HBsAg和GM-CSF融合表达质粒的构建

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pcDNA3．1( )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。

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

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.