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目的构建人淋巴细胞趋化因子(humanlymphotactin,hLptn)真核表达质粒,并在膀胱肿瘤细胞株BIU-87中的表达重组质粒。方法用RT-PCR法自活化的人外周血淋巴细胞扩增hLptn的含编码区序列的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pcD-NA3.1(+)-hLptn,用脂质体将其转染BIU-87细胞,经G418筛选稳定表达克隆,用免疫荧光染色技术鉴定经筛选的克隆。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;筛选获得了稳定表达转染hLptn的BIU-87细胞株。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中稳定表达,为研究hLptn基因介导的人膀胱肿瘤特异性主动免疫治疗奠定了基础。
Objective To construct eukaryotic expression plasmid of human lymphotactin (hLptn) and express the recombinant plasmid in bladder tumor cell line BIU-87. Methods The cDNA of hLptn coding region was amplified from human peripheral blood lymphocytes by RT-PCR and cloned into pGM-TEasyT vector. After sequencing, the target fragment was inserted into pcDNA3.1 (+) vector to obtain positive The pcD-NA3.1 (+) - hLptn was cloned and transfected into BIU-87 cells by lipofectamine. The clones were stably expressed by G418 selection and identified by immunofluorescence staining. Results The cloned cDNA sequence was different from the 225th nucleotide in the coding region of U23772 in GenBank, and was synonymous with it. The recombinant eukaryotic expression vector pcDNA3.1 (+) - hLptn was constructed and the recombinant plasmid was successfully transfected into hLptn BIU-87 cell line. Conclusion The successfully constructed hLptn eukaryotic expression system can stably express in human bladder tumor cell line BIU-87, which lays the foundation for the study of human bladder tumor-specific active immunotherapy mediated by hLptn gene.