癌变·畸变·突变 ›› 2003, Vol. 15 ›› Issue (1): 1-4.doi: 10.3969/j.issn.1004-616x.2003.01.001

• 论著 •    下一篇

pcEgr-IFNγ重组质粒的构建与体外X射线诱导表达特性

吴丛梅1,李修义2   

  1. 1.汕头大学医学院生物学教研室，广东　汕头　515031；　2.吉林大学公共卫生学院放射生物重点实验室，吉林　长春　130021
  • 收稿日期:2002-06-18 修回日期:2002-09-11 出版日期:2003-01-30 发布日期:2003-01-30
  • 通讯作者: 吴丛梅

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

WU Cong-mei 1,LI Xiu-yi 2   

  1. Department of Biology,Shantou University Medical college,Shantou 515031 ,China
  • Received:2002-06-18 Revised:2002-09-11 Online:2003-01-30 Published:2003-01-30
  • Contact: WU Cong-mei

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摘要： 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。

关键词: IFNγ表达, PCR, 质粒构建, X射线照射, 时程, 剂量效应

Abstract: Purpose:Cloning IFNγcDNA and constructing pcEgr-IFNγto study its expression intransfected B16 cells induced by X-ray irradiation indifferent doses,and to study the time course of the expression. Methods:IFNγcDNA was cloned by PCR and inserted into pcDNA3.1.CMV promoter of pcDNA3.1 was replaced by Egr-1 promoter to construct pcEgr-IFNγplasmid.The plasmids were transfected into B16 cells.The expression of IFNγof transfected B16 cells with X-ray irradiation indifferent doses and expression time course were detected by ELISA.Results:PCR product was sequenced,there sultindicated the sequence was correct.There combined plasmid was detected by electrophoresis.After X-ray irradiation indifferent doses,IFNγexpression of transfected B16 cells was 77.73 ～ 94.60pg / 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(90.00pg / ml),significantly higher than the control group(P<0.001).Conclusion:X-ray caninduce the recombine plasmid expressin B16 cells.The detection of doses and time course provides an experimental basis for invivo study infuture.

Key words: IFNγexpression, PCR, Construction of plasmid, X-ray irradiation, Time course, Dose effect

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