The Immune Antigen Chip Was Used To Study The Diagnostic Antigen Of Echinococcus Granulosus

Objective To screen and evaluate specific and sensitive diagnostic targets for echinococcosis granulosus by using protein microarray on a genome-wide scale.Methods 1) Establishment of a local protein database from the whole-genome sequence information of E. granulosus applicable in microarray construction. The local protein database include: ? all the identified cyst fluid proteins sequences from the literatures as of 2013; ? the predicted secreted proteins of E. granulosus by genome-wide prediction and bioinformatic approaches; ?the sequences containing amino acid tandem repeats by analyzing the E. granulosus transcriptome data using the Perl language; 2)High-throughput cloning and expression of the sequences in the local protein database. The approaches used are: ? preparation of the cDNA from the protoscolex and PCR amplification of the target genes; ? cloning of the amplified genes by In-Fusion method to obtain recombinant plasmids ? high throughput expression of the recombinant plasmids in a wheat germ cell-free system and verification of the expression status.; 3)Construction of protein chip with the expressed recombinant proteins for screening and evaluation of specific diagnostic antigens.Results 1) A local protein data base of E. granulosus was established, which containing 73 cyst fluid protein sequences and the secretome of 596 sequences, among them 83 proteins posses the catalytic activity found by GO analysis, and 33 proteins contain repeated sequences identified by the Perl analysis. 2) Of the 239 sequences in the local data base, 173 target fragments(72%) were PCR amplified successfully. From the 235 amplified sequences (173 currently amplified,62 available previously), 188 recombinant plasmids were obtained by In-Fusion method with a cloning efficacy of 80%. Of the 188 clones obtained, 174 soluble proteins were successfully expressed in the wheat germ cell-free system, and verified by Western blot. 3) A microarray containing 174 recombinant proteins was constructed and screened using a pooled serum probe of echinococcosis patients with the healthy sera as control,among them 75 were immunoreactive.Further analysis demonstrated that 6 highest sensitive antigens were EgP9, EgP164, EgP11, EgP44,EgP43 and EgP22, and the diagnostic efficacy was the highest when the EgP9, EgP11, EgP164 and EgP43 were used in combination showing a Youden's index 0.73, which was statistically higher than that of crude hadytid cyst fluid (Youden's index 0.60) under the same test conditions.Conclusion By using In-Fusion cloning, wheat germ cell-free expression and the microarray for high throughput screening based on the local protein data base of E.granulosus established on genome-wide searching, 6 diagnostic candidate antigens were identified from the 174 proteins screened. One of the proteins, EgP43 has not been reported previously. The present study provided a good base for further development of diagnostics for echinococcosis granulosus.