细粒棘球绦虫BAG3和EB1基因的克隆、表达及其在原头蚴细胞凋亡中的作用

doi:10.11843/j.issn.0366-6964.2022.05.024

摘要/Abstract

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^-1H₂O₂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。H₂O₂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H₂O₂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H₂O₂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。

关键词: 细粒棘球绦虫, BAG3, EB1, 凋亡, RNA干扰

Abstract: 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^-1H₂O₂ 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^-1H₂O₂ and the relative transcription levels of Eg-BAG3 and Eg-EB1 significantly increased in response to H₂O₂ 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 H₂O₂, 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.

Key words: Echinococcus granulosus, BAG3, EB1, apoptosis, RNA interference

中图分类号:

S852.734

何雪, 王凝, 华瑞其, 杜小迪, 杨光友. 细粒棘球绦虫BAG3和EB1基因的克隆、表达及其在原头蚴细胞凋亡中的作用[J]. 畜牧兽医学报, 2022, 53(5): 1562-1575.

HE Xue, WANG Ning, HUA Ruiqi, DU Xiaodi, YANG Guangyou. Cloning, Expression of Echinococcus granulosus BAG3 and EB1 and Their Roles in the Apoptosis of Protoscoleces[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(5): 1562-1575.

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