论文部分内容阅读
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 pSG300-m IFN-γ。通过 pSG300-m IFN-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1.0×10~9 pfu/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,Western blot 分析结果显示腺病毒的ElA 蛋白选择性在端粒酶阳性的肿瘤细胞中表达,ELISA 显示 CNHK300-mIFN-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 selective adenovirus vector system carrying anti-oncogene. Methods The murine interferon (IFN) -γ gene sequence was cloned and inserted into the viral genome of tumor-specific proliferating virus by molecular cloning to obtain the adenoviral vector plasmid pSG300-m IFN-γ. Recombinant virus CNHK300-mIFN-γ was obtained by homologous recombination of pSG300-m IFN-γ and plasmid pBHGE3 in 293 cells. Amplify, purify the virus and measure the virus titers using the TCID50 method. The proliferation of recombinant virus was observed by virus proliferation assay. The expression of adenovirus protein was detected by Western blot. The mIFN-γ of recombinant virus at different cells and phases was detected by double antibody sandwich enzyme-linked immunosorbent assay (ELISA) The amount of expression. Results The proliferating adenovirus CNHK300-mIFN-γ carrying the therapeutic gene was successfully constructed and its virus titer was 1.0 × 10 ~ 9 pfu / ml. Proliferation experiments confirmed that CNHK300-mIFN-γ could selectively express in telomerase positive tumors The results of proliferation and Western blot showed that the ElA protein of adenovirus was expressed selectively in telomerase positive tumor cells. ELISA showed that mHNN-γ was expressed after CNHK300-mIFN-γinfection of telomerase positive tumor Infection time expression also increased accordingly. Conclusion CNHK300-mIFN-γ provides a new strategy for the biological treatment of tumors.