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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。
Objective To study the construction of eukaryotic expression vector containing human Connexin26 (Cx26) gene and its stable and efficient expression in African monkey kidney cells (COS-7). Methods Human peripheral blood lymphocyte RNA was extracted and cDNA was prepared by reverse transcription. The specific primers were designed. The Cx26 gene was amplified from cDNA by PCR and inserted into eukaryotic expression vector pCI-neo to obtain pCI-Cx26 recombinant , Using enzyme digestion and sequencing identification. PCI-Cx26 was transfected into COS-7 cells by lipofectamine and then screened by G418 to obtain positive clones. The expression products were detected by RT-PCR and SDS-PAGE. Results Cx26 gene fragment of the expected size was amplified from cDNA prepared from human peripheral blood RNA and pCI-Cx26 was confirmed by double enzyme digestion sequencing. The results of RT-PCR and SDS-PAGE showed that pCI-Cx26 was stable and highly expressed in COS-7 cells. Conclusion The recombinant eukaryotic expression vector pCI-Cx26 of human connexin Cx26 gene was successfully constructed in this study. The pCI-Cx26 vector was transfected into COS-7 cells. The stable and highly efficient expression of Cx26 gene was observed in COS-7 cells. The results laid the experimental foundation for the future study of hereditary deafness caused by mutation of Cx26 gene in pCI-Cx26 gene therapy.