摘要
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 TEasyT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。
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.
出处
《医学研究生学报》
CAS
2005年第3期196-199,共4页
Journal of Medical Postgraduates
基金
国家自然科学基金资助项目(批准号: 30271300)