关键词:  , Hiwi基因；腺病毒；重叠延伸聚合酶链反应；Gateway克隆技术

Abstract:

ObjectiveTo obtain the  Hiwi gene encoding full-length and construct human Hiwi adenoviral vectors carryinggreen luorescence protein(GFP),and to establish  foundation for a further study on Hiwi function and mechanism of inducing leukemia stem cell differentiation and apoptosis.Methods All coding areas of human Hiwi gene full length were amplified using method of overlapping extension PCR technology，and  the full length coding aeras were inserted into the vector of Flag-IRES
-hrGFP carrying   GFP with Gateway technology to construct pDown- Hiwi-3×flag-IRES-hrGFP.The cloning vector pDown-Hiwi-3×flag-IRES-hrGFP and expression vector pAV.Des1d were used for homologous recombination reaction to obtain recombinant adenovirus vector pAV.Ex1d-Hiwi-3× flag-IRES-hrGFP.The positive clones were selected by PCR to extract the recombinant adenovirus plasmid and to pack into recombinant Ad-Hiwi-3×flag-IRES-hrGFP adenovirus.Results The human recombinant Hiwi was successfully cloned and the recombinant adenovirus vector pAV.Ex1d-Hiwi-3×flag-IRES-hrGFP was found to be successfully constructed via restriction enzyme digestion and sequencing methods.The adenovirus vector pAV.Ex1d-Hiwi-3×flag-IRES-hrGFP was transfected into the HEK293A cells using lipofectamine mediated gene transfection method.Under fluorescence microscope,the transfected cells with green fluorescence could be observed.Conclusion The expression plasmid of adenovirus vector pAV.Ex1d-Hiwi-3×flag-IRES-hrGFP is successfully constructed and  it can express in HEK293A cells.

Key words: Hiwi, gene adenovirus, overlapping extension,polymerase chain reaction, Gateway technology