title: [Cloning and expression and preliminary antigenicity identification for the diagnostic antigen of hepatitis E virus].
abstract: OBJECTIVE
  To clone, express and purify thioredoxin (named as N5), a specific diagnostic antigen of hepatitis E virus (HEV), and to initially evaluate its antigenicity and serological test performance.
  
  
  METHODS
  Based on the gene sequences of HEV-ORF2 and carboxyl terminal ORF3 on GenBank, the codon was optimized by the Escherichia coli codon preference, inserted it into prokaryotic expression vector M48 following total gene synthesization, and expressed in Escherichia coli fusion protein N5 recombined with Thioredoxin (TRX). Fusion protein was purified in affinity chromatography, evaluating its antigenicity with Western blot technology, then evaluating its serological test performance using the negative and positive serum samples confirmed of HEV infection with laboratory and clinical tests.
  
  
  RESULTS
  The recombinant plasmid expressing N5 diagnostic antigen was successfully established; high-level expression and purification to obtain soluble diagnostic antigens; Western blot results indicating fusion protein N5 can be bound specifically with the serum of HEV IgM antibody positive, showing satisfactory antigencity; using fusion protein N5 as the capture antigen to build indirect ELISA, testing 40 serum samples of HEV cases confirmed by pathogen detection and clinical diagnosis and 40 serum samples of healthy people, with the sensitivity and specificity of 95% (38/40) and 90% (36/40) respectively.
  
  
  CONCLUSIONS
  Recombinant plasmid expressing the HEV diagnostic antigen recombined with thioredoxin was successfully established, and soluble fusion protein N5 was obtained with high expression and strong antigenicity, promising in its future applications.
authors.affiliation: Department of Physiology, Xiangya School of Medical, Central South University, Changsha 410000, China; National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention.
authors.forename: Li
authors.initials: L
authors.lastname: Wan
authors.affiliation: National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention.
authors.forename: Qiudong
authors.initials: Q
authors.lastname: Su
authors.affiliation: National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention.
authors.forename: Yao
authors.initials: Y
authors.lastname: Yi
authors.affiliation: National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention; Email: bisl@ chinacdc.cn.
authors.forename: Shengli
authors.initials: S
authors.lastname: Bi
authors.affiliation: Department of Physiology, Xiangya School of Medical, Central South University, Changsha 410000, China; Email: guanchaxiang@csu.edu.cn.
authors.forename: Chaxiang
authors.initials: C
authors.lastname: Guan
mesh_terms: D015153:Blotting, Western; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D016752:Hepatitis E virus / Q000276:immunology*; D006801:Humans; D010957:Plasmids; D011994:Recombinant Proteins; D013879:Thioredoxins / Q000235:genetics*
chemical_list: D011994:Recombinant Proteins; C491203:TXN protein, human; D013879:Thioredoxins