hSALL4基因逆转录病毒载体的构建及在小鼠髓系祖细胞中的表达

Construction of a Recombinant Retroviral Vector Containing Human SALL4 cDNA and Its Expression in Mouse Myeloid Progenitor Cells

目的:构建含人SALL4B基因的逆转录病毒载体并证明其在小鼠髓系祖细胞中的表达。方法:①用PCR方法从质粒pcDNA3-hSALL4B扩增包括有全部编码序列的人SALl4BcDNA,将其定向克隆至逆转录病毒载体pLXSN,构建成含hSALL4的重组逆转录病毒载体;②将重组质粒在包装细胞系PT67中进行包装,用病毒上清感染32D细胞;③RT-PCR分析其mRNA表达;④Western blotting分析其蛋白质的表达。结果:①限制型内切酶酶切,PCR及DNA测序均证实hSALL4B成功地克隆入pLXSN;②转染的32D细胞在mRNA水平及蛋白水平均有SALL4B表达。结论:成功地构建了含人SALL4B的逆转录病毒表达载体;且表达于32D细胞中,为研究SALL4基因过表达对造血功能的作用及其机制奠定了基础。

Objective：To construct a retroviral vector carrying human SALIA cDNA and to verify the expression of this gene in mouse myeloid progenitor cells. Methods： ①SALL4 cDNA was obtained from pcDNA3-hSALL4B by PCR and cloned into a retroviral plasmid, pLXSN. ②Tbe recombinant plasmid was transected to the retrovirus packaging cell PT-67 by lipofectin. Mouse myeloid progenitor cell line 32D cells were transected by the retrovirns. ③The expression of the SALIAB was detected by RT-PCR and western blotting. Results： Recombinant pLXSN-hSALL4 was correctly constructed, as confirmed by restriction enzyme digestion analysis, PCR and DNA sequencing analysis. Successful transfection of 32D cells with this recombinant plasmid was verified by mRNA and protein expressions of SALL4B. Conclusion： hSALL4B was successfully cloned into retroviral vector pLXSN and was successfully expressed in 32D cells. This provided a good foundation for further studies on over-expression of SALIA gene in hematogenesis.