HBsAg和GM-CSF融合表达质粒的构建  

Construction of plasmid coexpressing hepatitis B surface antigen and granulocyte macrophage-colony stimulating factor

作　　者：卿玉玲[1] 赵嘉将[1] 任红[1] 

机构地区：[1]重庆医科大学病毒性肝炎研究所,重庆400010

出　　处：《重庆医科大学学报》2003年第3期261-263,266,共4页Journal of Chongqing Medical University

基　　金：重庆市科委应用基金资助项目 (渝科发计字 [2 0 0 1] 74号 )

摘　　要：目的 :构建乙肝HBsAg和GM -CSF的融合表达质粒 ,借助GM -CSF的免疫佐剂作用 ,提高乙型肝炎DNA疫苗的免疫效果。方法 :用PCR方法分别从 pEcob6和 pCD -hGM -CSF中扩增出基因S和GM -CSF ,克隆到真核表达质粒pcD NA3.1(+)中 ,构建pcDNA3.1-S及融合表达质粒 pcDNA3.1-GM -CSF -S ,然后以重组质粒转染真核细胞 ,研究重组质粒体外表达。结果 :经酶切鉴定及DNA序列证实重组质粒构建正确 ,细胞转染试验表明重组质粒能在COS - 7及HepG - 2内表达。结论 :乙肝HBsAg和GM -CSF的融合表达质粒构建成功 ,重组质粒能在真核细胞内表达。Objective:To construct plasmid coexpressing GM-CSF and HBsAg,enhance hepatitis B DNA vaccines antivirus effect.Methods:(1)The HBsAg-encoding fragment and GM-CSF-encoding fragment were created by PCR amplification from pEcob6 and pCD-hGM-CSF respectively,then were cloned into plasmid pcDNA3.1(+),pcDNA3.1-S and pcDNA3.1-GM-CSF(without stop codon)were constructed.(2)HBsAg DNA fragment (with stop codon) was amplified by PCR,then was subcloned into plasmid pcDNA3.1-GM-CSF.Results:Recombinant pcDNA3.1-S and pcDNA3.1-GM-CSF-S were confirmed by using restriction enzymes and DNA sequencing.HBsAg was detected in the lysates and supernatants from cells transfected with pcDNA3.1-S and in the lysates from cells transfected with pcDNA3.1-GM-CSF-S.Conclusion:Two eukaryotic expression plasmids were constructed successfully and could express HBsAg.

关 键 词：乙型肝炎 粒细胞、巨噬细胞集落刺激因子 质粒构建 

分 类 号：R392-33[医药卫生—免疫学]