携带治疗基因的增殖型腺病毒CNHK300-m干扰素-γ的构建

Construction and primary study of CNHK300-mIFN-γ, a conditionally replicating adenovirus driven by the human telomerase reverse transcriptase promoter and carrying an mouse interferon-γ gene

目的 研制出一种携带抗癌基因的选择增殖型腺病毒载体系统。方法 克隆鼠干扰素（IFN）-γ基因序列，利用分子克隆技术将其插入肿瘤特异性增殖病毒的病毒基因组中，得到腺病毒载体质粒pSC300-mIFN-γ。通过pSG300-mIFN-γ与质粒pBHGE3在293细胞中同源重组得到重组病毒CNHK300-mIFN-γ。扩增、纯化病毒，用TCID50方法测病毒滴度。通过病毒增殖实验观察重组病毒的选择性增殖能力，通过Western blot检测腺病毒蛋白的表达，通过双抗体夹心法酶联免疫吸附试验（ELISA）检测重组病毒在不同细胞及不同时相的mIFN-γ表达量。结果 成功构建了携带治疗基因的增殖型腺病毒CNHK300-mIFN-γ，病毒滴度为1．0×10^9pfu／ml，增殖实验证实CNHK300-mIFN-γ可以选择性地在端粒酶阳性肿瘤中增殖，Westernblot分析结果显示腺病毒的E1A蛋白选择性在端粒酶阳性的肿瘤细胞中表达，ELISA显示CNHK300-m IFN-γ感染端粒酶阳性肿瘤后有大量mIFN-γ的表达，并随着感染的时间表达量也相应上升。结论 CNHK300-mIFN-γ为肿瘤的生物治疗提供了一种新的策略。

Objective To develop a novel gene-viral therapeutic system which can replicate conditionally and express an antitumor gene. Methods An mouse interferon-γ gene was obtained from pCAl3-mIFN-γ and inserted into the genome of the conditionally replicating adenovirus CNHK300 by molecular cloning, to produce plasmid pSG300-mIFN-γ. The plasmid pSG300-mIFN-γ was co-transfected with pBHGE3 in 293 ceils to generate recombinant adenovirus CNHK300-mIFN-γ. The recombinant adenoviruses were verified by PCR, purified by cesium chloride density purification and propagated in 293 cells. Virus titer was measured by TCID50 method. Virus replication assay and Western blot were performed to evaluate the selective replication ability of CNHK300-mIFN-γ. The quantity of mIFN-γ protein in cells infected by CNHK300-mIFN-γ on day 3 and day 7 postinfection was detected with the enzyme-linked immunoabsordent assay （ELISA）. Results A new conditionally replicating adenovirus CNHK300-mIFN-γ was constructed and its titer was 1.0 × 10^9 pfu/ml. Proliferative test and Western blot revealed that CNHK300-mIFN-γ could selectively proliferate in telomerase-postive cancer cells. ELISA showed that mIFN-γ in CNHK300-mIFN-,y was expressed efficiendy in vitro and increased largely with the time prolongation in tumor cells. Conclusion CNHK300-mIFN-γ may provide a new strategy for tumor biotherapy.