弓形虫ROP18重组腺病毒载体的构建及其对神经干细胞C17.2凋亡的影响

发布时间：2018-06-23 22:12

本文选题：刚地弓形虫 + 棒状体蛋白　；参考：《中国人兽共患病学报》2016年03期

【摘要】：目的构建弓形虫棒状体蛋白ROP18重组腺病毒载体,研究其对神经干细胞C17.2凋亡的影响。方法将弓形虫ROP18从PEGFP-G2-ROP18重组质粒上亚克隆至腺病毒载体pHBAd-MCMV-GFP,重组腺病毒载体pHBAd-MCMVGFP-ROP18经PCR、测序鉴定正确后,与骨架质粒pHBAd-BHG共转染HEK293细胞进行包装,收获、扩增重组腺病毒并对其滴度进行测定。以MOI=70的重组腺病毒感染神经干细胞C17.2,转染不同时间荧光显微镜观察基因的表达情况。转染48h用CCK-8法检测C17.2的增殖情况;转染24h采用Annexin V-APC/PI双染流式细胞术检测细胞凋亡情况;Western blot法检测半胱天冬氨酸蛋白酶3(caspase-3)的蛋白水平。结果重组ROP18过表达腺病毒在C17.2神经干细胞内能有效表达。转染48h,与空载体对照组相比,C17.2细胞增殖活性明显降低(P0.05);转染24h,流式细胞术检测结果显示,ROP18基因转染组C17.2细胞凋亡率明显增加(P0.001);Western blot检测显示caspase-3活性片段的表达明显增加(P0.05)。结论成功构建了弓形虫ROP18重组腺病毒并在神经干细胞C17.2中表达,ROP18重组腺病毒能诱导C17.2神经干细胞凋亡。
[Abstract]:Aim to construct the recombinant adenovirus vector of Toxoplasma gondii ropiform protein ROP18 and to study the effect of ROP18 on apoptosis of neural stem cells (NSCs). Methods Toxoplasma gondii ROP18 was subcloned from PEGFP-G2-ROP18 recombinant plasmid into adenovirus vector pHBAd-MCMV-GFP.The recombinant adenovirus vector pHBAd-MCMVGFP-ROP18 was confirmed by PCR.The recombinant adenovirus vector pHBAd-MCMVGFP-ROP18 was cotransfected with the skeleton plasmid pHBAd-BHG into HEK293 cells for harvesting. The recombinant adenovirus was amplified and its titer was determined. C17.2 neural stem cells were infected with the recombinant adenovirus of MOIX70, and the expression of the gene was observed by fluorescence microscope at different time. The proliferation of C17.2 was detected by CCK-8 method at 48h, and the cell apoptosis was detected by Annexin V-APC / Pi double staining flow cytometry at 24h. The protein level of cysteine aspartate protease 3 (caspase-3) was detected by Western blot assay. Results Recombinant ROP 18 overexpression adenovirus could be expressed effectively in C 17.2 neural stem cells. After 48 h transfection, the proliferative activity of C17.2 cells was significantly lower than that of empty vector control group (P0.05), and the apoptotic rate of C17.2 cells was significantly increased (P0.001) Western blot assay showed that the expression of caspase-3 activity fragment was significantly increased (P0.05) in 24 h transfection group. Conclusion the recombinant adenovirus of Toxoplasma gondii ROP18 was successfully constructed and expressed in neural stem cell C17.2. The recombinant adenovirus can induce apoptosis of C17.2 neural stem cells.
【作者单位】：安徽医科大学病原生物学教研室;人兽共患病安徽省重点实验室;安徽医科大学生物化学教研室;
【基金】：国家自然科学基金(No.81572022)资助安徽医科大学青年拔尖人才支持计划项目(No.0113014104)资助~~
【分类号】：R382.5

【相似文献】