论文部分内容阅读
目的 构建一个含有绿色荧光蛋白（GFP）报告基因,并能够用于Ⅲ型酪氨酸激酶受体Flt3的配体（FL）基因修饰性肿瘤疫苗研究的质粒载体。方法 利用常规分子生物学方法设计并构建含有一个FL基因和一个GFP基因的pGFP-FL质粒。在该质粒中,以CMV启动子驱动FL基因,同时以肽链延长因子（EF-1α）启动子驱动GFP基因,并引入原核和真核选择基因KanR/neo。对所获得的pGFP-FL质粒以限制性内切酶酶切鉴定后,再将其导入Hepa1-6细胞内,直接在荧光显微镜下观察GFP基因的表达情况,同时以RT-PCR和PCR产物测序的方法对GFP阳性细胞内FL基因的表达进行检测。结果 质粒的酶切鉴定结果表明所构建的pGFP-FL与预期的结构一致,FL和GFP两者能同时在Hepa1-6细胞内表达。结论 本研究构建了一种新型的FL基因修饰肿瘤疫苗研究载体,位于该载体上的GFP的表达能够反映FL基因的转录表达情况,可以作为FL基因表达的报告基因。
OBJECTIVE: To construct a plasmid vector containing a green fluorescent protein (GFP) reporter gene that can be used in the study of ligand (FL) gene modified tumor vaccine of type III tyrosine kinase receptor Flt3. Methods The pGFP-FL plasmid containing one FL gene and one GFP gene was designed and constructed by conventional molecular biology methods. In this plasmid, the FL gene is driven by the CMV promoter while the GFP gene is driven by the peptide elongation factor (EF-la) promoter and the prokaryotic and eukaryotic selection gene KanR / neo is introduced. The pGFP-FL plasmids were identified by restriction endonuclease digestion and then introduced into Hepa1-6 cells. The expression of GFP gene was observed under fluorescent microscope directly, and RT-PCR and PCR products were sequenced Method to detect the expression of FL gene in GFP positive cells. Results Restriction analysis of plasmids showed that the constructed pGFP-FL was consistent with the expected structure. Both FL and GFP could be expressed simultaneously in Hepa 1-6 cells. Conclusion This study constructed a novel FL gene modified tumor vaccine research vector. The expression of GFP on this vector can reflect the transcriptional expression of FL gene and can be used as a reporter gene for FL gene expression.