cloning of the human Connexin26 gene. The human Cx26 cDNA containing the 678 bp whole coding region of the human Connexin26 gene was amplified by PCR using specific primers and cloned into the pCI-neo vector to construct the recombinant eukaryotic expression plasmid,pCI-Cx26. The recombinant plasmid was identified by restriction endonuclease digestion, and transfected into COS-7 cells by liposome. The expression of Cx26 mRNA and the protein were analyzed by RT-PCR and SDS-PAGE, respectively. Results Restriction endonuclease digestion analysis verified successful construction of the recombinant plasmid, pCI-Cx26. The expression of Cx26 mRNA and protein in the transfected COS-7 cells were detected by RT-PCR and SDS-PAGE, respectively. Conclusion The eukaryotic expression plasmid for human Cx26 has been constructed successfully with the capability of expression in COS-7 cells.