人CD81的克隆及在COS-7细胞中的表达

目的 从人外周血淋巴细胞中克隆出CD81基因，构建真核表达质粒，并在COS－7细胞中进行表达。方法 分离外周血淋巴细胞，提取细胞总RNA，采用RT－PCR扩增CD81基因。将CD81基因克隆至载体pcDNA3.1( )中，进行酶切及测序鉴定。以质粒pcDNA3.1-CD81转染COS－7细胞进行瞬时表达，并用免疫细胞化学染色法和流式细胞术检测蛋白的表达。结果 RT－PCR产物已插入载体pcDNA3.1( )构建成真核表达质粒pcDNA3.1-CD81。经双酶切和测序鉴定表明，克隆出的人CD81全长编码序列同GenBank收录的序列一致，并且真核表达质粒的构建正确。以脂质体转染COS－7细胞后，用免疫细胞化学染色法和流式细胞术检测表明，细胞可表达人CD81。结论 成功地构建真核表达载体pcDNA3.1-CD81，为进一步研究HCV和CD81的相互作用，以及建立可能的HCV细胞感染模型打下了基础。

Cloning of human CD81 and its expression in COS-7 cells

Aim To clone human CD81 gene from peripheral blood lymphocytes, and construct its eukaryotic expression vector, and then express it in COS-7 cells. MethodsCD81 gene was amplified by RT-PCR,using RNA extracted from peripheral blood lymphocytes as template, and was cloned into pcDNA3.1(+) vector .Constructed pcDNA3.1-CD81 was identified by endonucleases digestion and sequencing. The recombinant expression plasmid was transfected into COS-7 cells. Human CD81 expressed on membrane of COS-7 cells was detected by immunocytochemical staining and FACS. ResultsObtained full encoding sequence of CD81 was identitical with that included in Genbank, and the eukaryotic expression vector pcDNA3.1-CD81 was constructed correctly. Expression of human CD81 on COS-7 cells was proved by the immunological detection. ConclusionThe eukaryotic expression plasmid pcDNA3.1-CD81 was constructed successfully, which will contribute to further studies on interaction of CD81 with HCV and roles of CD81 in the process of HCV infection and to the establishment of possible better HCV cellular infection model.