Prokaryotic Expression And Diagnostic Value Of Fatty Acid-binding Protein And Galectin In Baylisascaris Schroederi

Baylisascaris schroederi is a common parasite of captive giant pandas which mainly parasitize in the small intestine.Being infected with B.schroederi the giant pandas often become mental depression,anorexia and emaciation and even end up in death,which makes B.schroederi a great threaten to giant pandas.The diagnosis of this ascariasis was carried out through sedimentation-floatation method or PCR to de-tect eggs in feces.Unfortunately these methods were unable to meet the goals of early diagnosis.In this study,we cloned and expressed the recombinant B.schroederi fatty acid-binding protein(rBs-FABP)and galectin(rBs-GAL)from the adult worm.West-ern blotting and immunolocalization were performed using the purified proteins.The ultimate purpose of this paper is to develop a rapid and accurate indirect ELISA assay for diagnosing of B.schroederi infections in giant pandas.1.Characterization of fatty acid-binding protein and its immunodiagnostic po-tential for detecting the Baylisascaris schroederiFatty acid-binding protein was a big family of cytoplasmic molecule,they can specifically combine fatty acids and some hydrophobic ligands,transport fatty acids from the cell membrane and promote esterification.In order to understand the molec-ular characterization of fatty acid-binding protein in B.schroederi,this study amplified fatty acid-binding protein gene from the total RNA of B.schroederi adult worm,the full length cDNA of fatty acid-binding protein was 438 bp,and the complete ORF of Bs-FABP isolated here encoded a polypeptide of 145 amino acids with a predicted Mw of 16.7 kDa.Its amino acid sequence share similarity with Ascaris suum up to 92%.Molecular mass of recombinant fatty acid-binding protein expressed in E.coli is about 34 KDa.Immunofluorescence staining showed that fatty acid-binding protein were mainly concentrated in gut and ovarian wall of adult worm,and also widely dis-tributed in other tissues.Building on good immunogenic properties,rBsFABP-based ELISA exhibited a sensitivity of 95.8%(23/24)and specificity of 100%(12/12)in detecting anti-FABP antibodies in the sera of naturally infected pandas.This study suggests that Bs-FABP has the potential to be as diagnostic antigen of ascariasis.2.Characterization of galectin and its immunodiagnostic potential for detecting the Baylisascaris schroederiGalectins are widely present in lots of animals including invertebrates and verte-brates and exist in all kinds of tissue cells.They play important roles in the cell adhe-sion,apoptosis,inflammatory response and many other physiological and pathologi-cal processes.This study amplified galectin gene from the total RNA of B.schroederi,the full-length of cDNA is 429 bp encoding 142 amino acids and the molecular weight of Bs-GAL is 16.1 kDa.Its amino acid sequence share a similarity of 76%with Asca-ris suum.Molecular mass of recombinant galectin expressed in E.coli is about 34 KDa.Immunofluorescence staining showed that galectin was mainly distributed in the body wall,uterus and eggs of adult worms.Building on good immunogenic properties,rBsGAL-based ELISA exhibited a sensitivity of 91.7%(22/24)and specificity of 100%(12/12)in detecting anti-GAL antibodies in the sera of naturally infected pan-das.