title: [Prokaryotic expression and identification of S-dsRNA gene from Cryptosporidium parvum virus].
abstract: OBJECTIVE
  To clone and express S-dsRNA gene of Cryptosporidium parvum virus, and investigate the reactogenicity of the recombinant.
  
  
  METHODS
  Total RNA was extracted from Cryptosporidium parvum and S-dsRNA gene was amplified by RT-PCR. The PCR product was cloned into pET-28a(+) expression vector. The recombinant plasmid pET-28a(+)-S was transformed into E. coli BL21 (DE3) and induced with IPTG. The expression situation of recombinant protein was analyzed by SDS-PAGE. Its reactogenicity was examined by Western blotting analysis.
  
  
  RESULTS
  pET-28a (+)-S was identified by PCR and double endonuclease digestion. SDS-PAGE result showed that the recombinant protein (M, 37,000) was expressed in the form of inclusion body. High level expression of recombinant protein was found at 1 mmol/L IPTG condition after incubation at 37 degrees C for 4 h and reached up to 72.6% of the total protein. The protein was recognized by the antisera from mice immunized with antigens from Cryptosporidium parvum oocysts.
  
  
  CONCLUSIONS
  The S-dsRNA gene of Cryptosporidium parvum virus has been expressed with adequate reactogenicity.
authors.affiliation: College of Animal Science and Veterinary Medicine, Jilin University, Changchan 130062, China.
authors.forename: Yu-mei
authors.initials: YM
authors.lastname: Diao
authors.forename: Peng-tao
authors.initials: PT
authors.lastname: Gong
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.forename: Li-bo
authors.initials: LB
authors.lastname: Su
authors.forename: Xiang-sheng
authors.initials: XS
authors.lastname: Huang
authors.forename: Jian-hua
authors.initials: JH
authors.lastname: Li
authors.forename: Xi-chen
authors.initials: XC
authors.lastname: Zhang
mesh_terms: D000818:Animals; D036022:Capsid Proteins / Q000235:genetics* / Q000302:isolation & purification*; D016785:Cryptosporidium parvum / Q000821:virology*; D005260:Female; D015870:Gene Expression; D005822:Genetic Vectors; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids; D012328:RNA Viruses / Q000235:genetics*; D012330:RNA, Double-Stranded; D012367:RNA, Viral / Q000235:genetics*
chemical_list: D036022:Capsid Proteins; D012330:RNA, Double-Stranded; D012367:RNA, Viral