Prokaryotic Expression And Preliminary Assessment Of LAP And TIM To Evaluation The Diagnostic Value In Echinococcus Granulosus

Cystic echinococcosis(CE)caused by the larval stage of Echinococcus granulosus,which parasitize in the liver and lungs and other organs of many intermediate host(livestocks and humans),a widespread parasitic zoonosis.Our country is one of the leading countries with CE in the word.In this study,we cloned and characterized leucine aminopeptidase(Eg-LAP)and triosephosphate isomerase(Eg-TIM),and western blotting,fluorescent immunohistochemical analysis were perform.Meanwhile,to evaluate the diagnostic value of these E.granulosus recombinant antigens,indirect ELISA method was established.Our studies provide technical support for comprehensive prevention and control of cystic hydatid.1.Cloning,expression and characteristic analysis of leucine aminopeptidase in Echinococcus granulosusLAPs cleave N-terminal proteins and peptides and also play major roles in regulating cellular catabolism and anabolism.In this study,we cloned and identified Eg-LAP.Through bioinformatics analysis found LAP contain the M17 members signature octapeptide NTDAEGRL and five metal binding.Phylogenetic analysis showed the Eg-LAP was clustered into a clade closely related to LAP from other cestode parasites,and closest relative with Echinococcus multilocularis.E.coli BL21(DE3)was induced to express insoluble rEg-LAP with a His-tag,then western blotting(WB)result show rEg-LAP possess immunogenicity.Fluorescence immunohistochemistry showed that native Eg-LAP was distributed in the tegument and hooks of PSCs,and widely distributed in the whole germinal layer and adult worm parenchymatous tissue,and only a few fluorescence was detected in the eggs.REg-LAP protein were used as an antigen to establish an indirect ELISA(iELISA)diagnostic method for sheep hydatid disease.The results showed that the diagnostic specificity of rEg-LAP were 79.09%(87/110);the diagnostic sensitivity of rEg-LAP protein were 95.8%(23/24).The cross reaction with Cysticercus tenuicollis and Coenurus cerebralis was 21.34%(3/14);We evaluate the diagnostic value of hydatid fluid antigen with the same serum found the specificity and sensitivity were 65.45%(72/110)and 41.7%(10/24)respectively.The rEg-LAP recombinant protein specificity and sensitivity is higher than fluid antigen respectively 13.64%and 54.1%,can be used as a candidate diagnostic antigens.2.Cloning,expression and characteristic analysis of triosephosphate isomerase in Echinococcus granulosusTriosephosphate isomerase play an irreplaceable role in glycolysis transformation between the two subunits the process,at the same time also prompted sugar glycolysis reaction to produce a lot of energy.In this research,no transmembrane regions or signal peptide sequences were predicted in the Eg-TIM amino acid sequence while triosephosphate isomerase contain 8 alpha helix,8 beta folding,interphase arrangement,10 highly conserved amino acid sequences(AYEPVWAIGTG)contain active site.Phylogenetic analysis showed the Eg-TIM was clustered into a clade closely related to TIM from other cestode parasites,especially closely with Echinococcus multilocularis.E.coli BL21(DE3)was induced to express insoluble rEg-TIM with a His-tag,then western blotting(WB)result show rEg-TIM possess immunogenicity.Fluorescence immunohistochemistry showed that native Eg-TIM was distributed in the the part which need to evaginate and hooks of PSCs,and widely distributed in the whole germinal layer and adult worm parenchymatous tissue.REg-TIM protein were used as an antigen to establish an indirect ELISA(iELISA)diagnostic method for sheep hydatid disease.The results showed that the diagnostic specificity of rEg-LAP were 53.6%(59/110);the diagnostic sensitivity of rEg-LAP protein were 87.5%(21/24).