小鼠趋化因子CXCL10真核表达载体的构建 (pdf)

    【摘要】 应用RT-PCR方法，从小鼠脾脏T细胞的mRNA中，扩增获得编码小鼠趋化因子CXCL10的cDNA，并加上人单核细胞趋化蛋白1的信号肽基因，将其克隆到Pucm-T载体，经酶切及测序鉴定，进而构建CXCL10重组真核表达载体。采用脂质体法体外转染COS-7真核细胞，经Western blot和趋化指数检测，转染细胞能够表达CXCL10蛋白，其上清对淋巴细胞具有趋化作用。CXCL10的真核表达载体构建成功，为进一步研究其在肿瘤治疗中的作用奠定了基础。

    【关键词】 CXCL10；基因克隆；真核表达载体

    Gene cloning and eukaryotic expression vector construction of murine CXCL10

    LI Bo, LI Qing, ZHAO Qing-li,et al.Department of Urology, Qianfoshan Hospital of Shandong Province, Jinan，Shandong 250014, China

    【Abstract】 The cDNA fragment encoding the murine CXCL10 was obtained from mRNA of mouse T cells by using reversal transcript-polymerase chain reaction (RT-PCR)and contained a signal peptide derived from the human monocyte chemoattractant protein 1, then it was cloned into the Pucm-T vector. By partial nucleotide sequencing, the cDNA was consistent with the reported mCXCL10 cDNA in the gene bank, and then was inserted into the eukaryotic expression vector pcDNA3.1(+). The recombinant plasmid was transfected into COS-7cell line by DOTAP liposome. The expression of the target molecule was assayed by Western blot and chemotaxis assay.It was found that the cells transfected with plasmid vector could express mCXCL10 protein and it’s culture supernatants could attract T cells markedly. The successful construction of mCXCL10 eukaryotic expression vector lays the foundation for it’s further researching in treatment of tumor. ......