Construction and conifrmation of a recombinant adenovirus of human survivin

FAN Zaiwen; ZHANG Shuai; LIU Yuwei; WU Juanjuan

Abstract：ObjectiveTo construct a recombinant adenovirus vector containing human survivin gene siRNA, and to provide reference for gene therapy of hypoxic pulmonary hypertension.MethodsSurvivin-siRNA template DNA sequences were designed before they were cloned into the expression vector to construct the survivin-siRNA expression vector. After verification, the survivin-siRNA expression vector and the plasmid pBHGE3 containing the right arm of adenovirus were co-transfected into human 293A cells. Ad-survivin was abundantly amplified and virus titer was evaluated. Smooth muscle cells of the pulmonary artery were cultured under hypoxic conditions for 24 h. Five groups were designed: control group (without adenovirus infection), negative control group (infected with Ad-LacZ), short hairpin groups (infected with Ad-survivin sh1 or sh2 or sh3). The Ad-survivin was used to infect the pulmonary artery smooth muscle cells exposed to hypoxia. The expression level of the survivin protein in smooth muscle cells of the pulmonary artery was determined by Western blot.Results ①The siRNA fragment ligated with plasmid pAd-U6-CMV was sequenced. The results suggested that the target gene fragment was inserted properly;②Ad-survivin-shRNA1, Ad-survivin-shRNA2 and Ad-survivin-shRNA3 titers were 1×1010, 1.2×1010 and 1×1010 respectively by TCID50 assay;③The level of survivin protein expression of smooth muscle cells of the human pulmonary artery in sh2 group under hypoxic conditions was significantly lower than in other groups (P<0.05).ConclusionThe construction of three kinds of adenoviral recombinants is successful and identification of packing is completed. A recombinant adenovirus vector containing human survivin is constructed successfully, which will contribute to studies on the role of survivin gene in smooth muscle cells of the hypoxic pulmonary artery.