Objective:In order to lay the foundation for quantitative detection of adenovirus neutralizing antibodies,a luciferase gene recombinant adenovirus vector is aimed to be constructed. Methods:The adenoviral expression system,ViraPowerTM Adenoviral Expression System,was used to construct the recombinant adenovirus vector. Using PCR method,luciferase gene was amplified from the pGL3 plasmid and CACC was added at the 5 end. After ligation and transformation,the luciferase gene was cloned into the entry vector,pENTR /D-TOPO,and identified by sequencing and PCR. LR ClonaseTM Ⅱ Enzyme Mix was used to recombine entry-cloning with recombinant expression vector(pAd-CMV/V5-DEST)to generate expression vector plasmid,rAd-Luci. After identification by PCR,the rAd-Luci plasmid was linearized by the restriction enzymes,PacⅠ,and transfected HEK293A packaging cells. After amplification of the recombinant adenovirus,limiting dilution method was used to detect the virus titer. Western blot was used to detect the expression of luciferase protein. Results:The luciferase gene was obtained by PCR method. The adenoviral entry and expression clones were identified correctly by PCR and sequencing. Those correct recombinant adenoviral expression plasmids transfected HEK293A cells to obtain the recombinant adenovirus with virus titer of 1.8 × 1011 pfu/ml. This recombinant virus was able to express functional luciferase protein correctly. Conclusion:The construction of luciferase gene recombinant adenovirus vector was successful and this study has laid an experimental basis for quantitative detection of adenovirus neutralizing antibodies.