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目的: 构建表达人类晶状体来源的上皮生长因子 p52(LEDGFp52)重组腺病毒载体,检测 LEDGFp52 重组腺病毒能否在真核细胞中有效表达目的基因。方法: 将 LEDGFp52 基因片段亚克隆到腺病毒穿梭质粒pAdTrack- CMV上构建腺病毒穿梭载体 pAdTrack- CMV-LEDGFp52,将腺病毒穿梭载体 pAdTrack- CMV- LEDGFp52 与5 型腺病毒骨架质粒 pAdeasy- 1 共转染 BJ5183 细菌, 经细菌内同源重组产生携带 LEDGFp52 基因的重组腺病毒载体 pAd- LEDGFp52, 将 pAd- LEDGFp52 经脂质体法转化293 细胞包装产生重组腺病毒 Ad- LEDGFp52, 将 Ad-LEDGFp52 体外转染 293 细胞, CPE 法及 westernblot 观察目的基因的表达。结果: 构建了表达 LEDGFp52 基因的重组腺病毒质粒, E.coli 内成功同源重组腺病毒, 在 293 细胞内包装产生重组腺病毒 Ad- LEDGFp52, 滴度可达 5×1012 pfu/L。在真核细胞中有效表达目的基因 LEDGFp52, 出现显著的 CPE 效应。结论: 成功构建表达 LEDGFp52 的重组腺病毒载体, 为进一步进行 LEDGF p52 基因功能的研究提供了实验基础。
OBJECTIVE: To construct a recombinant adenovirus vector expressing human lens epithelial growth factor p52 (LEDGFp52) and to detect whether recombinant adenovirus carrying LEDGFp52 can efficiently express the target gene in eukaryotic cells. METHODS: The adenovirus shuttle vector pAdTrack-CMV-LEDGFp52 was subcloned into the adenovirus shuttle plasmid pAdTrack-CMV and the adenovirus shuttle vector pAdTrack-CMV-LEDGFp52 was co-transfected with adenovirus backbone plasmid pAdeasy-1 The recombinant adenovirus vector pAd-LEDGFp52 carrying LEDGFp52 gene was produced by homologous recombination in bacteria. The recombinant adenovirus Ad-LEDGFp52 was transformed into 293 cells by lipofectamine. Ad-LEDGFp52 was transfected into Ad-LEDGFp52 in vitro 293 cells, CPE method and western blot to observe the expression of the target gene. Results: The recombinant adenovirus plasmid expressing LEDGFp52 gene was successfully constructed. Recombinant adenovirus was successfully constructed in E. coli and packaged in 293 cells to produce recombinant adenovirus Ad-LEDGFp52 with a titer of 5 × 1012 pfu / L. Eukaryotic cells effectively expressed the target gene LEDGFp52, a significant CPE effect. Conclusion: The recombinant adenovirus vector expressing LEDGFp52 was successfully constructed and provided the experimental basis for the further study on the function of LEDGF p52 gene.