Abstract: Abstract: Objective To establish a recombinant adenovirus vector with hRb94 by λ phage-site specific recombination systems．Methods Total RNA was extracted from human embryo and reversed transcript to get object cDNA, the hRb94 gene fragment was amplified by PCR.The attBflanked PCR primers were designed and used to amplify hRb94 gene by PCR．An entry clone was performed by a BP recombination reaction with attB-PCR products and donor vector pDONR^TM221. Then the entry clone and the target vector Ad／CMV／V5-DEST with attR1 and attR2 sites was recombined together in vivo to create the expression clone (Ad-hRb94)by an efficient LR recombination reaction．After the expression clone was confirmed by PCR and sequencing.Ad-hRb94 was digested with Pac I and transferred into 293A cells to be packaged into adenovirus stock．Ad-hRb94 was amplified by infection of 293A cells and the titer was measured. ResultsThe target gene of hRb94 was transferred into Ad／CMV／V5-DEST vector correctly with the right ORF (open reading frame) by LR recombination reaction and it was confirmed by PCR and sequencing．The expression clone Ad-hRb94 was packaged into maturated adenovirus successfully．The titer of Ad-hRB94 was 9. 41×1010pfu／ml． Conclusion Ad-hRB94 was constructed with Gateway^TM clone technology，which lays an experimental foundation for the further research on the genetic therapy of hRb94．