丙型肝炎病毒核心区与IgG Fc段融合基因疫苗的构建及表达

Construction and expression of chrimeid plasmid pcDNA3HCV-Fc

目的 :构建能同时表达丙型肝炎病毒核心区与IgGFc段的融合基因真核表达载体 pcDNA3HCVC Fc ,为下一步修饰和转染树突状细胞 ,制备能高效表达HCVC和Fc基因的树突状细胞疫苗做准备。 方法 :用分别含有XholⅠ和XbaⅠ双酶切位点的人免疫球蛋白 (IgG)Fc区基因上、下游引物 ,以含有人IgG基因序列的质粒pCMVsFc为模板 ,通过PCR扩增获得Fc区基因片段 ,基因片段回收后 ,以XholⅠ和XbaⅠ双酶切 ,定向插入到含HCVC基因的质粒PcDNA3HCVC下游双粘端位点之间 ,获得重组表达质粒pcDNA3HCVFc。通过酶切、PCR及插入片段序列测定对质粒进行鉴定。以抗HCVC和抗Fc单克隆抗体为一抗 ,利用间接免疫荧光法检测 pcDNA3HCV Fc在人肝癌细胞 772 1中的瞬时表达。 结果 :酶切、PCR及测定鉴定证实 ,pcDNA3HCV Fc插入片段为Fc区基因片段 ,免疫荧光法检测表明其可以在 772 1细胞中瞬时表达。 结论 :构建的质粒 pcDNA3HCV Fc可以在772 1细胞中瞬时表达Fc基因 ,为研究HCVC Fc融合基因修饰的树突状细胞的功能奠定了基础。

Objectives:To construct a recombinant cherimal plasmid of HCV Fc which can express HCV core gene and IgG Fc gene.This should be useful to transfect dendritic cells and changed into dendritic cell vaccine. Methods:The IgG Fc gene derived from the plasmid pCMVsFc by using polymerase chain reaction(PCR) was inserted into the backward position of cytomegalovirus(CMV) immediate early poromotor element of HCV C plasmid(pcDNA3 HCV C) then the recombinant plasmid pcDNA3.HCV Fc was obtained. Results:The insert DNA of pcDNA3HCV Fc was conformed HCV core and Fc gene by endonuclease,PCR and sequencing.HCV core gene and Fc gene was expressed transiently with lipofectamine 2000 coated in human hepatoblastoma 7721 cells which conformed by immunofluorescence. Conclusions:Recombinant cherimal plasmid vector pcDNA3. HCV Fc can express HCV core and Fc gene.Transiently in 7721 cells.This should be useful to transfect dendritic cells and changed into dendritic cell vaccine.