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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。
Objective To clone CD81 gene from human peripheral blood lymphocytes, construct eukaryotic expression plasmid and express in COS-7 cells. Methods Peripheral blood lymphocytes were isolated and total cellular RNA was extracted. CD81 gene was amplified by RT-PCR. CD81 gene was cloned into the vector pcDNA3.1 (+), digested and sequenced. The recombinant plasmid pcDNA3.1-CD81 was transfected into COS-7 cells for transient expression. Immunocytochemistry and flow cytometry were used to detect the protein expression. Results RT-PCR products were inserted into pcDNA3.1 (+) vector to construct eukaryotic expression plasmid pcDNA3.1-CD81. Double enzyme digestion and sequencing showed that the cloned human CD81 full-length coding sequence was identical with that of GenBank, and the eukaryotic expression plasmid was constructed correctly. After transfection of COS-7 cells with liposomes, immunocytochemical staining and flow cytometry showed that the cells could express human CD81. Conclusion The eukaryotic expression vector pcDNA3.1-CD81 was successfully constructed, which laid the foundation for the further study of the interaction between HCV and CD81 and the establishment of a possible HCV infection model.