Prokaryotic Expression Of CPI,TPx,TIM,PGK And EN Of D.immitis And Evaluation Of Diagnostic Value Of These Five Recombinant Proteins

Dirofilaria immitis also known as heartworm disease,spreads via mosquito vectors,is mainly parasitic in pulmonary arteries and right atrium of dogs and cats.When human beings are infected with D.immitis,it principally causes pulmonary embolism,and then forms solidary pulmonary nodule.This disease is a worldwide-distributed zoonosis,espicially in tropical and subtropical regions.Currently,the heartworm disease has been reported in 27 provinces crossing five districts in China,seriously endangering public health,which has no ideal measures for diagnosis and prevention.In this study,we have cloned and expressed the cysteine protease inhibitors(Di-CPI),thioredoxin peroxidase(Di-TPx),triose phosphate isomerase(Di-TIM),Phosphoglycerate kinase(Di-PGK)and Enolase(Di-EN)of D.immitis.We also evaluated potential serological diagnostic value of these five recombinant proteins by the indirect ELISA methods in order to screen suitable diagnostic antigen for heartworm disease,which can provide technical support for comprehensive prevention and control of this disease.The results showed that the specificity of rDi-CPI,rDi-TPx,rDi-TIM,rDi-PGK and rDi-EN were 75.0%(18/24),79.2%(19/24),66.7%(16/24),83.3%(20/24)and 95.8%(23/24),respectively.The sensitivity of of rDi-CPI,rDi-TPx,rDi-TIM,rDi-PGK and rDi-EN were 91.7%(22/24),87.5%(21/24),58.3%(14/24),75.0%(18/24)and 62.5%(15/24),respectively.Cross-reaction with 27 canine sera including infection with Echinococcus granulosus(8 samples),Taenia hydatigena(2 samples),hookworm(5 samples),Toxocara canis(12 samples)showed that rDi-CPI had cross-reactions with 9 sera(including 2 E.granulosus,2 T.hydatigena,1 hookworm and 4 T.canis positive sera);rDi-TPx had cross-reactions with 7 sera(including 2 E.granulosus,1 T.canis positive serum).There were cross-reactions between rDi-TIM and 6 sera(including 2 E.granulosus and 4 T.canis positive sera);rDi-PGK and rDi-EN both only had cross-reactions with 1 E.granulosus.In summary,the indirect ELISA method based on rDi-TPx as antigen had high specificity and sensitivity in diagnosis,and did not cross-react with the positive serum of hookworm,so it's the most suitable antigen among these five recombinant proteins for the diagnosis of heartworm disease.