Objective:To construct the recombinant luciferase reporter plasmids containing series of truncated sequences of Kaposi’s sarcoma-associated herpesvirus (KSHV) ORF50 promoter and seek the specific response sites sequences of ORF50 promoter acted by HSV-1. Methods:Series of truncated sequences of KSHV ORF50 promoter were amplified using PCR from KSHV genome. The PCR products were further cloned into pGL3 basic vector to construct the recombinant luciferase reporter plasmids. The recombinant plasmids were confirmed by restriction enzymes digestion and sequence analysis. After infected with HSV-1, Vero cells were transfected with the ORF50 promoter-driven luciferase constructs to induce luciferase gene expression. Relative luciferase activity unit (RLU) was calculated and compared. Results:Series of truncated sequences of KSHV ORF50 promoter were isolated and cloned successfully. The promoter-driven luciferase expression of pGL3-95, pGL3-46 and pGL3-17 were declined significantly in Vero cells infected with HSV-1 when compared with pGL3-1500, pGL3-750, pGL3-375 and pGL3-185. Conclusion:The recombinant luciferase reporter plasmids containing series of truncated sequences of KSHV ORF50 promoter were constructed successfully. The specific response sites sequences of ORF50 promoter acted by HSV-1 are between -185 bp and -95 bp.