Indentification Of Conserved Hypothetical Protein(CHP) And Its Interaction With TERT In Giardia Duodenalis

Giardia duodenalis is an eukaryotic parasite identified worldwide.WHO has listed it as one of the most important pathogens that can threaten the health of human beings and animals.The parasite has wide hosts and geographic ranges and has become a threat to public health.The irregular outbreaks of giardiase brought both people and animals lots of health problems and economic losses.Although Giardia has simple two-stage life cycle,its molecular characteristics,celllular mechanisms,and pathogenesis is still poorly understood.In addition,it is very important to conduct some researches on Giardia proteomics and try to find some novel drug targets.Telomere is a small molecule locating at the ends of eukaryotic chromosomes.The process of replication can be terminated by the insertion of telomerase,which leads to programmed cell death.Among factors that affect telomere mechanism,the most critical and important one is telomerase.Therefore,to study the telomerase related proteins is crucial to understanding the mechanism of telomere.In recent years,some relative researches has been conducted on the telomeres of Leishmania and Trypanosoma.Giardia,one of the most ancient eukaryotes,the study of its telomere-associated mechanism is also very important.Such studies about the tetramer chain of telomere inhibitor,the telomerase-associated disulfide isomerase,the subunits of replication factor have been performed for Giardia.To better undertand the telomerase-associated proteins,in this study,the yeast GAL4-based two-hybrid system was used to screen the potential preteins that interact with the RNA binding domain of the TERT protein of Giardia.GST pull-down and Co-IP assay were used to validate the Protein-Protein Interaction between them.The total RNA of Giardia was extracted by Trizol method and c DNA was obtained by RT-PCR.Then,the bait plasmid p GBKT7-Gl_TRBD was constructed and transfected into Y187 yeast,no toxicity and self activation were found.The proteins that interacts with Giardia TERT were screened from the establised c DNA library of Giardia by the yeast GAL4-based YTH system.Finally,two potential proteins were selected and identified as CHP?accession number XM₀01707200.1,full length 846 bp?and VSP?accession number XM₀01705026.1,full length2280 bp?.Due to the hypermutation of VSP,we just selected CHP for pull-down GST and Co-IP analysis.According to the hydrophobicity analysis of proteins,293 T cells were used to express the Gl_TRBD gene,and the CHP was expressed by p GEX4T-1 vector.The target protein CHP was successfully captured by the TRBD-binded glutathione-affinity resin,and the interaction between CHP and TERT was verified by Western Blot analysis.CHP and TERT were then transfected into293 cell lines and examined using specific tag antibodies.Their interection was verified by thefollowing Western Blot analysis.The findings will provide research basis for the study of the CHP function and the regulatory mechanism of telomerase-related proteins.