FoxO3腺病毒载体的构建及对脑缺血损伤中自噬相关蛋白的影响  

Construction of FoxO3 adenovirus vector and the effect on the autophagy related protein in cerebral ischemia injury

作　　者：汪鑫[1] 马黎敏 王守艳[1] 嵇再雄 邵永佳 陈霞[1] 

机构地区：[1]南通大学医学院基础医学研究室,南通226001

出　　处：《南通大学学报（医学版）》2016年第3期165-170,共6页Journal of Nantong University(Medical sciences)

基　　金：南通市自然科学应用计划项目(BK2014039);南通大学研究生创新科研实践项目(YKC14051)

摘　　要：目的:构建转录因子FoxO3的过表达腺病毒载体AV-FoxO3,包装产生高滴度腺病毒;观察其感染氧糖剥夺(oxygen glucose deprivation,OGD)损伤小鼠海马细胞系HT22细胞自噬相关蛋白表达的影响。方法:在Gen Bank中查询FoxO3基因及其上下游的序列,设计合成引物并在引物中引入HA序列,逆转录聚合酶链式反应(reverse transcriptionpolymerase chain reaction,RT-PCR)体外扩增目的基因,用限制性内切酶NheⅠ-HF和Af1Ⅱ双酶切表达载体p AdenoEF1-BGH-MCMV-EGFP-3FLAG,使用无缝克隆试剂盒将目的基因构建进表达载体中,转化大肠杆菌DH5α感受态细胞,挑取转化子进行菌落PCR鉴定获得阳性克隆(FoxO3重组质粒)并测序;利用Ad Max系统在HEK293A细胞中分别包装FoxO3重组质粒与对照质粒,得到AV-FoxO3和CMV-GFP-A.D.并测定病毒滴度;用AV-FoxO3和CMV-GFP-A.D.感染HT22细胞,荧光显微镜观察感染是否成功,Western Blot检测HT22细胞中HA蛋白(重组FoxO3序列中所含的标签蛋白)的表达;Western Blot检测OGD损伤HT22细胞中自噬相关蛋白LC3和Beclin-1的表达。结果:菌落PCR和测序结果均表明FoxO3重组质粒构建成功;与辅助质粒共包装感染细胞获得腺病毒颗粒,测定滴度分别为6.32×1010 Ifu/m L和4×1011Ifu/m L;荧光观察及Western Blot检测均说明AV-FoxO3成功地感染了HT22细胞;FoxO3过表达促进自噬相关蛋白LC3和Beclin-1的表达。结论:AV-FoxO3构建成功并可用于HT22细胞的感染,增加细胞内FoxO3表达;FoxO3参与调控自噬相关蛋白LC3和Beclin-1的表达。Objective： To construct the ov erexpressed transcription factor FoxO3 adenovirus vector, package high titer adenovirus and observe its effect on the expression of autophagyrelated protein in mouse hippocampal cell lines（HT22） after oxygen glucose deprivation（OGD）. Methods： Query the sequence of FoxO3 gene and its upstream and downstream sequence in Gen Bank, then design and synthesize primers and introduction of HA in the primer sequences, reverse transcription-polymerase chain reaction（RT-PCR） amplification the target gene in vitro, use endonuclease Nhe Ⅰ-HF and Af1Ⅱ double digestion the expression vector p Adeno-EF1-BGH-MCMV-EGFP-3FLAG and then connected seamless with the target gene fragment, transform E.coli DH5α a competent cells,positive clones were sequenced. After package in HEK293 A cell with Ad Max system, we got recombinant adenovirus vector Av-FoxO3 and CMV-GFP-A.D., then the titer of adenovirus vector were determined. Av-FoxO3 and CMV-GFP-A.D. infected HT22 cells, immunofluorescence was used to detect the infection whether transfection was successful. Western Blot was used to test the expression of HA（a flag protein of recombinant FoxO3sequence）and FoxO3. Western Blot was used to test the expression of autophagy related protein LC3 and Beclin-1 after OGD. Results： Results of sequencing and PCR proved that FoxO3 recombinant plasmid was constructed successfully;adenovirus vector were acquired in plasmid co-transfected secondary packaging cells, the titer of adenovirus were 6.32×1010Ifu/m L and 4 ×10^11Ifu/m L respectively; both f luorescence microscopy and Western Blot results suggested that AV-FoxO3 infected HT22 cells successfully. Overexpressed FoxO3 promote the expression of autophagy related protein LC3 and Beclin-1.Conclusion： AV-FoxO3 was constructed successfully, which could infect cells successfully and increased the expression of FoxO3. FoxO3 involved in regulation of autophagy related protein LC3 and Beclin-1.

关 键 词：转录因子FoxO3 腺病毒 HT22细胞 氧糖剥夺 自噬 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学]