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  

Construction of Toxoplasma ROP18 recombinant adenovirus vector and its effect on the apoptosis of C17.2 stem cells

作　　者：张贤[1,2] 甘小凤[1,2] 程正阳[1,2] 都建[3] 罗庆礼[1,2] 沈继龙[1,2] 余莉[1,2] 

机构地区：[1]安徽医科大学病原生物学教研室,合肥230032 [2]人兽共患病安徽省重点实验室,合肥230032 [3]安徽医科大学生物化学教研室,合肥230032

出　　处：《中国人兽共患病学报》2016年第3期234-239,共6页Chinese Journal of Zoonoses

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~~

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(P<0.05);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(P<0.001);Western blot检测显示caspase-3活性片段的表达明显增加(P<0.05)。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。To study the effect of Toxoplasma rhoptry kinase 18（ROP18）on the apoptosis of C17.2neural stem cells,Toxoplasma gondii ROP18 was subcloned from PEGFP-G2-ROP18 into pHBAd-MCMV-GFP.The recombinant adenovirus vector pHBAd-MCMV-GFP-ROP18 was comfirmed by PCR and sequencing,and the identified recombinant adenovirus vector was transfected into 293 cells for packaging with skeleton plasmid.After amplification,ROP18 recombinant adenovirus（MOI=70）was transfected into C17.2neural stem cell.The proliferation of C17.2cells at 48 horus Post-treatment was detected using CCK-8kit and the apoptosis of C17.2cells at 24 hours Post-treatment was detected by flow cytometer.Western blot was used to determine the expression levels of caspase-3.ROP18 recombinant adenovirus was expressed effectively in C17.2neural stem.Compared with the control group,proliferation of C17.2cells was significantly decreased（P〈0.05）in the ROP18 recombinant adenovirus group at 48 hours while the apoptosis of C17.2cells was significantly increased in the ROP18 recombinant adenovirus（P〈0.001）at 24 hours Post treatment and the caspases-3expression levels was significantly increased（P〈0.05）.ROP18 recombinant adenovirus was successfully constructed and was highly expressed in C17.2neural stem cells.ROP18 recombinant adenovirus induced the apoptosis of C17.2neural stem cells.

关 键 词：刚地弓形虫 棒状体蛋白18 神经干细胞C17.2 重组腺病毒 细胞凋亡 

分 类 号：R382.5[医药卫生—医学寄生虫学]