摘要
目的为解决杆状病毒作为基因传递载体瞬时表达缺陷,拟构建睡美人(SB)转座子介导的杆状病毒表达载体,通过检测绿色荧光蛋白EGFP的表达变化来评价该系统在哺乳动物体内外的表达效率。方法以pFastBac DUAL质粒为骨架,将pPh启动子替换为CMV-SB100X-SV40 PA表达元件,然后在其下游插入IR/DR-CMV-EGFP-SV40 PA-IR/DR表达元件,获得的质粒命名为pBacSB-CE;同时作为对照构建pBac-CE,在pPh启动子下游插入CMV-EGFP表达元件。将构建好的重组质粒转化大肠杆菌DH10Bac,经蓝白斑筛选后转染Sf-9细胞,获得重组杆状病毒BacSB-CE和Bac-CE。将重组病毒转导人胶质瘤U87细胞,通过MTT、倒置荧光显微镜和流式细胞仪检测细胞的增殖以及EGFP的表达效率;建立裸鼠皮下胶质瘤肿瘤模型,通过组织免疫荧光进一步观察重组病毒在体内的转导效率。结果经PCR鉴定成功获得了重组病毒BacSB-CE和Bac-CE;MTT结果证实重组病毒对U87细胞的增殖无副作用;倒置荧光显微镜和流式结果显示,EGFP在BacSB-CE转导的U87细胞中至少能表达60 d,在转导15 d后仍能检测到约10^(9) a.u.总荧光强度,而在Bac-CE转导的细胞中,15 d后已检测不到荧光细胞和荧光信号;体内组织免疫荧光检测进一步显示BacSB-CE组在第10天时仍能检测到绿色荧光细胞,而Bac-CE组在第5天时基本看不到荧光细胞。结论该研究成功构建了SB转座子介导的杆状病毒基因传递系统,获得的重组病毒对哺乳动物细胞具有良好的安全性,并能介导EGFP在哺乳动物体内外持续稳定表达。
Objective In order to solve the baculovirus as a gene delivery vector transient expression defect,this study intends to construct a sleeping beauty(SB)transposon-mediated baculovirus expression vector,using green fluorescent protein gene EGFP as the target gene,to evaluate the system expression efficiency in mammals.Methods Using the pFastBac DUAL plasmid as the skeleton,the pPh promoter was replaced with the CMVSB100X-SV40 PA expression element along with the IR/DR-CMV-EGFP-SV40 PA-IR/DR sequence,and the obtained plasmid was named pBacSB-CE.Meanwhile,as a control,pBac-CE was constructed by inserting the CMVEGFP expression element downstream of pPh promoter.The constructed recombinant plasmids were transformed into DH10Bac competent cells,and the recombinant bacmids were obtained by blue-white screening.Then the recombinant bacmids were transfected into Sf-9 insect cells by liposome,resulting in recombinant baculovirus BacSB-CE and Bac-CE.U87 cells were transduced with recombinant virus,the cell proliferation and the expression efficiency of EGFP were detected by MTT,inverted fluorescence microscopy and flow cytometry.The subcutaneous glioma tumor model of nude mice was established and the transduction efficiency of recombinant virus in vivo was further observed by immunofluorescence.Results The results showed that the recombinant viruses BacSB-CE and Bac-CE were successfully obtained by PCR identification.MTT results showed that the recombinant virus had no side effect on the proliferation of U87 cells.The results of inverted fluorescence microscopy and flow cytometry showed that EGFP could be expressed for at least 60 d in BacSB-CE transduced U87 cells and 10^(9 )a.u.total fluorescence intensity could still be detected after 15 d of transduction,while in Bac-CE transduced cells,no fluorescence cells and fluorescence signal could be detected after 15 d.Conclusion In this study,the SB transposon-mediated baculovirus gene delivery system was successfully constructed.The recombinant virus has a good biosafety and c
作者
王志昇
李梦婷
姬勇敢
杨文
杨丽
杨萌萌
Wang Zhisheng;Li Mengting;Ji Yonggan(School of Pharmacy,Ningxia Medical University,Yinchuan 710004;Laboratory Animal Center,Ningxia Medical University,Yinchuan 710004)
出处
《安徽医科大学学报》
CAS
北大核心
2021年第3期386-391,共6页
Acta Universitatis Medicinalis Anhui
基金
国家自然科学基金(编号:31460242)
宁夏自然科学基金(编号:NZ17067)。
关键词
睡美人转座子
杆状病毒
U87细胞
持续表达
sleeping beauty transposon
baculovirus
U87 cells
sustained expression
