Bioinformatics Analysis and Validation of PCNA Protein in Echinococcus granulosus sesensu lato
[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.
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