摘要
目的研究堆型艾美尔球虫延伸因子1-α(EaEF-1α)在堆型艾美尔球虫的感染期间调控细胞凋亡的主要机制。方法从球虫裂殖子侵染DF-1细胞实验的上清液中分析筛选得到EaEF-1α,与质粒pEGFP-N1重组得到重组质粒pEGFP-N1-EaEF-1α,转染入DF-1细胞中,Western bolt及荧光检测蛋白表达,通过流式细胞术检测脂多糖组、pEGFP-N1组、pEGFP-N1-EF-1α组和空白组DF-1细胞凋亡情况,酶联免疫吸附实验检测脂多糖组、pEGFP-N1组、pEGFP-N1-EF-1α组和空白组DF-1细胞中凋亡因子Bax、Bcl-2、Caspase3、Caspase8表达水平。结果流式细胞术检测发现pEGFP-N1-EF-1α组细胞凋亡率显著低于pEGFP-N1组和脂多糖组。DF-1细胞裂解液中Bax表达水平降低,Bcl-2及Caspase3表达水平上调,pEGFP-N1-EF-1α组Bcl-2/Bax的值显著高于pEGFP-N1组和脂多糖组。Caspase8表达水平无差异。结论EaEF-1α体外过表达通过线粒体通路显著抑制细胞凋亡。
Objective To investigate the main mechanism by which Eimeria acervulina elongation factor 1-α(EaEF-1α)regulates apoptosis during Eimeria acervulina infection.Method EaEF-1αwas isolated from the supernatant of Eimeria acervulina schizozoites infecting DF-1 cells.It was then recombined with the plasmid pEGFP-N1 to obtain the recombinant plasmid pEGFP-N1-EaEF-1α,which was transfected into DF-1 cells.Western blot and fluorescence detection were used to verify the expression.Apoptosis of DF-1 cells in the lipopolysaccharide group,pEGFP-N1 group,pEGFP-N1-EF-1αgroup,and blank group was detected by flow cytometry.The expression levels of apoptosis factors Bax,Bcl-2,Caspase3,and Caspase8 in DF-1 cells in the lipopolysaccharide group,pEGFP-N1 group,pEGFP-N1-EF-1αgroup,and blank group were detected by enzyme-linked immunosorbent assay(ELISA).Result Flow cytometry showed that the apoptosis rate of the pEGFP-N1-EF-1αgroup was significantly lower than that of the pEGFP-N1 group and the lipopolysaccharide group.The expression level of Bax in the lysates of DF-1 cells decreased,while the expression levels of Bcl-2 and Caspase3 increased.The value of Bcl-2/Bax in the pEGFP-N1-EF-1αgroup was significantly higher than that in the pEGFP-N1 group and the lipopolysaccharide group.There was no difference in the expression level of Caspase8.Conclusion Over-expression of EaEF-1αin vitro significantly inhibits apoptosis through the mitochondrial pathway.
作者
李治翰
刘永宁
单乙戈
安健
LI Zhihan;LIU Yongning;SHAN Yige;AN Jian(College of Animal Science and Technology,Beijing University of Agriculture,Beijing 102206)
出处
《实验动物科学》
2025年第1期38-44,共7页
Laboratory Animal Science
基金
北京市特色高水平骨干专业群项目-动物医学专业群项目-技术平台与社会服务建设项目(PXM2020-157102-000060-15)。
