Bioinformatics analysis and preliminary validation of polymerase ζ for DNA damage repair in Echinococcus granulosus sensu lato(EgPolζ)
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.
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