摘要
旨在探究细粒棘球绦虫BAG3(Eg-BAG3)和EB1(Eg-EB1)的分子特征及在原头蚴细胞凋亡过程中的作用,采用原核表达方法获得重组Eg-BAG3和Eg-EB1,利用生物信息学、蛋白免疫印迹及免疫荧光定位方法对Eg-BAG3和Eg-EB1的分子特征进行了初步探究,随后利用10 mmol·L^(-1)H_(2)O_(2)诱导原头蚴细胞凋亡,qRT-PCR方法检测Eg-BAG3和Eg-EB1的转录水平,并通过RNA干扰技术进一步探究二者与原头蚴细胞凋亡之间的关系。结果显示,Eg-BAG3含有BAG蛋白家族特征性的BAG结构域,Eg-EB1含有内吞蛋白家族特征性的BAR结构域,二者分别属于典型的BAG蛋白和内吞蛋白。免疫荧光定位结果显示Eg-BAG3广泛分布于细粒棘球绦虫的各个发育时期,Eg-EB1主要定位于原头蚴皮层、吸盘及顶突和包囊生发层,而在成虫未见特异性分布。H_(2)O_(2)可以成功诱导原头蚴细胞凋亡,且原头蚴中Eg-BAG3和Eg-EB1的相对转录水平都随着H_(2)O_(2)处理时间的延长而显著上升,在8 h后的相对转录水平与0 h相比具有显著性差异(P<0.05)。RNA干扰结果显示,Eg-BAG3干扰组原头蚴细胞凋亡率显著高于未干扰组(P<0.05),而Eg-EB1干扰组原头蚴细胞凋亡率低于未干扰组(P<0.05),表明Eg-BAG3在H_(2)O_(2)诱导的原头蚴细胞凋亡过程中起到抗凋亡作用,而Eg-EB1起到促凋亡作用。Eg-BAG3可以抑制氧化应激诱导的原头蚴细胞凋亡而Eg-EB1促进细胞凋亡,且二者可能参与调控不育囊的形成。
To explore the molecular characteristics of Echinococcus granulosus BAG3(Eg-BAG3)and EB1(Eg-EB1)and their roles in the apoptosis of protoscoleces,recombinant Eg-BAG3 and Eg-EB1were expressed in E.coli BL21(DE3),and bioinformatic analysis,Western blotting and immunofluorescence localization were conducted to explore the molecular characteristics of Eg-BAG3 and Eg-EB1.The apoptosis of protoscoleces was induced by 10 mmol·L^(-1)H_(2)O_(2) and the transcription levels of Eg-BAG3 and Eg-EB1 were detected by qRT-PCR,and their roles in the apoptosis of protoscoleces was further verified by RNA interference(RNAi).Eg-BAG3 contained the characteristic BAG domain of BAG protein family and Eg-EB1 contained the characteristic BAR domain of endophilin protein family,suggesting that Eg-BAG3 and Eg-EB1 were the typical BAG protein and endophilin protein,respectively.Immunolocalization showed that Eg-BAG3 was widely distributed in all development stages of E.granulosus,Eg-EB1 was mainly located in the tegument,suckers and rostellum of protoscoleces and germinal layer,while no positive signal was observed in adults.Protoscoleces apoptosis was successfully induced by 10 mmol·L^(-1)H_(2)O_(2) and the relative transcription levels of Eg-BAG3 and Eg-EB1 significantly increased in response to H_(2)O_(2) treatment.RNA interference(RNAi)analysis showed that the apoptosis rate of protoscoleces in the Eg-BAG3 interference group was significantly increased compared with non-interference group(P<0.05),while the Eg-EB1 interference group showed a lower apoptosis rate of protoscoleces than non-interference group(P<0.05),suggesting that Eg-BAG3 inhibited protoscoleces apoptosis induced by H_(2)O_(2),while Eg-EB1 promoted protoscoleces apoptosis.In conclusion,Eg-BAG3 could inhibit protoscoleces apoptosis induced by oxidative stress,while Eg-EB1 promotes apoptosis,and both Eg-BAG3 and Eg-EB1 are possibly involved in regulating the formation of infertile cysts.
作者
何雪
王凝
华瑞其
杜小迪
杨光友
HE Xue;WANG Ning;HUA Ruiqi;DU Xiaodi;YANG Guangyou(College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China)
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2022年第5期1562-1575,共14页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家自然科学基金(31672547)
四川省科技厅重点研发项目(2020YFN0030)。
