CDNA Cloning, Genomic Structure And Recombinant Protein Expression Of The Duck IL-8

In order to clarify molecular character of duck IL-8, and to obtain its recombinant protein, and then to promote the research of avian interleukin-8(IL-8), we cloned duck Il-8(Anpl-IL-8) firstly and elucidated its genomic structure. Finally recombinant protein of Anpl-IL-8(rAnpl-IL-8) was expressed and identified.Firstly, a duck cDNA PCR library was constructed based on separating peripheric lymphocyte from duck blood, stimulated with Con A and isolation total RNA from the cells stimulated. According to the sequences alignment of chicken, human and other animals, a pair of degenerate primer was designed to amplify a fragment of the duck interleukin-8(Anpl-IL-8). RACE-PCR was adopted to amplify the 5' end of Anpl-IL-8, and then Anpl-IL-8 full cDNA was cloned by Anchor-PCR. At the same time, the genome of Anpl-IL-8 was cloned and analyzed its structure. Finally a pair of expressed primer was designed to sub-clone the mature peptide of Anpl-IL-8, which was inserted into pQE30 expression system to construct pQE30/Anpl-IL-8 prokaryotic system.The results show that the cloned Anpl-IL-8 coded 103 amino acids peptide, including 21 amino acids of signal peptide and 82 amino acids mature protein. It shares an 80.9% nucleotide acids homology and a 90.3% amino acids homology with the chicken. Genome of Anpl-IL-8 was cloned and analysised. The results show that the genenom include 4 extrons and 3 introns, and the sequence of the extrons was the same to the cDNA of the Anpl-IL-8. The phylogenetic tree of IL-8 indicates the evolutionary pathways to other animals. At the same time, a prokaryotic expression system of the pQE30/Anpl-IL-8 was constructed and used to express Anpl-IL-8 .The cells were centrifuged, sonicated in order to get inclusion bodies, and the inclusion bodies were purified by the Ni-NTA affinity. The results of Tricine DSD-PAGE show that the rAnpl-IL-8 had a molecular weight of about 9.0kDThe research on Anpl-IL-8 provide the dates for further study the mechanism of inflammatory and immunity responses of the Anpl-IL-8...