Aim: 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.
Conclusion: The novel swine gene P58(IPK) has been successfully cloned and its polyclonal antibody has been prepared.