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目的 构建人FLT3配基 (FL)逆转录病毒载体 ,并观察其在人骨髓基质细胞中的表达。方法 用基因重组技术 ,将FLcDNA插入逆转录病毒载体pLXIN ,获得重组载体pLFIN。采用脂质体法将重组质粒pLFIN转染包装细胞PA317,G418筛选抗性克隆 ,用抗性克隆上清液感染人骨髓基质细胞 ,RT PCR和基因组DNA PCR检测外源基因mRNA的转录及染色体的整合 ,ELISA法和小鼠CFU GM集落形成法检测FL蛋白表达量和生物学活性。结果 酶切鉴定表明成功构建重组逆转录病毒载体pLFIN ;染色体基因组中整合有外源基因FL和Neo;在mRNA和蛋白质水平均可检测到FL表达 ;2 4hFL蛋白表达量为 4.35ng ml,活性测试结果显示转染的骨髓基质细胞分泌FL。结论 逆转录病毒介导的FL在骨髓基质细胞中获得表达 ,并具有良好的生物学活性 ,为进一步研究转基因骨髓基质细胞对造血调控的影响提供了实验依据
Objective To construct human FLT3 ligand (FL) retroviral vector and observe its expression in human bone marrow stromal cells. Methods FLcDNA was inserted into retroviral vector pLXIN using gene recombination technique to obtain recombinant vector pLFIN. The recombinant plasmid pLFIN was transfected into packaging cells PA317 by liposome method. The resistant clones were screened by G418 and transfected into human bone marrow stromal cells with the resistant clone supernatants. RT and PCR were used to detect the transcriptional and chromosomal The protein expression level and biological activity of FL protein were detected by ELISA, ELISA and CFU GM colony formation assay. Results Restriction endonuclease digestion showed that the recombinant retroviral vector pLFIN was successfully constructed. FL and Neo were integrated into the chromosomal genome. The expression of FL was detected at both mRNA and protein levels. The level of 4FL protein was 4.35 ng ml, The results show that transfected bone marrow stromal cells secrete FL. CONCLUSION: Retrovirus-mediated FL is expressed in bone marrow stromal cells and has good biological activity, providing an experimental basis for further study of the effect of transgenic bone marrow stromal cells on hematopoietic regulation