细粒棘球绦虫DNA损伤修复聚合酶ζ生物信息学分析及初步验证
Bioinformatics analysis and preliminary validation of polymerase ζ for DNA damage repair in Echinococcus granulosus sensu lato(EgPolζ)
许少全 1麦尔哈巴·麦麦提艾力 1周润 1吕国栋 1赵金龙 1吴洁茹 1赵军2
作者信息
- 1. 新疆医科大学药学院,乌鲁木齐 830017
- 2. 新疆医科大学第一附属医院药学部临床药学科,乌鲁木齐 830054
- 折叠
摘要
目的 对细粒棘球绦虫DNA损伤修复聚合酶ζ(DNA damage repair polymerase ζof Echinococcus granulosus sensu lato,EgPolζ)进行生物信息学分析,并分析去氢骆驼蓬碱(Harmine,HM)及其衍生物H-2-168、H-2-104干预后对EgPolζ的影响.方法 提取细粒棘球绦虫总RNA并逆转录合成cDNA,利用RT-PCR技术克隆EgPolζ,通过测序获得EgPolζ全长序列.采用生物信息学在线网站、在线数据库与分析软件等工具预测和分析EgPolζ相关生物学信息,构建系统进化树进行同源性比较,HM及其衍生物H-2-168和H-2-104与EgPolζ蛋白进行分子对接.Western Blot检测HM及其衍生物H-2-168和H-2-104干预细粒棘球绦虫后EgPolζ含量变化.结果 经生物信息学预测分析EgPolζ为亲水性蛋白,缺乏信号肽位点,但具有一个跨膜区域;该蛋白具有多个酸磷酸化位点;其结构域包含DNA聚合酶δ催化亚单位、3'-5'核酸外切酶结构域超家族与DNA聚合酶B型家族催化域;EgPolζ二级结构中与抗原表位相关的β-折叠约占4.59％;分析EgPolζB细胞抗原表位且对其进行评估后得到8条预测表位;EgPolζ与多房棘球绦虫(Echinococcus multilocularis,Em)的Polζ序列相似性为93.56％,二者同属于绦虫纲,进化关系较近,与智人和家鼠等哺乳动物的进化关系较远.HM、H-2-168和H-2-104与EgPolζ蛋白结合能分别为-5.91、-5.71与-5.05 kcal/mol;Western Blot结果分析:与DMSO组相比,HM组与H-2-104组EgREV3蛋白含量均降低(P＜0.05,P＜0.01);H-2-168组中EgREV3蛋白含量无显著变化(P＞0.05).结论 成功克隆了 EgPolζ全长序列,分析预测EgPolζ对细粒棘球绦虫的DNA修复具有调控作用,且HM与H-2-104抑制EgPolζ蛋白表达,从而影响细粒棘球绦虫DNA损伤修复功能.
Abstract
Objective Bioinformatics analysis of DNA damage repair polymerase ζ of Echinococcus granulo-sus sensu lato(EgPolζ)was performed.The effects of Harmine(HM)and its derivatives H-2-168 and H-2-104 on EgPol ζ after intervention were analyzed.Methods Total RNA of Echinococcus granulosus was extracted and cDNA was synthesized by reverse transcription.EgPolζ was cloned by RT-PCR,and the full length of EgPolζ was obtained by sequencing.Bioinformatics online websites,online databases and analysis software were used to predict and analyze the biological information related to EgPolζ.Phyloge-netic tree was constructed for homology comparison.Molecular docking of HM and its derivatives H-2-168 and H-2-104 with EgPolζ protein was performed.Western Blot analysis of EgPolζ content of HM and its derivatives H-2-168 and H-2-104 after intervention with Echinococcus granulosus.Results According to bioinformatics prediction analysis,EgPolζ was a hydrophilic protein and lacking signal peptide sites,but it has a transmembrane region;The protein had multiple acid phosphorylation sites;Its structural domain contained DNA polymerase 8 Catalytic subunits,3'-5'exonuclease domain superfamily and DNA polymer-ase B-type family catalytic domains;Antigen epitope related secondary structures in EgPolζ β-Folding ac-counted for approximately 4.59％;After analyzing the antigenic epitopes of EgPolζ B cells and evaluating them,8 predicted epitopes were obtained;The similarity of the Pol sequence between EgPolζ and Echino-coccus multilocularis was 93.56％.Both belong to the tapeworm class and were closely related to evolu-tionary relationship with mammals such as Homo sapiens and mice.The binding energies of HM,H-2-168 and H-2-104 to EgPolζ protein were-5.91,-5.71 and-5.05 kcal/mol,respectively;Western Blot a-nalysis:Compared with the DMSO group,the content of EgREV3 protein in HM group and H-2-104 groups were decreased(P＜0.05,P＜0.01);The EgREV3 protein content in H-2-168 group was not sig-nificant changed(P＞0.05).Conclusion The full-length sequence of EgPolζ was cloned successfully,and the analysis predicted that EgPolζ had a regulatory effect on DNA repair of Echinococcus granulosus,and HM and H-2-104 inhibited the expression of EgPolζ protein,thus affecting the DNA damage repair func-tion of Echinococcus granulosus.
关键词
细粒棘球绦虫/聚合酶ζ/基因克隆/生物信息学分析Key words
Echinococcus granulosus sesensu lato/polymerase ζ/gene cloning/bioinformatics analysis引用本文复制引用
出版年
2024
NETL
NSTL
万方数据