Analysis Of Genes Expression Profiles In Different Developmental Stages And Screening For Specific Antigens In Echinococcus Granulosus

Objective:To screen specific antigens of protoscolex(PSC) stage of Echinococcus granulosus(Eg),we elicit bioinformatics methods to compare specific antigens in different developmenalt stages of Eg expression profile and find differential antigens in PSC stage, so as to screen the cumulative data of specific diagnostic antigen which lay the foundation for the early diagnosis and prevention of Eg in human and livestock.Method:1. Analysis of differential antigen expression in different developmental stages: using Eg m RNA transcriptome sequencing data which published in http://chgc.sh.cn/Eg by China National Center for Human Genome Research(CHGCS), we screened differential molecules which is expressed in PSC but not in oncosphere according to the article reads and RPKM in different developmental stages.2. Using Uniprot and Softberry Prediction software to locate these differential molecules, membrane and secretory proteins were screened out. Then DNAstar, NCBI/BLAST public database and www.predictprotein.org online database were used to predict epitopes, analyze secondary structure and homology comparison of these specific molecules. Finally, a range of candidates were selected for the following research.3. Expression one of candidate molecule in Escherichia coli(E. Coli): Isolation of target gene(1) : PSCs were isolated from clinical operation, of which total RNA were extracted by TRIZOL. Then target gene was obtained by RT-PCR; The target gene was cloned into p(2) GEM-T vector and transformed into E. coli JM109;(3)The target gene was cloned into p ET-28 a expression vector and transformed into E. coli BL21(DE3) to induce the expression of the recombinant protein(r EG-01883), then the recombinant protein was purified by His-bind resin column containing Ni2+. At last, r EG-01883 was identified by western blot.4. Analysis of immunological characteristics :(1) The recombinant protein was used to immunize animals, and subsequently antiserum was obtained.(2) The immunological characteristics of recombinant proteins were analyzed and identified by ELISA and western blot.5. Preliminary evaluation target molecular for potential value of diagnostic antigen: Binding activity of recombinant proteins with cystic echinococcosis(CE) patients was detected by ELISA, compared with healthy donors.Results:1. We obtained 319 specific molecules of protoscolex from 7218 Eg genes.2. Finally 26 molecules encoding membrane and secretory proteins were identified as candidate by bioinformatics methods.3.The selected target gene EG-01883 was obtained successfully by RT-PCR. Then vector EG-01883/p ET-28a/BL21(DE3) was successfully constructed. The target protein r EG-01883 was successfully expressed, purified and identified by his-Tag antibody. Molecular weight of r EG-01883 was as predicted as about 36 k D.4. Immunological characteristics of r EG-01883: ELISA showed that ? The purified recombinant protein r EG-01883 induced specific Ig G antibody response; ? The levels of IFN-? and IL-4 in vaccinated group were significantly higher than that in adjuvant group or control group(p<0.05); ? Western blot results showed that r EG-01883 was recognized by immunized and infected serum of mice.5. Recombinant protein r EG-01883 was recognized by sera of CE patients and the binding activity was significantly higher, compared with sera of healthy control(p<0.05).Conclusion:We firstly confirmed that PSC-specific EG-01883 showed immunogenicity and antigenicity, which could recognize the sera of CE patients, revealing that it possess the potential value of diagnostic antigen.