Screening And Identification Of Circulating Antigen In Serum From Rabbit Infected With Schistsoma Japonicum

Diagnosis is critical to prevention and treatment of schistosomiasis. A selection of candidate antigen for diagnosis of schistosomiasis always has an important influence on the monitoring and control of schistosomia-sis. Circulating antigens secreted by live schistosomes in body fluids were used to indicate the infection intensity and to assess cure. Identification of Schistosoma candidate circulating antigens with high sensitivity and specificity for diagnosis of schistosomiasis is necessary.In our previous researches, we confirmed that SIEA26-28kDa SjscFv with special targeted-effect can identify Schistosoma japonicum from female to immature eggs,mature eggs.Therefor,we took advantage of specific targeting immune recognition ability of SjscFv and regard SjscFv as a probe to recognize Schistosoma circulating antigen in rabbit serum by Western Blot. The selected circulating antigen based on NanoLC-MS/MS mass spectrometry and bioinformatic analysis might be useful diagnosetic targets against schistosomiasis, which laid the foundation of establishing a high sensitivity and specificity method of detecting circulating antigen.Objective1.Construct pET43.1a/SjscFv prokaryotic plasmid, express,purify and identify SjscFv protein.2.Screen the Schistosoma circulating antigen in rabbit serum recognized by SjscFv.3.NanoLC-MS/MS mass spectrometry and bioinformatics analysis circulating antigen, in order to find new ways for the development of anti-schistosomiasis diagnostic antigen.Methods1.The construction of Prokaryotic plasmid pET43.1a/SjscFv and expression,purification and identification SjscFv proteinWe designed upstream and downstream primer according to the open reading frame (ORF), and regarded the pET28a/SjscFv plasmid as template to amplify the gene fragment of SjscFv, ligated the digested fragments of pET43.1a and PCR amplification. After identificated by broth PCR, double digestion analysis and DNA sequencing,SjscFv plasmid was cloned into the vector pET43.1a and transformed into the E.coli BL21(DE3). SjscFv protein were abundantly expressed on the expression conditions of optimum IPTG concentration, time and temperature, and were identified by Western blot. The protein have a protein concentration measurement after purified by Ni-NTA agars.2.1dentification of the circulating antigen of Schistosoma in rabbit serum recognized by SjscFv.Separate Schistosoma japonicum infection rabbit serum from2to10weeks post-challenge by SDS-PAGE and Native-PAGE gel electrop-horesis, and then find specific combination of gel with recombinant proteins pET43.1a/SjscFv by Western Blot. After rabbit serum circulating antigen components recognized by SjscFv were purified by electroelution, rabbits were immunized with the mixture of purified antigen and the same volume adjuvant, then collect immune rabbit serum with the high antibody titer. Extract crude antigen of adults and eggs, collected S. japonicum adult and the liver of rabbit infected S. japonicum for paraffin sections, Western Blot and immunohistochemistry were used to analysis expression and localization of SjscFv protein.3.Analysis and identify the circulating antigen recognized by SjscFv by NanoLC-ESI-MS/MS mass spectrometry,bioinformatics analysis were used to explore the structure and function of circulating antigen which have a good diagnostic antigen application prospect.Results1.Both PCR,double enzyme digestion and DNA sequencing all confirmed that the fragment carried by the constructed plasmid pET43.1a/SjscFv corresponded to the target gene.The target protein largely were expressed on91kDa, the results of optimizing prokaryotic expression conditions were when cultured at28?and0.05mmol/L IPTG overnight.The concentration of protein purified by column chromatography can reached to0.339mg/mL and Western Blot showed it have a good immunreactivity.2.pET43.1a/SjscFv showed specific target binding bands with Schistosoma japonicum infection rabbit serum from2to10weeks post-challenge on95kDa by Western blot. Western Blot and immunohi-stochemistry showed the SjscFvT protein largely expressed in the adult body surface.3.NanoLC-ESI-MS/MS mass spectrometry analysis identified six proteins,they are heat shock protein, actin, Importin-?1, Na+/K+transporting ATPase subunit alpha, SjCHGC04905protein, SjCHGC06108protein, and relative molecular weight of Schistosomiasis Importin-beta protein was101214.1KDa, corresponding with results of SDS-PAGE, Importin-beta1mainly has HEAT repeat domain,IBB_N domain ARM repeat domain,the tertiary structure of Importin-beta made up of alpha helix can freely stretch or shrink, no transmembrane area, there are abundant antigen epitope, which made it to be a good candidate diagnosis molecules.ConclusionsLpE43.1a/SjscFv was successfully constructed;the target protein SjscFv were expressed and purified.2.Found six associated circulating antigen, the circulating antigen abundantly expressed in the adult body surfaced and were inferred it was associated antigen in the early of Schistosoma japonicum infection.3.Structural and functional analysis of importin-?1showed it may be a good candidate diagnosis antigen.