Prokaryotic Expression Of GAPDH Gene And Tpx Gene Of Baylisascaris Schroederi And The Evaluation Of Diagnostic Value Of Recombinant Antigens

Giant panda as a kind of endangered rare animal is known as national treasure and living fossil and Has been rated as the country-level key protect animal.Baylisascariosis of pandas is a parasitic disease which is caused by Baylisascaris schroederi parasitized in intestine of pandas.Baylisascaris schroederi is a common parasite in captive pandas and would make hosts indigestive,vomitive,diarrheal,leptystic,tussive,anaemic and dull.The adults would also cause the blocking of the gastrointestinal tract,biliary and pancreatic duct or even death.At present,the diagnosis of the disease mainly rely on brinefloatation and PCR method,but these two methods both can't carry on early diagnosis of Baylisascariosis of pandas.This study was designed to amplify Glyceraldehyde-3-phosphate dehydrogenase(GAPDH)gene and Thioredoxin peroxidase(Tpx)gene of Baylisascaris schroederi,purified the protein,carried out immunoblotting and immunofluorescence localization.In addition,indirect ELISA was established to evaluate the diagnistic values of the two recombinant protein,the results laid the foundation of early diagnosis and comprehensive prevention and control of this disease.1.Characteristic analysis and the evaluation of diagnostic value of the Glyceraldehyde-3-phosphate dehydrogenase(GAPDH)in Baylisascaris schroederiGlyceraldehyde-3-phosphate dehydrogenase(GAPDH or G3PDH)is a multifunctional enzyme in the process of metabolish of parasites and it's also the key enzyme in the process of glycolysis.some studies have showed that GAPDH could be a potential candidate antigen for parasitic disease.This study amplified GAPDH gene from the total RNA of Baylisascaris schroederi and this protein contained 360 amino acid residues with a predicted molecular mass of 40KDa.The amino acid sequence homology between the population and Ascaris suum was 98%and Molecular mass of the recombinan protein was about 57KDa.Western blot showed that the recombinant protein could react with the positive sera of pandas infected with Baylisascaris schroederi.Fluorescent immunohistochemical results showed that GAPDH was widely distributed in whole tissue,including gut,ovarian,uterus,muscle and eggs.The content of GAPDH in Body wall and ovary wall was more than that in other parts.Moreover,Bs-GAPDH was used as an antigen to establish an indirect ELISA diagnostic method for detecting pandas infected with Baylisascaris schroederi.The results showed that the diagnostic sensitivity and specificity of Bs-GAPDH were 95.83%and 100%respectively.This study suggested that Bs-GAPDH could be a diagnostic candidate antigen of Baylisascariosis of panda.2.Characteristic analysis and the evaluation of diagnostic value of the Thioredoxin peroxidase(Tpx)in Baylisascaris schroederiParasites as a kind of aerobic organisms have to face the reactive oxygen species(ROS)pressure from metabolize and host immunity.For adapting to the double pressure,parasites have to resist these oxidative injury from ROS by evolving a series of antioxidant system.Thioredoxin peroxidase(Tpx)is exactly one of the most important antioxidant enzymes which is widely distrubuted in prokaryote and eucaryon.This study amplified Tpx gene from the total RNA of Baylisascaris schroederi and this protein contained 195 amino acid residues with a predicted molecular mass of 22KDa.The amino acid sequence homology between the population and Ascaris suum was 95.38%and Molecular mass of the recombinant protein was about 39KDa.Western blot showed that the recombinant protein could react with the positive sera of pandas infected with Baylisascaris schroederi.Fluorescent immunohistochemical results showed that Tpx was widely distributed in whole tissue,including gut,ovarian,uterus,muscle and eggs.The content of Tpx in Body wall,ovary wall and eggs were more than that in other parts.Bs-Tpx was used as an antigen to establish an indirect ELISA diagnostic method for detecting pandas infected with Baylisascaris schroederi.The results showed that the diagnostic sensitivity and specificity of Bs-Tpx was 75%and 91.7%respectively.This study suggested that Bs-Tpx was unsuited to be a diagnostic candidate antigen of Baylisascariosis of panda.