| The zoonotic importance of giardiasis has been proposed and established by WHO since the late 70's.Giardia duodenalis is an important parasitic protist that is transmitted by the faecal-oral route and causes diarrhea in vertebrates.The parasite infects a wide range of mammals including humans,livestock,and wildlife.Additionally,Giardia can also affect fish,amphibians,reptiles,birds,and rodents.Giardia infections commonly cause diarrhea in humans,especially children,traveler,and immunodeficient or immunocompromised people,accompanied by some other clinical symptoms including abdominal pain,flatule nce,intestinal disease,weight loss and malabsorption syndrome.In addition,Giardia can cause morbidity in livestock that leads to corresponding economic losses.For all,Giardia is regarded as an important zoonotic parasite.The identification and characterisation of Giardia is critical to investigating and understanding the epidemiology of giardiasis.To establish a rapid and effective diagnostic method has become the key point of the prevention and treatment of giardiasis.There is a strongly biologic link between the cytoskeleton and virulence in Giardia.With the aid of cytoskeleton,Giardia can move on the intestine and attach to the epithelium to avoid being swept away.Giardia is a highly polarised protist,and the cytoskeleton is dominated by several structures,including four pairs flagellum,the ventral disk,and median body.Giardins are unique and abundant cytoskeletal proteins for Giardia,including ?-giardin,?-giardin,?-giardin and ?-giardin,that range in size from 29-38 k Da.?-giardins are a class of calcium-dependent membrane-binding proteins,comprising a polygene family with 21 members.The diversity of ?-giardin reveals the importance of the biological function of Giardia.Thus far,there have been a huge number of documented researches on ?-giardins,while the research on ?-giardin and ?-giardin is relatively less.Herein,based on the nucleotide sequence of ?-giardin and ?-giardin in Genbank,the PCR primers were designed.c DNA has been obtained by RT-PCR from the extracted total RNA of G.duodenalis assemblages A.The ?-giardin and ?-giardin genes were amplified from c DNA.The recombinant expression plasmid p ET-28a-?-giardin and p ET-28a-?-giardin were constructed and then transformed into Escherichia coli Rosseta(DE3)strain for the prokaryotic expression of the recombinant proteins induced by IPTG.The pure fusion protein was obtained by KCl-stained gel-cutting purification methoed and urea was used as a denaturant to dissolve inclusion bodies for dialysis renaturation.Subsequently,polyclonal antibodies were prepared against the protein,and its distribution in Giardia was determined by immunofluorescence localization.The results show that the ?-giardin gene has a length of 882 bp and expresses 293 amino acids.The ?-giardin gene is 936 bp in length,which encodes a polypeptide of 311 amino acids.The recombination proteins were about 36 k Da and 38 k Da in weight,respectively,mainly exist as inclusion bodies.However,due to the low expression of ?-giardin,the concentration of purified protein was not enough to immune animals.ELISA and Western-Blotting analysis identified that the antibodies against ?-giardin possessed good antigenic specificity and prominent binding activities.In addition,?-giardin was showed to be mainly located in the ventral disk of G.duodenalis trophozoites by immunofluorescence techniques.The aim of this study is to provide a theoretical basis for the establishment of ELISA detection methods and provide theoretical basis for the study of pathogenic mechanism,clinical diagnosis and treatment of G.duodenalis. |
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