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 polymeraseζfor DNA damage repair in Echinococcus granulosus sensu lato(EgPolζ)

目的对细粒棘球绦虫DNA损伤修复聚合酶ζ(DNA damage repair polymeraseζof 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。

Objective Bioinformatics analysis of DNA damage repair polymeraseζof Echinococcus granulo-sus sensu lato(EgPolζ)was performed.The effects of Harmine(HM)and its derivatives H-2-168 and H-2-104 on EgPolζafter intervention were analyzed.Methods Total RNA of Echinococcus granulosus was extracted and cDNA was synthesized by reverse transcription.EgPolζwas cloned by RT-PCR,and the full length of EgPolζwas 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 EgPolζprotein was performed.Western Blot analysis of EgPolζcontent of HM and its derivatives H-2-168 and H-2-104 after intervention with Echinococcus granulosus.Results According to bioinformatics prediction analysis,EgPolζwas 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 polymeraseδCatalytic subunits,3'-5'exonuclease domain superfamily and DNA polymer-ase B-type family catalytic domains;Antigen epitope related secondary structures in EgPolζβ-Folding ac-counted for approximately 4.59%;After analyzing the antigenic epitopes of EgPolζB cells and evaluating them,8 predicted epitopes were obtained;The similarity of the Pol sequence between EgPolζand 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 EgPolζprotein 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 EgPolζwas cloned successfully,and the analysis predicted that EgPolζhad a regulatory effect on DNA repair of Echinococcus granulosus,and HM and H-2-104 inhibited the expression of EgPolζprotein,thus affecting the DNA damage repair func-tion of Echinococcus granulosus.