【作者】 徐彤；

【导师】 郑明学； 吕晓玲；

【作者基本信息】 山西农业大学 ， 兽医学， 2023， 硕士

【摘要】 为了解柔嫩艾美耳球虫(Eimeria tenella,E.tenella)顶膜抗原1(Apical membrance protein 1,Et AMA1)在E.tenella调控宿主细胞凋亡中的作用,本研究采用酵母真核和原核表达Et AMA1蛋白,MTT法测定其活性,蛋白免疫印迹法(Western Blot)和间接免疫荧光确定该蛋白表达和定位;通过原代鸡胚盲肠上皮细胞培养、Annexin V-FITC/PI、H.E.染色法、分光光度法、q PCR、Western Blot和抑制阻断等方法,测定Et AMA1蛋白对宿主细胞感染率、凋亡率以及死亡受体和线粒体凋亡信号通路关键因子表达与激活的动态变化。结果显示:(1)构建了Et AMA1蛋白酵母表达系统和原核表达系统,表达获得58 k Da Et AMA1重组蛋白。Et AMA1可与抗E.tenella鸡阳性血清反应,分布于裂殖体和子孢子表面及其胞内。真核表达蛋白浓度在0-0.1μg/m L,细胞活性随着Et AMA1浓度增加而升高,蛋白浓度为0.1μg/m L时达峰值,之后下降。工作浓度为0.1μg/m L时,作用24 h细胞增殖活性极显著(P<0.01)高于4 h、48 h和空白对照组(Control组)。表明具有细胞增殖活性的Et AMA1蛋白表达系统构建成功,该蛋白天然存在于E.tenella中。(2)E.tenella接种4-96 h,E.tenella+Et AMA1组感染率极显著(P<0.01)高于E.tenella组,极显著(P<0.01)低于E.tenella+Et AMA1+Caspase-9抑制剂(ZLEHD-FMK)组和E.tenella+Et AMA1+Caspase-8抑制剂(Z-IETD-FMK)组。表明添加Et AMA1蛋白可提高E.tenella感染率,同时添加线粒体通路或死亡受体通路抑制剂感染率会更低。(3)E.tenalla接种4-96 h,Et AMA1组宿主细胞早期凋亡率和晚期凋亡/坏死率均极显著(P<0.01)低于Control组,极显著(P<0.01)高于Z-IETD-FMK+Et AMA1组、Z-LEHD-FMK+Et AMA1;E.tenalla+Et AMA1组宿主细胞早期凋亡率和晚期凋亡/坏死率均极显著(P<0.01)低于E.tenalla组,极显著(P<0.01)高于Z-IETD-FMK+Et AMA1+E.tenalla组和Z-LEHD-FMK+Et AMA1+E.tenalla组。表明添加Et AMA1蛋白可降低宿主细胞凋亡率,同时添加线粒体或死亡受体通路抑制剂,宿主细胞凋亡率会更低。(4)E.tenalla接种4-96 h,Et AMA1组Caspase-8、Caspase-9、Caspase-3、Fas和TNFR1 m RNA相对表达量极显著(P<0.01)或显著低于(P<0.05)Control组,极显著(P<0.01)或显著(P<0.05)高于Z-IETD-FMK+Et AMA1组或Z-LEHD-FMK+Et AMA1组,而Bcl-2/Bax比值极显著(P<0.01)高于Control组;E.tenalla+Et AMA1组Caspase-8、Caspase-9、Caspase-3、Fas和TNFR1 m RNA相对表达量极显著(P<0.01)或显著低于(P<0.05)E.tenella组,极显著(P<0.01)或显著(P<0.05)高于Z-IETD-FMK+Et AMA1+E.tenalla组或Z-LEHD-FMK+Et AMA1+E.tenalla组,而Bcl-2/Bax比值极显著(P<0.01)高于E.tenella组。表明添加Et AMA1蛋白可降低感染和非感染E.tenalla宿主细胞Caspase-8、Caspase-9、Caspase-3、Fas和TNFR1 m RNA表达量,增加Bcl-2/Bax比值,同时添加线粒体或死亡受体通路抑制剂此变化更显著。(5)E.tenalla接种4-96 h,Et AMA1组Caspase-8、Caspase-9和Caspase-3活性均极显著(P<0.01)或显著(P<0.05)低于Control组,显著(P<0.05)或极显著(P<0.01)高于Z-IETD-FMK+Et AMA1组或Z-LEHD-FMK+Et AMA1组,TNFR1和Fas蛋白表达量极显著(P<0.01)低于Control组,而Bcl-2/Bax比值极显著(P<0.01)高于Control组;E.tenalla+Et AMA1组Caspase-8、Caspase-9和Caspase-3活性均极显著(P<0.01)或显著(P<0.05)低于E.tenalla组,显著(P<0.05)或极显著(P<0.01)高于E.tenalla+Z-IETD-FMK+Et AMA1组或E.tenalla+Z-LEHD-FMK+Et AMA1组;TNFR1和Fas蛋白表达量极显著(P<0.01)低于E.tenalla组,而Bcl-2/Bax比值极显著(P<0.01)高于E.tenalla。表明添加Et AMA1蛋白可降低感染和非感染E.tenalla宿主细胞Caspase-8、Caspase-9和Caspase-3蛋白活性及Fas和TNFR1蛋白表达量,增加Bcl-2/Bax比值,同时添加线粒体或死亡受体通路抑制剂此变化更显著。综上所述,E.tenalla可通过Et AMA1抑制死亡受体凋亡和线粒体凋亡通路激活,下调TNFR1和Fas关键蛋白表达,上调Bcl-2/Bax比值,降低Caspase-9、Caspase-8和Caspase-3活性,进而抑制E.tenalla宿主细胞凋亡。更多还原

【Abstract】 The objective was to understand the role and molecular mechanism of E.tenella Apical membrance protein 1(Et AMA1)in the regulation of E.tenella host cell apoptosis.In this study,Et AMA1 protein was expressed in yeast eukaryotic and Escherichia coli,and its activity was measured by MTT assay.Western Blot and indirect immunofluorescence were used to determine the expression and localization of the protein.The dynamic changes of Et AMA1 protein on host cell infection rate,apoptosis rate,expression and activation of key factors of death receptor and mitochondrial apoptosis signaling pathway were determined by primary chick embryo cecal epithelial cell culture,Annexin V-FITC/PI,H.E.staining method,spectrophotometry,q PCR,Western Blot and inhibition blocking methods.The results showed that:(1)The yeast and prokaryotic expression system of Et AMA1 protein were constructed,and the 58 k Da recombinant protein was obtained.Et AMA1 could react with the positive serum against E.tenella chicken and was distributed on the surface of schizont and sporozoite as well as in the cytoplasm.The eukaryotic expression protein concentration of Et AMA1 was 0-0.1 μg/m L,and the cell viability increased with the increase of Et AMA1 concentration.The cell viability reached the peak at 0.1 μg/m L protein concentration,and then decreased.The cell proliferation activity was extremely significant(P < 0.01)at 4 h,48 h and Control group when the working concentration was 0.1 μg/m L.These results indicated that the expression system of Et AMA1 protein with cell proliferation activity was successfully constructed,and the protein is naturally present in E.tenella.(2)The infection rate of E.tenella + Et AMA1 group was significantly higher than that of E.tenella group 4-96 h after inoculation(P < 0.01).Compared with the E.tenella + Et AMA1 + Caspase-9 inhibitor(Z-LEHD-FMK)group and the E.tenella + Et AMA1 + Caspase-8 inhibitor(Z-IETD-FMK)group,the expression of Et AMA1 in the E.tenella + Et AMA1 + caspase-8 inhibitor(Z-IETD-FMK)group was significantly decreased(P < 0.01).These results indicated that Et AMA1 protein could increase the infection rate of E.tenella,and the inhibitors of mitochondrial pathway or death receptor pathway could reduce the infection rate.(3)E.tenalla inoculation for 4-96 h,the early apoptosis rate and late apoptosis/necrosis rate of host cells in Et AMA1 group were significantly lower than Control group(P < 0.01).It was significantly higher than Z-IETD-FMK + Et AMA1 group and Z-LEHD-FMK + Et AMA1 group(P < 0.01);The early apoptosis rate and late apoptosis/necrosis rate of E.tenalla + Et AMA1 group were significantly lower than those of E.tenalla group(P < 0.01).It was significantly higher than that in Z-IETD-FMK + Et AMA1 + E.tenalla and Z-LEHD-FMK + Et AMA1 + E.tenalla groups(P < 0.01).These results indicated that Et AMA1 protein could reduce the apoptosis rate of host cells,and the mitochondrial or death receptor pathway inhibitors at the same time could reduce the apoptosis rate of host cells.(4)The relative m RNA expression of Caspase-8,Caspase-9,Caspase-3,Fas and TNFR1 in Et AMA1 group were extremely significant(P < 0.01)or significantly lower(P < 0.05)than those in Control group from 4-96 h after inoculation.The ratio of Bcl-2/Bax was significantly higher than that of Control group(P< 0.01),but significantly higher than that of Z-IETD-FMK + Et AMA1 group(P < 0.05).The relative m RNA expression of Caspase-8,Caspase-9,Caspase-3,Fas and TNFR1 in E.tenalla + Et AMA1 group were extremely significant(P < 0.01)or significantly lower than those in E.tenella group(P < 0.05).The ratio of Bcl-2/Bax was significantly higher than that of E.tenella group(P < 0.01).It was extremely significant(P < 0.01)or significantly(P < 0.05)higher than Z-IETD-FMK + Et AMA1 + E.tenalla group or Z-LEHD-FMK + Et AMA1 + E.tenalla group.The results showed that Et AMA1 protein reduced the m RNA expression of Caspase-8,Caspase-9,Caspase-3,Fas and TNFR1 in infected and non-infected E.tenalla host cells,and increased the Bcl-2/Bax ratio.The changes were more significant when mitochondria or death receptor pathway inhibitors were added at the same time.(5)E.tenalla inoculation for 4-96 h,the activities of Caspase-8,Caspase-9 and Caspase-3 in Et AMA1 group were extremely significant(P < 0.01)or significantly(P < 0.05)lower than those in Control group.It was significantly higher(P < 0.05)or extremely significantly(P < 0.01)than Z-IETD-FMK + Et AMA1 group or Z-LEHD-FMK + Et AMA1 group,and significantly lower(P < 0.01)than Control group in TNFR1 and Fas protein expression.The Bcl-2/Bax ratio was significantly higher than that of the Control group(P < 0.01).The activities of Caspase-8,Caspase-9 and Caspase-3 in E.tenalla + Et AMA1 group were significantly lower than those in E.tenalla group(P < 0.01)or significantly lower than those in E.tenalla group(P < 0.05).It was significantly(P < 0.05)or extremely significantly(P < 0.01)higher than that in E.tenalla + Z-IETD-FMK + Et AMA1 or E.tenalla + Z-LEHD-FMK + Et AMA1;The protein expression of TNFR1 and Fas in E.tenalla group was significantly lower than that in E.tenalla group(P <0.01),while the ratio of Bcl-2/Bax was significantly higher than that in E.tenalla group(P < 0.01).Et AMA1 decreased the activities of Caspase-8,Caspase-9 and Caspase-3 proteins and the expression of Fas and TNFR1 proteins,and increased the ratio of Bcl-2/Bax in E.tenalla cells.The changes were more significant when mitochondria or death receptor pathway inhibitors were added at the same time.In conclusion,E.tenalla could regulate cell apoptosis by inhibiting the activation of key factors in death receptors and mitochondrial apoptosis pathway through Et AMA1.更多还原