人淋巴细胞趋化因子真核表达质粒的构建及体外转染的生物学活性测定

目的：构建人淋巴细胞趋化因子(HLptn)真核表达质粒，在膀胱肿瘤细胞株BIU-87中表达重组质粒，并检测趋化活性。方法：用RT-PCR法自活化的人外周血淋巴细胞扩增HLptn含编码区序列的cDNA，克隆至pGM-T Easy T载体，测序正确后，将目的片段插入pcDNA3．1( )载体，获得阳性克隆pcDNA3．1( )．HLptn；用脂质体介导其转染BIU-87细胞，用Western-blot检测转染后细胞中HLptn的表达；取转染后的上清液，采用Boyden小室法检测表达的HLptn趋化CD4^ 、CD8^ T淋巴细胞的生物学活性。结果：克隆的cDNA序列与GenBank中U23772的序列编码区内第225位碱基不同，系同义突变，构建了真核重组表达载体pcDNA3．1( )-HLptn；转染的BIU-87细胞表达HLptn，其培养上清对CD4^ 、CD8^ T淋巴细胞具有趋化活性。结论：成功构建的HLptn真核表达系统可在人膀胱肿瘤细胞株BIU-87中表达。

Construction of human lymphotactin eukaryotic expression vector and detecting of chemotactic activity after being transfected in vitro

Objective: To construct an eukaryotic expression vector containing human lymphotactin (HLptn) cDNA and to express it in human bladder carcinoma cell (BCC)line BIU-87 and detect chemotactic activity. Methods: HLptn cDNA was amplified by RT-PCR from stimulated human peripheral blood mononuclear cells and cloned to vector pGM-T Easy. After confirming the sequence,the cDNA was inserted into pcDNA3.1(+) and the postive clone pcDNA3.1(+)-HLptn was obtained. The recombinant plasmid was transfected into BIU-87 cells with Lipofectamine TM2000 and the expression was detected by Western blot. Its culture supernatants were used to chemotaxis assay of HLptn attracting CD4 + T cells and CD8 + T cells with Boyden Chemotaxis Chamber. Results: The sequence of cloned HLptn cDNA was different from NO.U23772 at No.325 base in ORF,it was same sense mutation; Mammalian recombinant expression plasmid pcDNA3.1(+)-HLptn was constructed; It can express in BIU-87 with Lipofectamine TM2000. Its culture supernatants was chemotactic for CD4 + T cells and CD8 + T cells. Conclusion: The eukaryotic expression system of HLptn constructed with our method could express in human BCC cell line BIU-87.