pcEgr-IFNγ重组质粒的构建与体外X射线诱导表达特性

STUDY ON CONSTRUCTION OF pcEgr-IFNγ AND EXPRESSION PROPERTY OF X-RAY INDUCTION IN VITRO

目的 :扩增小鼠IFNγcDNA全长并构建pcEgr_IFNγ重组质粒 ,研究该重组质粒在不同剂量X射线作用下在B16细胞中的表达及时程。 方法 :利用PCR方法扩增小鼠IFNγcDNA全长 ,插入 pcDNA3.1质粒的多克隆位点 ,用Egr_1的启动子替代pcDNA3.1质粒的CMV启动子 ,构建成 pcEgr_IFNγ重组质粒 ;通过脂质体转染法将重组质粒转染入B16细胞 ,利用ELISA试剂盒检测在不同剂量X射线作用下 ,重组质粒的表达量及表达时程。 结果 :IFNγcDNA的扩增产物经测序 ,结果基本与已知序列相符 ,重组质粒经酶切鉴定 ,得出相应的特异带。不同剂量X射线照射后 ,pcEgr_IFNγ在B16细胞中表达量为77.73～94.60pg/ml ,明显高于对照组(P<0.05～P<0.01) ;2GyX射线照射后6h ,pcEgr_IFNγ表达量最高 ,为90.00pg/ml,明显高于对照组(P<0.001)。 结论 :用本实验克隆的IFNγcDNA所构建的 pcEgr_IFNγ重组质粒 ,经X射线的诱导 ,在被转染的B16细胞中表达量明显增高。对重组质粒X射线诱导表达的剂量时程的检测 ,为将来pcEgr_IFNγ基因治疗合并放疗的体内研究奠定了基础。

Purpose: Cloning IFNγ cDNA and constructing pcEgr_IFNγ to study its expression in transfected B16 cells induced by X_ray irradiation in different doses, and to study the time course of the expression. Methods: IFNγ cDNA was cloned by PCR and inserted into pcDNA3.1. CMV promoter of pcDNA3.1 was replaced by Egr_1 promoter to construct pcEgr_IFNγ plasmid. The plasmids were transfected into B16 cells. The expression of IFNγ of transfected B16 cells with X_ray irradiation in different doses and expression time course were detected by ELISA. Results: PCR product was sequenced, the result indicated the sequence was correct. The recombined plasmid was detected by electrophoresis. After X_ray irradiation in different doses, IFNγ expression of transfected B16 cells was 77.73 ～ 94.60 pg / ml, significantly higher than the control group(P<0.05 ～ P<0.01). Six hours after 2 Gy X_ray irradiation, the expression was the highest(90.00 pg / ml), significantly higher than the control group (P<0.001). Conclusion: X_ray can induce the recombine plasmid express in B16 cellls. The detection of doses and time course provides an experimental basis for in vivo study in future.[