重组人survivin腺病毒的构建与鉴定

目的构建人survivin基因shRNA重组腺病毒载体，为缺氧性肺动脉高压（Hypoxic pulmonary hypertension， HPH）的基因治疗研究提供实验基础。方法构建survivin-siRNA表达质粒，测序鉴定正确后，将survivin-siRNA表达质粒与含有腺病毒右臂的骨架质粒pBHGE3共转染至人293A细胞，包装成腺病毒Ad-survivin并扩增，TCID50法测定病毒滴度。将人肺动脉平滑肌细胞缺氧培养24 h，并将细胞分为空白对照组（Con组），阴性对照组（Ad-LacZ组），不同Ad-survivin序列的干扰组（sh1组、sh2组、sh3组）。Ad-survivin感染缺氧人肺动脉平滑肌细胞，Western blot检测survivin蛋白，验证重组腺病毒干扰效果。结果①将shRNA片段与pAd-U6-CMV连接后的质粒进行测序，结果提示目的基因片段插入成功；②TCID50法测定病毒Ad-survivin-shRNA1、Ad-survivin-shRNA2和Ad-survivin-shRNA3的滴度分别是1×1010，1.2×1010，1×1010；③sh2组缺氧人肺动脉平滑肌细胞内survivin蛋白表达明显低于其他4组(P＜0.05)。结论本研究成功构建人survivin腺病毒载体，为研究survivin基因在缺氧性肺动脉高压中的作用提供了实验基础。

Construction and conifrmation of a recombinant adenovirus of human survivin

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.