title: [Eukaryotic expression and identification of the recombination protein of rat decay-accelerating factor with Pichia pastoris expression system].
abstract: OBJECTIVE
  To construct yeast eukaryotic expression vector carrying the gene of rat decay-accelerating factor (DAF), and to induce the expression of recombinant protein.
  
  
  METHODS
  The cDNA of rat DAF was amplified by RT-PCR from the fresh hepatic tissue of rat. The PCR product was inserted into the Pichia pastoris vector pPIC9K. Then the recombinant plasmid pPIC9K-DAF was transformed into yeast GS115 through electroporation. The positive high copy number transformants were rapidly selected by using G418-YPD and were induced by methanol. The induced product was analyzed by DNA sequencing, SDS-PAGE and Western blot. Finally, the bio-activity of recombinant rat DAF was examined.
  
  
  RESULTS
  The rat DAF cDNA was successfully cloned in pPIC9K. The positive clone in pPIC9K expression vector (pPIC9K-DAF) was sieved, and the pPIC9K-DAF showed the same seqencing result as reported in GenBank. And the recombination protein of rat DAF (relative molecular mass is 70 x 10(3)) has natural rat immunogenicity and bio-activity.
  
  
  CONCLUSIONS
  Successfully cloning and high-level expression of rat DAF recombination protein in Pichia pastoris laid a foundation for further research on immunity and complement system.
authors.affiliation: Department of Pathology, West China Second Hospital, Sichuan University, Chengdu 610041, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.forename: Li
authors.initials: L
authors.lastname: Zhang
authors.forename: Peng
authors.initials: P
authors.lastname: Zhang
mesh_terms: D000818:Animals; D018958:CD55 Antigens / Q000096:biosynthesis* / Q000235:genetics; D003001:Cloning, Molecular; D018274:Electroporation; D005260:Female; D005822:Genetic Vectors / Q000235:genetics*; D008099:Liver / Q000737:chemistry; D008297:Male; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection
chemical_list: D018958:CD55 Antigens; D011994:Recombinant Proteins