title: [Cloning, prokaryotic expression of novel swine gene P58IPK and its polyclonal antibody preparation].
abstract: OBJECTIVE
  To clone a novel swine gene P58(IPK)[58-kDa(inhibitor of protein kinase) protein] and prepare its polyclonal antibody for further research of influenza and host interaction.
  
  
  METHODS
  The swine P58(IPK); gene was first identified in silico through homology searching in the swine EST database. Then this gene was amplified by reverse transcription polymerase chain reaction (RT-PCR). The cDNA of the gene contained the complete open reading frame(ORF) of 1 518 bp, and encoded 505 amino acid residues (Accession No.HQ287801). The gene was first analyzed using bioinformatics methods. Then P58(IPK) was cloned into a prokaryotic expression vector pET-32a to construct a recombinant plasmid named as pET-P58(IPK). The fusion protein his-P58(IPK) was expressed in E.coli BL21 and purified using a his-tag protein purification column. Subsequently rabbits were immunized with the purified protein.
  
  
  RESULTS
  Specific polyclonal antibody against the fusion protein his-P58(IPK) was obtained. The activity of the antibody was determined through double-immunodiffusion test. The titer of the antibody was 1:20 000 as shown by ELISA. specifically recognized the protein P58(IPK) by Western blot and immunofluorescence assay.
  
  
  CONCLUSIONS
  The novel swine gene P58(IPK) has been successfully cloned and its polyclonal antibody has been prepared.
authors.affiliation: Investigation Group of Molecular Virology, Immunology, Oncology & Systems Biology, Center for Bioinformatics, Northwest A & F University, Yangling 712100, China.
authors.forename: Jun-ge
authors.initials: JG
authors.lastname: Wen
authors.forename: Hao
authors.initials: H
authors.lastname: Song
authors.forename: Yan
authors.initials: Y
authors.lastname: Sun
authors.forename: Yuan
authors.initials: Y
authors.lastname: Gao
authors.forename: Zhi-kun
authors.initials: ZK
authors.lastname: Xu
authors.forename: Lian-feng
authors.initials: LF
authors.lastname: Bo
authors.forename: De-li
authors.initials: DL
authors.lastname: Zhang
mesh_terms: D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D050956:HSP40 Heat-Shock Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D007114:Immunization; D010957:Plasmids; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D013552:Swine
chemical_list: D000906:Antibodies; D050956:HSP40 Heat-Shock Proteins; D011994:Recombinant Proteins