乙型肝炎HBsAg和GM-CSF融合表达质粒的构建及表达

Construction and expression of plasmid co-expressing HBsAg and GM-CSF

目的 构建乙型肝炎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方法及分子克隆技术构建融合表达质粒pcDNA3.1 S GM CSF和pcDNA3.1 GM CSF S ,并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 乙型肝炎HBsAg和GM CSF的融合表达质粒构建成功 。

Objective To construct plasmids co expressing GM CS F and HBsAg and study the expression of plasmids in vitro . Me thods ①The HBsAg encoding fragment and GM CSF encoding fragm ent were created by PCR amplification and cloned into plasmid pcDNA3.1 (+);② HBsA g D NA fragment and GM CSF DNA fragment were amplified by PCR and subcloned into pl asmid pcDNA3.1 GM CSF or pcDNA3.1 S respectively;③HepG2 cells and COS 7 cells were transfected with plasmids. HBsAg in culture supernatants and cell lysates were a ssayed by ELISA analysis. GM CSF expressed by cells transfected with plasmids w ere detected using immunocytochemistry. Results Recombinant wer e confirmed using restriction enzymes and DNA sequencing, HBsAg was detected in the lysates from cells transfected with pcDNA3.1 GM CSF S, GM CSF were d etec ted in cells transfected with plasmids pcDNA3.1 GM CSF and pcDNA3.1 GM CSF S. Conclusions All eukaryotic expression plasmids have been con s tructed successfully. pcDNA3.1 GM CSF S could express HBsAg and GM CSF in vitro .