Study On The Localization And Function Of Telomerase Interaction Protein RFC1 In Giardia Duodenalis

Giardia duodenalis(syn.Giardia intestinalis,Giardia lamblia)is a unicellular eukaryotic anaerobe with dual nuclei and usually causes diarrhoeal disease worldwide.The Giardia of the Duodenalis is widely distributed,and Giardia disease is endemic or outbreak in the world.It is one of the ten parasitic diseases that mainly affect human health.The most common form of dissemination of Giardia duodenalis is the spread of water,which often occurs in travelers.It is therefore called “traveller's diarrhea”.The Giardia virus can infect humans and most mammals,so it is internationally recognized as an important human-animal parasitic disease.The body structure of Giardia duodenalis is simple.It has only a very simple intimal system and a special dual-cell nuclear structure.It is able to achieve "immortalized" in vitro pure culture and passaging.It belongs to early eukaryotes and has a history of biological evolution.It is an ideal "model organism" with an important position.The immortalization of cells is one of the hot spots in current cell biology research.The current study suggests that cell immortalization is closely related to telomeres.Telomere is a special nuclear protein complex that stabilizes the chromosome structure.With further research on telomeres and telomerase,it has been found that some telomerase-associated proteins play an important role in the regulation of telomerase activity.The duodenary size of Giardia guinea trophozoites are similar,and studies have shown that the DNA content is the same between them,and they are synchronous in the life activities of replication,transcription,and mitosis.However,this still does not mean that the two cell nuclei are exactly the same.For example,whether or not telomerase and its activity regulation mechanism are all still unclear.In the previous study of this laboratory,the yeast two-hybrid screening system was used to discover that the replication factor C-subunit 1(RFC1)of Giardia duodenalis can interact with telomerase in vitro and obtain the in vitro protein interaction technology.verification.Therefore,this study further explored the interaction between RFC1 and telomerase in vivo validation and RFC1 regulation of telomerase activity,providing insights into telomerase activity regulation,cell cycle regulation,or survival strategies for Giardia duodenalis.Experimental support.Interaction between RFC1 and telomerase in cells: By modifying the bimolecular fluorescence complement system to construct the recombinant plasmids pbTRBD-HA-KN151 and pbRFC1-Myc-LC151 and transfect it into 293 T cells by electroporation.After 24 hours culture,the laser Confocal microscopy observed red fluorescence in the nucleus of 293 T cells,indicating that RFC1 and telomerase can interact in the cell.Interaction of RFC1 and telomerase in Giardia duodenalis: By preparing rabbit-derived RFC1 and murine telomerase polyclonal antibodies using Duolink? PLA technology to validate RFC1 in vivo with Giardia duodenalis The interaction of telomerase and the discovery of red fluorescence in the duodenal Duodenalis of the Duodenalis showed the simultaneous presence of telomerase in both nucleus of the Duodenalis,further demonstrating the equivalence of the two cores.Study on Cutting Efficiency of Ribozymes In vitro: According to the characteristics of chimeric hammerhead ribozymes,ribozymes targeting the RFC1 transcript were designed to reduce the expression of RFC1.In the in vitro RFC1 transcript cleavage assay,the constructed ribozyme of the Giardia duodenalis viral vector mediated its cleavage efficiency to 87.26%.It shows that the ribozyme system can effectively reduce the expression of RFC1 and can meet the following experimental requirements.Regulation of telomerase activity by RFC1: Electroporation of a chimeric hammerhead ribozyme mediated by a Giardia virus vector into the Duodenalis Giardia and obtaining hygromycin resistance The Giardia duodenalis strain.The amount of RFC1 transcripts in pC631-RFC1(A)-pac strains was significantly reduced by Real-time PCR,only 31.2% of the wild strains,and the results showed that the growth rate was significantly reduced.Detection of telomerase activity showed that the telomerase activity of the pC631-RFC1(A)-pac strain was significantly lower than that of the wild strain.The experimental results showed that the reduction of RFC1 content in Giardia duodenalis could reduce the reproduction rate of trophozoites and decrease the activity of telomerase.In summary,we found that the presence of telomerase in the two cell nucleus through the interaction of protein in the body of Giardia duodenalis further demonstrated the equivalence of the two cores.For the first time,RFC1 protein was found to regulate telomerase activity,laying an important foundation for further investigation of telomerase activity regulation mechanism of Giardia duodenalis and prevention of giardiasis.