| Giardia duodenalis distributes globally,about 200 million people in Asia,Africa and Latin America are infected with Giardia each year and exhibit certain clinical symptom.Because of its zoonotic nature,it is listed as one of the ten major parasitic diseases that endanger human health all over the world.Hosts are infected with infective Giardia cysts and the infection can cause diarrhea,dizziness,and weight loss.Hosts that Giardia can infect range widely,including a variety of vertebrates such as humans,non-human primates,cattle,sheep,pigs,birds,dogs,cats,foxes,tigers,etc..Giardia can also infect fishes,amphibians,reptiles,and rodents.With the development of tourism,Giardia has become a common pathogenic agent of traveler’s diarrhea.Therefore,as an important zoonotic pathogen,Giardia is getting more and more attention.Cytoskeleton is one of the important features specific to eukaryotes.Among the cytoskeletal proteins found in Giardia,giardins play an important role in adsorption,invasion,and infection.Giardia is also a model to study eukaryotes.Two Giardia strains(with virus and without virus)were referred in this study.Giardia lamblia(formally G.duodenalis)virus(GLV)is a kind of double-stranded RNA specific to Giardia.GLV can infect Giardia strain without GLV.There were lots of molecular and epidemiological researches present for Giardia,while this is not the case for GLV.Infection with a dose of 5×10~5GLV will not affect the normal state of the Giardia.Therefore,in recent years,the use of GLV as an exogenous-gene carrier has become more and more widespread.Both the GLV and its transcripts are infectious.Coat protein is the main protein expressed by GLV.Alpha giardin is the protein unique to Giardia and there might be certain relationship between GLV infection and alpha giardin expression.In this study,we designed primers based on the sequence of alpha-15-giardin obtained from GenBank,cultured Giardia trophozoites to full of the bottle,collected them and extracted the total RNA,and then obtained the cDNA by reverse transcription.We used cDNA as the template to amplify alpha-15-giardin gene,successfully constructed the recombinant expression plasmid pGEX-4T-α-15,and it was transformed into the BL21 competent cells.The prokaryotic expression of pGEX-4T-α-15 fusion protein was induced by IPTG method.SDS-PAGE was used to analyze its expression style and the pure fusion protein was obtained by using the gel extraction method.The purified protein was used to immunize mice,the polyclonal antibody was prepared,and the immune serum with high potency and specificity was obtained.The protein was located in the cytoplasm of Giardia and near the cytoplasmic membrane.No distribution of the protein was observed at the abdominal flagellum.Then,we extracted the total proteins of two kinds of Giardia strains for Western blot analysis and qPCR analysis.Based on the results,it can be inferred that alpha-15-giardin had an effect on resistance to the GLV.The data might be helpful in understanding the alpha-15-giardin biological features and the relationships between alpha-15-giardin expression and GLV infection,searching for some new drug targets,and the diagnosis and prevention of giardiosis. |
