携带人Ⅸ因子基因逆转录病毒载体的构建及其在小鼠骨髓细胞中的表达  

Construction of retroviral vector MFG Ⅸ and its expression in hematopoietic celis

作　　者：杨建民[1] 卢大儒[2] 王健民[1] 于敏[1] 薛京伦[2] 邱信芳[2] 孟沛霖[1] 

机构地区：[1]上海长海医院血液科,上海200433 [2]上海复旦大学遗传学研究所,上海200433

出　　处：《中国实验血液学杂志》1998年第2期133-136,共4页Journal of Experimental Hematology

摘　　要：构建携带人Ⅸ因子。cDNA的逆转录病毒载体MFGⅨ,并观察其在造血细胞中的表达。应用DNA重组技术将人Ⅸ因子cDNA构建入MFG载体,转导入包装细胞系PA317细胞,应用病毒上清转染造血细胞,并用PCR,ELISA及免疫组化方法检测其表达。研究结果表明,酶切鉴定成功构建MFGⅣ逆转录病毒载体,转入HT1080细胞系及小鼠骨髓细胞后,用PCR,ELISA及免疫荧光染色和免疫细胞化学染色证实Ⅸ因子的表达,且ELISA结果显示应用MFGⅨ转染Ⅸ因子蛋白及活性均高于LNCⅨ。结论提示MFGⅨ载体能较好转染造血细胞。To construct retroviral vector MFG K carrying human factor K and observe its expression in hematopoietic celis, the hFK cDNA was inserted into the retroviral vector MFG, the DNA of MFG K was transduced the DNA into the packaging celi line PA317, the hematopoietic celis were transduced with the supernatant from MFG K/PA317, its expression was detected by PCR analysis, ELISA and immunohistochemistry methods. Results showed that the retroviral vector MFG K was successfully constructed, HT1080 and murine bone mar-row celi were transduced with the supernatant from MFG K/PA317. PCR, ELISA analysis, immunocytoche-mistry and immunofluorescence examination confirmed the gene transfer and in vitro expression of hFIX deter-mined by ELISA was higher than the control group. It is concluded that hematopoietic celis can be successfully transduced by retroviral vector MFG K .

关 键 词：Ⅸ因子 逆转录病毒载体 MFGⅨ 造血细胞 骨髓细胞 

分 类 号：Q782[生物学—分子生物学]