细粒棘球绦虫PCNA蛋白生物信息学分析及验证

doi:10.16431/j.cnki.1671-7236.2024.11.030

摘要/Abstract

摘要： 【目的】分析细粒棘球绦虫(Echinococcus granulosus sensu lato，Eg)增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)蛋白的生物信息学特性，以及去氢骆驼蓬碱(HM)及其衍生物(H-2-168与H-2-104)对EgPCNA蛋白含量的影响，以期治疗囊型棘球蚴病并为药物靶点的筛选奠定基础。【方法】运用PCR扩增并克隆EgPCNA基因全长序列，采用生物信息学软件预测和分析EgPCNA蛋白相关生物学信息。利用Mega 7.0构建蛋白序列系统发育树，并运用Western blotting分析HM及其衍生物(H-2-168与H-2-104)对EgPCNA蛋白含量的影响。【结果】 EgPCNA基因全长783 bp，编码260个氨基酸，EgPCNA蛋白分子质量为28.36435 ku，等电点为4.62，脂肪指数为96.81，亲水性值为－0.015，为亲水性蛋白，无跨膜结构域。亚细胞定位预测结果显示，蛋白分布于细胞质。该蛋白含有PCNA超家族结构与PCNA保守功能结构域，二级结构主要为无规则卷曲，其次是α-螺旋，有2条可靠的B细胞抗原表位，与人和家鼠等哺乳动物的亲缘关系较远。Western blotting分析结果显示，与DMSO组相比，HM组EgPCNA蛋白表达量极显著上调(P＜0.01)，H-2-168与H-2-104组EgPCNA蛋白表达量均显著下调(P＜0.05)。【结论】本研究成功克隆了EgPCNA全长基因。EgPCNA蛋白对细粒棘球绦虫的DNA复制和修复具有调控作用，H-2-168与H-2-104具有下调EgPCNA蛋白含量的功能。

关键词: 细粒棘球绦虫; 增殖细胞核抗原; 克隆; 生物信息学

Abstract: 【Objective】 The objective of this study was to analyze the bioinformatic properties of proliferating cell nuclear antigen (PCNA) protein in Echinococcus granulosus sensu lato (Eg) and effect of harmine (HM) and its derivatives (H-2-168 and H-2-104) on the content of protein EgPCNA,in order to treat cystic echinococcosis and establish a basis for the hunt for potential therapeutic targets. 【Method】 The full length sequence of EgPCNA gene was amplified by PCR and cloned.Biological information related to EgPCNA was predicted and analyzed by bioinformatics softwares.Mega 7.0 was used to construct phylogenetic tree of EgPCNA protein,and the effects of HM and its derivatives (H-2-168 and H-2-104) on EgPCNA protein content were analyzed by Western blotting. 【Result】 The total length of EgPCNA gene was 783 bp,encoding 260 amino acids,the molecular weight of protein EgPCNA was 28.36435 ku,the isoelectric point was 4.62,and the fat index was 96.81.With a hydrophilic value of －0.015,it was a hydrophilic protein with no transmembrane domain.The subcellular localization prediction results showed that the protein was distributed in the cytoplasm.It contained PCNA superfamily structure and PCNA conserved functional domain.The secondary structure of this protein was mainly random coil,followed by alpha helix,and had two reliable B-cell epitopes,which was far from the relationship with mammals such as Homo sapiens and Mus musculus.Western blotting analysis showed that compared with DMSO group, the expression of EgPCNA protein in HM group was extremely significantly up-regulated (P＜0.01),and the expression of EgPCNA protein in H-2-168 and H-2-104 groups was significantly down-regulated (P＜0.05). 【Conclusion】 In this study,the full length of EgPCNA gene was successfully cloned.Bioinformatics analysis predicted that protein EgPCNA had a regulatory role in DNA replication and repair of Echinococcus granulosus,and H-2-168 and H-2-104 could down-regulate protein EgPCNA content.

Key words: Echinococcus granulosus sesensu lato; proliferating cell nuclear antigen; cloning; bioinformatics

中图分类号:

S852.73

许少全, 麦尔哈巴·麦麦提艾力, 吕国栋, 周润, 赵金龙, 李婧, 夏衣旦木·吐尼牙孜, 赵军. 细粒棘球绦虫PCNA蛋白生物信息学分析及验证[J]. 中国畜牧兽医, 2024, 51(11): 4956-4963.

XU Shaoquan, MAIERHABA·Maimaitiaili, LYU Guodong, ZHOU Run, ZHAO Jinlong, LI Jing, XIAYIDANMU·Tuniyazi, ZHAO Jun. Bioinformatics Analysis and Validation of PCNA Protein in Echinococcus granulosus sesensu lato[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(11): 4956-4963.

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