title: Cloning and characterization of an Eimeria necatrix gene encoding a gametocyte protein and associated with oocyst wall formation.
abstract: BACKGROUND
  Gametocyte proteins of Eimeria (E.) spp. are important components of the oocyst wall and some have been used to develop transmission-blocking vaccines against avian coccidiosis.
  
  
  METHODS
  Total RNA isolated from E. necatrix gametocytes was utilized as templates for RT-PCR amplification and sequencing of cDNA encoding a gametocyte protein using gene-specific primers. The cDNA was cloned into the bacterial expression vector pET28a(+) and expressed in E. coli BL21 cells. The antigenicity of the recombinant gametocyte protein and its localization in different E. necatrix life-cycle stages were determined by western blot and indirect immunofluorescence analyses, respectively.
  
  
  RESULTS
  A 731-nucleotide sequence of cDNA [GenBank: KF649255] of E. necatrix had 97.7% identity to that of Etgam22 of E. tenella. The cDNA ORF encoded a 186-amino acid protein containing a histidine-proline-rich region. The recombinant gametocyte protein (rEnGAM22) was predominately expressed in the insoluble inclusion body and recognized by antiserum from chickens immunized with oocysts of E. necatrix, E. maxima and E. tenella. A specific antibody to the rEnGAM22 protein recognized the wall-forming bodies in macrogametocytes and the walls of oocysts and sporocysts.
  
  
  CONCLUSIONS
  The gene cloned from E. necatrix gametocytes is an ortholog to Etgam22 of E. tenella and presents a potential target for future recombinant subunit vaccines against coccidiosis.
authors.forename: Dandan
authors.initials: D
authors.lastname: Liu
authors.forename: Liqin
authors.initials: L
authors.lastname: Cao
authors.forename: Yulan
authors.initials: Y
authors.lastname: Zhu
authors.forename: Changjing
authors.initials: C
authors.lastname: Deng
authors.forename: Shijie
authors.initials: S
authors.lastname: Su
authors.forename: Jinjun
authors.initials: J
authors.lastname: Xu
authors.forename: Wenjie
authors.initials: W
authors.lastname: Jin
authors.forename: Jingui
authors.initials: J
authors.lastname: Li
authors.forename: Lili
authors.initials: L
authors.lastname: Wu
authors.affiliation: Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Ministry of Education Key Lab for Avian Preventive Medicine, Key Lab of Jiangsu Preventive Veterinary Medicine, College of Veterinary Medicine, Yangzhou University, 12 East Wenhui Road, Yangzhou, Jiangsu 225009, People's Republic of China. yzjptao@126.com.
authors.forename: Jianping
authors.initials: J
authors.lastname: Tao
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004539:Eimeria / Q000235:genetics*; D015870:Gene Expression; D005854:Germ Cells / Q000378:metabolism*; D008969:Molecular Sequence Data; D033661:Oocysts / Q000378:metabolism*; D021381:Protein Transport; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D011994:Recombinant Proteins; D016415:Sequence Alignment
chemical_list: D015800:Protozoan Proteins; D011994:Recombinant Proteins