Screening And Identification Of Protein Interacting With RNA-dependent RNA Polymerase In Giardia Duodenalis

Giardia duodenalis,also known as Giardia lamblia(Giardia),as parasitic protozoa,mainly causes clinical symptoms such as diarrhea in humans and animals,and is prevalent worldwide,seriously threatening the health of animals and humans.However,there is still lack of effective drugs or vaccines for prevention and control.The main reason is that the pathogenesis of Giardia is not fully clear.Giardia is a model organism,so it is very important to study the mechanism of Giardia.The parasitic viruses is exclusively parasitic in parasites and studies have shown that the protozoan virus can affect the pathogenicity and virulence of protozoa.Relevant reports indicate that the infection of Giardiavirus can inhibit the growth of Giardia and reduce the pathogenicity of Giardia,thereby reducing the clinical symptoms of Giardia disease.Although the invasion of Giardiavirus can change the growth characteristics of giardia,the mechanism of interaction between Giardia and Giardiavirus needs further study.RNA-dependent RNA polymerase(Rd Rp)is the main protein encoded by ds RNA viruses.Studies have shown that Rd Rp can interact with other components of the virus or host factors when it plays an important role.However,little research has been conducted on Rd Rp of the Giardiavirus.Therefore,in this study,the interaction proteins of Rd Rp were screened by constructing Giardia yeast two-hybrid c DNA library.GST pull-down,Co-Immunoprecipitation,bimolecular fluorescence complementation and Duolink proximal ligation assay techniques were used to verify the interaction between Rd Rp and interacting proteins.Construction of a yeast two-hybrid c DNA Library of Giardia:The RNA of Giardia trophozoite(WBc2 strain)was extracted by Trizol method and reverse-transcribed into c DNA.The two-hybrid c DNA plasmid library of giardia yeast was constructed by SMART technology.The results showed that the capacity of the constructed library was 3.7×106 cfu,and the recombination rate of the library was 100% and the insertion fragments ranged from 400 bp to 2 kbp when the insertion fragments of 60 monoclonal colonies selected randomly.Screening of Rd Rp Interacting Proteins of Giardiavirus: The bait plasmid p GBKT7-Rd Rp was constructed by recombining the Rd Rp fragment of Giardiavirus amplified by PCR with bait vector p GBKT7,and the bait vector was transformed into Y2 HGold yeast competent cells.The results showed that the bait protein was successfully expressed in yeast cells without obvious toxicity and self-activation.The Rd Rp-interacting proteins were screened by co-transformation of p GBKT7-Rd Rp with the plasmid of the c DNA library.The Rd Rp-interacting protein was preliminarily screened.The candidate gene was chaperone protein dna J.The interaction between Rd Rp and chaperone protein dna J was preliminarily verified by alpha-galactosidase assay.Verification of the interaction between Rd Rp and chaperone protein dna J in vitro: Prokaryotic expression vectors p ET-32a-Rd Rp and p GEX-4T-1-chaperone protein dna J were constructed to induce expression and purify the recombinant protein.The results showed that both expressed proteins were soluble.The purified recombinant protein Rd Rp was co-incubated with chaperone protein dna J.GST pull-down technique was used to verify the interaction between the two proteins in vitro.Verification of the Interaction between Rd Rp and chaperone protein dna J in Cells: The eukaryotic expression vectors pc DNA-His-Rd Rp and pc DNA-HA-chaperone protein dna J were constructed and co-transfected into HEK-293 T cells.Western blot was used to detect the expression of protein.The results showed that both Rd Rp and chaperone protein dna J could be expressed in HEK-293 T cells.The results of Co-Immunoprecipitation showed that there was interaction between the two proteins.The constructed bimolecular fluorescent vector pb Fos-Rd Rp and pb Jun-chaperone protein dna J were co-transfected into HEK-293 T cells.The interaction between Rd Rp and chaperone protein dna J was verified by bimolecular fluorescence technique.Verification of the interaction between Rd Rp and chaperone protein dna J in Giardia duodenalis:The antisera of rabbit-derived Rd Rp protein and mouse-derived chaperone protein dna J were prepared,and the interaction between Rd Rp and chaperone protein dna J in Giardia was verified by Duolink proximal ligation assay technique.Red fluorescence was found in the cytoplasm of Giardia.The results showed that there was interaction between Rd Rp and chaperone protein dna J in Giardia.Preliminary Function studies of chaperone protein dna J:Giardia trophozoites were treated with KNK437,an inhibitor with concentrations of 100 μM and 200 μM,respectively,to reduce the gene expression of chaperone protein dna J,and to detect the effects of KNK437 on Giardia proliferation and Giardiavirus replication.The results indicated that the decrease of chaperone protein dna J expression could reduce the proliferation of Giardia trophozoites and the number of Giardiavirus copies.It preliminarily indicated that chaperone protein dna J had a positive regulatory effect on Giardiavirus.In conclusion,the protein that interacts with Rd Rp is chaperone protein dna J,which was preliminarily screened by constructing the Giardia yeast two-hybrid c DNA plasmid library.The interaction between Rd Rp and Chaperone protein dna J was verified by GST pull-down,Co-Immunoprecipitation and bimolecular fluorescence complementary technique.The interaction between Rd Rp and chaperone protein dna J in the cytoplasm of Giardia was further verified by Duolink proximal ligation assay technology.After decreasing the expression of chaperone protein dna J,the function of chaperone protein dna J was preliminarily explored and its positive regulatory effect on Giardiavirus was found,which provided a theoretical basis for further study on the role of Giardia chaperone protein dna J on Giardiavirus.