增强型绿色荧光蛋白逆转录病毒载体的构造和表达

逆转录病毒载体被广泛用作对造血细胞进行基因转移的工具，转导方法的改进有赖于应用可快速分析并被高效选择的基因标志，为此，我们克隆了增强型绿色荧光蛋白（ＥＧＦＰ）基因，并构建可表达ＥＧＦＰ的逆转录病毒载体ＬＧＳＮ，通过脂质体转梁和交互感染方法建立高滴度的逆转录产病毒细胞，用以分析对造血细胞标记ＥＧＦＰ基因的可行性，流式细胞术和荧光显微镜检测发现，ＥＧＦＰ病毒转录的ＧＰ＋ｅｖｎＡｍ１２细胞和Ｋ５６２细胞均可发出稳定的绿色荧光信号，阳性率可分别达９７％和８６％，聚合酶链反应分析显示ＬＧＳＮ转子细胞内有原病毒的整合，上述结果提示，作为新一代选择性标志，ＥＧＦＰ是适于研究对造血细胞基因转移与报告基因，对促进人类基因治疗的研究有重要意义。

Construction and Expression of Retroviral Vector Encoding Enhanced Green Fluorescent Protein

Retroviral vectors are wildly used as vehicles for gene transfer into hematopoietic cells based on its potency for efficient gene delivery and integration of transgene in host genome. The development of better transduction protocols depends on gene markers that allow a rapid detection and effective selection of genetically transduced cells. In this study, the enhanced green fluorescent protein(EGFP), a gene that is optimized for detection and expression in mammalian cells, was firstly amplified and cloned by high-fidelity PCR. The vector LGSN carrying EGFP gene was then constructed and the retroviral producer cell lines that yield high titers of LGSN vector in supernatants were developed by liposome-mediated transfection in combination with cross infection. Both GP envAm12 murine fibroblasts and K562 leukemic cells transduced with EGFP virus demonstrated a stable green fluorescence signal readily detectable by flow cytometry or fluorescence microscopy in up to 97% and 86% of examined cells, respectively. The integration of LGSN provirus in transduced cells was confirmed by PCR analysis. These results indicate EGFP is a suitable reporter molecule for gene transfer and expression in hematopoietic cells. Therefore, the bright and long-term expression of EGFP in living cells will advance the study of gene therapy in vitro and in vivo, particularly for human applications.