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目的:以牛生长激素(bGH)为模型,建立逆转录病毒载体转移系统,体外转染鼠成纤维细胞株NIH3T3和人胚肺成纤维细胞2BS,以获得bGH表达。方法:将PCR扩增的bGH基因片段插入逆转录病毒载体pSXLG-MDR相应酶切位点,获得的重组逆转录病毒质粒。应用脂质体方法转染重组质粒进入包装细胞PA317,筛选长春新碱抗性克隆株,收集上清液感染NIH3T3细胞和人胚肺成纤维细胞2BS,筛选携带bGH基因的成纤维细胞的克隆株3T3/bGH和2BS/bGH。结果:RT-PCR分析表明基因转染组的3T3/bGH细胞和2BS/bGH细胞在相当于680bp处有一清晰的条带,而空质粒对照组无相应条带出现。RIA分析表明转染基因细胞的培养液及胞浆蛋白有bGH蛋白表达,而作为对照的3T3/mdr细胞和NIH/3T3细胞均无此蛋白表达。结论:应用逆转录病毒IRBS载体系统转染bGH基因进入成纤维细胞,该系统易筛选,转染效率较高。
OBJECTIVE: To establish a retroviral vector transfer system using bGH as a model, and to transfect rat fibroblast cell line NIH3T3 and human fetal lung fibroblast 2BS in vitro to obtain bGH expression. Methods: The bGH gene fragment amplified by PCR was inserted into the corresponding restriction site of retroviral vector pSXLG-MDR to obtain the recombinant retroviral plasmid. The recombinant plasmids were transfected into packaging cells PA317 by liposome method. Vincristine-resistant clones were screened. The supernatant was collected to infect NIH3T3 cells and 2BS of human embryonic lung fibroblasts. The cloned fibroblasts carrying bGH gene 3T3 / bGH and 2BS / bGH. Results: RT-PCR analysis showed that the 3T3 / bGH cells and the 2BS / bGH cells in the gene transfected group had a clear band at 680bp, whereas no blank band appeared in the empty plasmid control group. RIA analysis showed that bGH protein was expressed in the culture medium and cytoplasm of transfected cells, but not in 3T3 / mdr cells and NIH / 3T3 cells as control. CONCLUSION: The bGH gene is transfected into fibroblasts by retroviral vector IRBS. The system is easy to select and has high transfection efficiency.