乙肝病毒前S1基因与截短C基因穿梭表达载体构建

目的构建HBV前S1基因和截短C基因分泌型大肠杆菌和分枝杆菌穿梭质粒。方法以含HBV基因组质粒pCP10序列为模板,PCR扩增前S1基因和C基因截短片段,依次克隆至分泌型穿梭表达载体pDE22,电穿孔转化法将重组质粒导入耻垢分枝杆菌,经潮霉素抗性筛选及PCR鉴定,阳性克隆热休克诱导表达,表达产物经SDS-PAGE分析。结果扩增到了预期长度的2个目的基因,构建了分泌型穿梭载体,SDS-PAGE分析可见相对分子质量约23000蛋白表达条带。结论已成功构建耻垢分枝杆菌分泌表达前S1基因和截短C基因的穿梭载体,为研究含前S基因和截短C基因的重组BCG疫苗奠定了基础。

Construction of Shuttle Expression Vector of Pre-S1 Gene and Truncated Core Gene of Hepatitis B Virus

Objective To construct the shuttle plasmid for the secretory expression of pre-S1 gene and truncated core gene of hepatitis B virus(HBV).Methods Amplify the pre-S1 and truncated core gene fragments by PCR using the sequence of HBV genomic plasmid pCP10.Clone the amplified gene fragments into shuttle expression vector pDE22.Transform the recombinant plasmid to M.smegmatics by electroporation.Screen positive clones with hygromycin and identify by PCR.Induce the expression of target protein by heat shock.Identify the expressed product by SDS-PAGE.Results The two target gene fragments at the expected lengths were amplified,and the shuttle vector for secretory expression was constructed.SDS-PAGE profile showed a protein band with a relative molecular weight of 23 000.Conclusion The shuttle vector for secretory expression of pre-S1 and truncated core gene of HBV in M.smegmatics was successfully constructed.It laid a foundation of development of recombinant BCG vaccine containing pre-S and truncated core gene.